Involvement of Secondary Metabolites and Extracellular Lytic Enzymes Produced by Plant Growth Promoting Rhizobacteria in Inhibiting the Soilborne Pathogens in Faba Bean Plants

Research Site

All experiments were carried out at the Agricultural Research Station in the New Valley, New Valley Governorate and Egypt.

Source of Fungal Pathogen

The highly pathogenic isolate each of R. solani, F. solani, F. oxysporum and M. phaseolina isolated from diseased chickpea plants collected from New Valley Governorate was used in this study Khalil SM, et al. [9].

Preparation of Fungal Inoculum

The inoculum of pathogenic fungi was prepared from one week old culture grown on 50 mL potato dextrose broth (PDB) medium in conical flask (250 mL) and incubated at 25 ± 1°C. The content of flask was homogenized in a blender for one min. Plastic pots were filled with sterilized soil and mixing with fungal inocula at rate of 100 mL homogenized culture per pot, seven days before planting.

Source of the PGPR

Plant growth promoting rhizobacteria (PGPR) namely, B. megaterium ((isolate BMM5), B. subtilis (isolate BSM1) and B. cereus (BCM8) and Ps. fluorescens (PFM1) were obtained from the Laboratory. of Plant Pathology Department, New Valley Agric. Res. Station.

In Vitro Screening of Inhibitory Effect of PGPR

The tested isolates of antagonistic PGPR were streaked at one side on PDA medium in plates and incubated for 24 hours at 25°C±1, then one disc (7 mm in diameter) of tested pathogenic fungi viz. R. solani, F. solani, M. phaseolina and F. oxysporum was placed on the opposite side [10]. The inoculated plates with pathogenic fungus only were used as control. After 3-7 days incubation, linear growth of pathogenic fungi in all treatments was recorded. The decrease of percentage that occurred in linear growth of the pathogenic fungi was determined at the end of the experiment using formula as follows: Reduction in linear growth = [(R1- R2)/R1] ×100 R1= the radius of normal growth in control plates; R2= the radius of inhibited growth

Production of Indole Acetic Acid (IAA)

The production of IAA was determined by colorimetric measurement at 530 nm using Salkowski’s reagent. Bacteria were grown under shaking (120 rpm) for 2 days at 30ºC in Luria Bertani broth medium (Tryptone 10.0 g, Yeast Extract 5.0 g Sodium Chloride 10.0 g) supplemented with tryptophan (1 mg mL−1) as IAA precursor. After incubation, the cells were centrifuged (3,000 rpm for 10 min at 4°C) and 1 mL of supernatant was combined with 2 mL of Salkowski’s reagent (150 mL of 95-98% H2SO4, 7.5 mL of 0.5 M FeCl3•6H2O, and 250 mL distilled water) and incubated for 30 min at room temperature. The quantification of IAA was carried out using a standard curve of pure IAA (Sigma−Aldrich, Co.).

Production of Hydrogen Cyanide (HCN)

Bacterial strains were grown at 25±2°C on a rotary shaker in Tryptic Soy Broth (TSB). Filter paper (Whatman No. 1) was cut into uniform strips of 10 cm long and 0.5 cm wide saturated with alkaline picrate solution and placed inside the conical flasks in a hanging position. After incubation at 25±2°C for 48 h, the sodium picrate present in the filter paper was reduced to reddish compound in proportion to the amount of hydrocyanic acid evolved. The developed color was eluted in 10 mL of distilled water and the absorbance was measured at 625 nm.

Production of Siderophore

Bacterial strains were grown in KB broth for 3 days at 25±2°C and centrifuged at 3000 rpm for 10 min and the supernatants were collected. The pH of the supernatant was adjusted to 2.0 with diluted HCl and equal quantity of ethyl acetate was added in a separating funnel, mixed well and ethyl acetate fraction was collected. Five milliliters of ethyl acetate fraction were mixed with 5mL of Hathway’s reagent (1.0 mL of 0.1M FeCl3 in 0.1 N HCl to 100 mL distilled water1.0 mL of potassium ferricyanide). The absorbance for dihydroxy phenols was read at 700 nm. A standard curve was prepared using dihydroxy benzoic acid. The quantity of siderophore synthesized was expressed as μmol benzoic acid/mL of culture filtrate.

Ammonia Production

Freshly grown culture was inoculated into peptone water and incubated for 7, 10, and 13 days at 27±2°C. Broth was collected, centrifuged and the amount of ammonia in the supernatant was estimated by means of Nesslerization reaction 1 mL Nessler’s reagent was added to 1 mL of supernatant and volume of this mixture was made up to 10 mL by addition of ammonia-free distilled water. Development of brown to yellow color was a positive test for ammonia production and optical density was measured by spectrophotometer at 450 nm [11]. The concentration of ammonia was estimated based on a standard curve of ammonium sulfate ranging from 0.1 to 1 μmol mL-1.

Phosphate Solubilization

To detect the ability of organisms to solubilize phosphate, isolates were spot inoculated using sterile tooth picks onto Pikovskaya’s Agar medium containing (per liter) tricalcium phosphate 2.5 g, glucose 13 g, (NH4) SO4 0.5 g, NaCl 0.2 g, MgSO4.7H2O 0.1 g, KCl 0.2 g, yeast extract 0.5 g, MnSO4 trace, FeSO4.7H2O trace, agar 15 g, pH adjusted to 7.2. Plates were incubated for three days at 28 ± 2°C [12]. The strains showing halo zone of tricalcium phosphate solubilization were considered positive. Phosphate solubilization index (SI) was calculated by measuring the diameter of halo zone and bacterial colony using the formula Edi-Premoto M, et al. [13].

SI (Solubility Index) = colony diameter + halo zone diameter/colony diameter

Assay of Chitinase

PGPR strains were cultured at 25±2 °C for 96 hr. on a rotary shaker in 250 mL conical flasks containing 50 mL of chitin–peptone medium (glucose 0.5%, peptone 0.2%, colloidal chitin 0.2%, K2HPO4 0.1%, MgSO4 .7H2O 0.05% and NaCl 0.05%, pH 6.8). The cultures were centrifuged at 12,000 g for 20 min at 4°C and the supernatant was used as enzyme source. Colloidal chitin was prepared from crab shell chitin according to Berger LR, et al. [14]. The reaction mixture contained 0.25 mL of enzyme solution, 0.3 mL of 1M sodium acetate buffer (pH 5.3) and 0.5 mL of colloidal chitin (0.1%). The reaction mixture was incubated at 50°C for 4 h in a water bath. Chitinase activity was determined by measuring the release of reducing sugars by the method of Nielsen MN, et al. [15]. One unit of chitinase was determined as 1 nmol of N-acetyl-β-D-glucosamine (GlcNAc) released per minute per mg of protein.

Assay of β-1, 3-glucanase

Bacterial strains were grown at 25±2°C for 96 hr. on a rotary shaker in 250 mL conical flasks containing 50 mL of peptone medium containing laminarin (0.2%) (Laminaria digitata; Sigma). The cultures were then centrifuged at 12,000 g for 20 min at 4°C and the supernatant was used as enzyme source. The reaction mixture contained 0.25 mL of enzyme solution, 0.3 mL of 0.1M phosphate buffer (pH 5.5) and 0.5 mL of laminarin (0.2%). The reaction mixture was incubated at 40°C for 2 h in water bath. β- 1,3-glucanase activity was determined as 1 nmol of glucose released/min./ mg of protein.

Protease Production

Bacterial strains were streaked on casein agar and/ or incubated at 28 ± 2°C for four days. At the end of the incubation, the plates were observed for halo zone around the colonies, which indicates the presence of protease. The halo zone diameters were recorded.

Cellulase Production

Carboxy methyl cellulose (CMC) agar plates were prepared by screening for cellulose enzyme production according to the method by Kasana RC, et al. [16]. A sterile paper disc was dipped into microbial culture and transferred onto the CMC agar plates. The plates were incubated overnight at 33°C. After incubation, the plates were flooded with Congo red solution for 15min; followed by de-staining with the salt solution for 15min. unstained areas indicate where the CMC has been degraded due to production of cellulose by the bacterial strain. The halo zone diameters were recorded.

Amylase Production

Amylase production was determined by the method described by Alariya SS, et al. [17]. Fresh bacterial cultures were inoculated on Starch Agar plate and incubated at room temperature for 24-48 hr. Plate was flooded by Iodine solution after incubation. The hydrolysis of starch is observed as colorless zone around growth with violet background and is reported as trace to 4.

Determination of Extracellular Compounds

A 0.2 μm cellophane membrane was placed on PDA plates and 200μL of antagonistic bacterial suspension (1 × 107 cfu/mL) were inoculated in the center of plates. The plates were incubated at 25±2°C for 48 h, then the membrane with the grown bacterial strains was removed and the plate was inoculated in the middle with a five mm disk of a pure culture of R. solani, F. solani, M. phaseolina and F. oxysporum, individually. Plates were incubated at 25±2°C for 7 days and the radial growth of the pathogen was measured. Sterile double-distilled water replaced the bacterial suspension in control plates. There were four replicates for each treatment [18, 19].

Production of Volatile Antibiotics

Firstly, 200μL of bacterial suspension (1 × 10 7 cfu/mL) from each strain were spread on the surface of a Petri plate containing nutrient agar medium and incubated at 25±2°C for two days. In another Petri plate containing PDA medium, a 5 mm disk of a 7-days-old culture of tested pathogenic fungi viz. R. solani, F. solani, M. phaseolina and F. oxysporum was placed at the center. Then both half plates were placed face to face preventing any physical contact between the pathogen and the bacterial suspension [20]. The plates were sealed with parafilm. In the control plates, pathogenic fungi were placed at the center. Plates were incubated at 25±2°C for 48 hr. and the percentage of inhibition was calculated for each isolate in four replicates for each treatment.

Statistical Analysis

Analyses of variance were carried out using MSTATC, 1991 program (Ver. 2.10). Least significant difference was employed to test for significant difference between treatments at p ≤ 0.05 [21].

In vitro Screening Inhibitory Effect of PGPR

Plant growth promoting rhizobacteria viz., B. megaterium, B. subtilis, B. cereus and Ps. fluorescens strains were evaluated for antagonistic effect against R. solani, F. solani, M. phaseolina and F. oxysporum on Petri dishes containing PDA medium. Data in Figure 1 show that all PGPR strains succeeded in reducing the radial growth of all tested pathogenic fungi. Ps. fluorescens followed by B. megaterium were more active than B. subtilis and B. cereus for suppressing redial growth of tested fungi. On the other hand, F. solani followed by F. oxysporum was more affecting with PGPR, while R. solani and M. phaseolina was the lowest one. Such results confirm previous reports indicating that PGPR has the potential to inhibit growth of soil borne pathogens [22, 23, 24]. Glick BR, et al. [25] reported that the most effective mechanism that a PGPR can employ to prevent proliferation of phytopathogens in the synthesis of antibiotics.

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Enzymatic Activities and Secondary Metabolites of the PGPR Strains

Results of testing 4 PGPR strains (B. megaterium, B. subtilis, B. cereus, Ps. fluorescens) in vitro against tested pathogenic fungi showed that different strains exhibited different combinations of antimicrobial metabolites such as HCN, Siderophore, IAA, Ammonia production and Phosphate solubilisation. Pseudomonas fluorescens produced the highest levels of cyanide hydrogen and ammonia followed by B. subtilis. While, the higher level of IAA was produced B. subtilis followed by Ps. fluorescens. On the other hand, B. megaterium is the most PGPR strain produced of siderophore followed by Ps. fluorescens. On the other hand, all the tested PGPR strains successfully solubilized inorganic phosphate on Pikovskya’s agar medium (Table 1), indicated by forming a clear halo around their PGPR colony. The maximum phosphate solubilization after 72 hr. for in plates inoculated with B. megaterium (21.3mm) followed by Ps. fluorescens (18.7mm), while plates inoculated with B. subtilis and B.

cereus where recorded phosphate solubilization 13.8 and 14.8 mm, respectively.

On the other hand, all plant growth promoting rhizobacteria strains (PGPR) were able to produce mycolytic enzymes viz. cellulase, chitinase β-1,3-glucanase, amylase and protease in growth media except B. cereus and B. megaterium not able to produce protease and amylase in media growth, respectively (Table 1). B. megaterium recorded the highest activities of chitinase, β-1,3-glucanase in media growth compared with the other PGPR strains, whereas produced 23.12 and 80.12 nmol/min/mg protein, respectively. While, Ps. fluoresces produced the highest protease and amylase in media growth (20.08 and 14.75 halo zone diameter). B. subtilis produced the highest cellulase level in media growth (16.35 halo zone diameter). On the other hand, B. cereus produced the lowest levels of all tested enzymes (chitinase, β-1,3-glucanase, protease, cellulase and amylase) in media growth).

The results agree with Sarhan EAD, et al. [24]; Kumar S, et al. [26]; Choudhary SR, et al. [27]. Arora NK, et al. [28] found that the lytic enzymes produced by bio-control agents interfere with the growth and activities of pathogen by hydrolyzing the polymeric compounds, including chitin, proteins, celluloses, hemicelluloses, etc., and consequently killing or suppressing the growth of pathogens. Hydrolytic enzymes showed their ability to control plant pathogens. A hydrolytic enzyme like chitinase, glucanase, protease and cellulase can degrade the fungal cell-wall and causes the cell lysis of fungal pathogens. Also, Abdel-Monaim MF, et al. [20] found that PGPR strains viz. B. subtilis and B. megaterium were able to produce indole acetic acid (IAA), siderophore, hydrogen cyanide (HCN), extracellular compound and volatile antibiotics in vitro. In addition, both PGPR strains produced mycolytic enzymes viz. chitinase, β-1, 3-glucanase and protease in growth media. B. megaterium produced greater amounts of secondary metabolites than B. subtilis.

Effect of Extracellular Metabolites

Data present in Figure 2 show that all plant growth promoting rhizobacteria (B. megaterium, B. subtilis, B. cereus and Ps. fluorescens produced extracellular metabolites. Ps. fluorescens and B. megaterium was significantly the highest produce extracellular metabolites against all tested pathogenic fungi. While B. cereus and B. subtilis recorded the lowest produce extracellular metabolites in case of all pathogenic fungi. On the other hand, F. solani was the most affecting with extracellular metabolites produced with PGPR, while M. phaseolina was the least affecting ones in this respect.

These results revealed that certain extracellular metabolites generated by PGPR affected mycelial growth and germ tube elongation. Some Bacillus species are known to produce numerous antimicrobial compounds which have been well characterized genetically and biochemically in vitro Arguelles-Arias A, et al. [29]. Among these antagonistic compounds, the predominant lipopeptides of the surfactin, iturin and fengycin families have been well reported for their potential against a wide range of plant pathogens Pathak KV, et al. [30].

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Effect of Volatile Antibiotics

ANOVA results showed that the colony diameter of tested pathogenic fungi (R. solani, F. solani, M. phaseolina and F. oxysporum varied significantly (P ≤ 0.05) depending on antagonistic treatments tested. In fact, data given in Figure 3. Revealed that all tested pathogenic growth decrease, due to the inhibitory effects of volatile metabolites from the rhizobacteria strains tested were varied by differently PGPR strains and pathogenic fungi. Ps. fluorescens was recorded the highest inhibitory effect against all tested pathogenic fungi followed by B. megaterium, while B. subtilis and B. cereus gave the lowest ones against all pathogenic fungi in this respect. Volatile inhibitors that have been identified include ethylene, ammonia, allyl alcohol, acrylic acid, trimethylamine, benzaldehyde, and N,N-dimethyloctylamine [31, 32]. The spectrum of volatile compounds produced appears to depend not only on environmental conditions, such as nutrients, temperature, pH and moisture, but also on the organism itself. Ryu CM, et al. [33] demonstrated significant growth promotion of Arabidopsis by B. subtilis strain GB03 and B. amyloliquefaciens strain IN937a. Among VOCs released from strain GB03, 2,3-butanediol was found as a major component that promoted plant growth and elicited ISR against Erwinia carotovora subsp. carotovora. Several mutant lines of Arabidopsis, including brassinosteroid- and gibberellic acid-insensitive mutants, auxin-transport- deficient mutants, and cytokinin receptor-deficient mutants, were used to elucidate the signaling pathways that promote growth.

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