Abiotic Conditions on Growth of Pseudomonas fluorescens (DS17R) and Its Ability to Produce Secondary Metabolites (Including Phenazines) Against Phytophthora colocasiae, the Causal Agent of Taro Leaf Blight

Research Article

Austin J Biotechnol Bioeng. 2018; 5(2): 1095.

Abiotic Conditions on Growth of Pseudomonas fluorescens (DS17R) and Its Ability to Produce Secondary Metabolites (Including Phenazines) Against Phytophthora colocasiae, the Causal Agent of Taro Leaf Blight

Ntyam Mendo SA1,2, Kouitcheu Mabeku LB¹, Tounkara Lat S², Tchameni Nguemezi S³, Ngono Ngane RA³ and Sameza Modeste L³*

¹Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon

²Biotechnology Unit, Institute of Food Technology (ITA), Senegal

³Department of Biochemistry, Faculty of Science, University of Douala, Cameroon

*Corresponding author: Sameza Modeste Lambert, Department of Biochemistry, Faculty of Science, University of Douala, P.O. Box 24157 Douala, Cameroon

Received: March 06, 2018; Accepted: April 27, 2018; Published: May 04, 2018

Abstract

Control of Taro Leaf Blight (TLB) by chemical pesticides remains ineffective in Cameroon. Alternative methods of control are required to fight against the causative agent of this epidemy. Fluorescent Pseudomonas is the main rhizobacteria widely used against plant pathogens because they produce a broad range of antimicrobial secondary metabolites (including phenazines) involved in the biocontrol mechanism. However many ecological factors could influence the production of compounds involved in biocontrol. In the present study, we evaluated the abiotic conditions of P. fluorescens DS17R growth, phenazine production and antimicrobial activity against Phytophthora colocasiae the causal agent of TLB. The abiotic factors were pH, temperature, carbon and nitrogen sources, and osmotic stress. The bacterium was grown on King B medium and monitored by spectrophotometer. The secondary metabolites were extracted with chloroform and the phenazines content estimated by the MnO2- based reduction assay. The antimicrobial activity of each extracts was evaluated by the well diffusion method. Results showed that the growth, phenazine production and the antimicrobial activity of secondary metabolites extracted from P. fluorescens DS17R was influenced by different abiotic conditions. Overall, glycerol was the best carbon source while peptone and NH4Cl were the best nitrogen sources. These best sources of growth promoted significant phenazine production in correlation with the antimicrobial activity observed. In addition the optimum pH and temperature for phenazine production was 6 and 28°C respectively. On other hand, the positive correlation between growth, antimicrobial activity and phenazines production of the extract was observed at 2.5% NaCl. These findings show that Pseudomonas fluorescens DS17R extract could be positively optimized by certain abiotic factors and has the potential to be further developed as natural antimicrobial agent against P. colocasiae.

Keywords: Pseudomonas fluorescens; Phenazine; Antimicrobial activity; Phytophthora colocasiae; Taro leaf blight

Introduction

Taro Leaf Blight (TLB) disease caused by Phytophthora colocasiae is the main constraint of Taro production worldwide. This disease can spread rapidly to other plant part and has resulted in yield loses of up to 80-100% in many countries [1-3]. Several methods have been used to solve this major problem in Cameroon. One of them is the use of systemic pesticides such as metalaxyl and mancozeb. Unfortunately, the resistance developed by the pathogen strains, accumulation of residues and environmental problems can occur when these chemicals are repeatly applied. Moreover the appearance of the disease during the rainy season makes pesticides spraying ineffective [4]. Therefore, alternative ecofriendly treatment is needed in order to control the disease. In this respect, rhizobacteria have received considerable attention as an alternative approach to control plant diseases. Many rhizobacteria can actively colonize roots [5,6] and suppress phytopathogens by the production of siderophores or secondary metabolites like phenazines antibiotics [7,8]. Among benefit rhizobacteria, fluorescent Pseudomonas is non-pathogenic microorganisms, which suppress the soil-borne pathogens through multiple mechanisms [9] including the production of secondary metabolites [10-12]. Among these secondary metabolites, phenazines are highlight of biological significance. Chemically, they belong to the alkaloid class of compounds, which contain a basic amino group in their structure. Phenazines are water-soluble and are secreted into media at concentrations as high as grams per liter of bacterial culture [13]. Most species synthesize two or more species-specific phenazines except Pseudomonas fluorescens, which, so far, is known to produce only Phenazine-1-Carboxylic Acid (PCA) [14]. Many ecological factors such as carbon and nitrogen source, temperature, pH, osmotic stressetc, could influence the production of this metabolite [15-18]. In the previous work Ntyam et al. [19] demonstrated the antimicrobial activity of Pseudomonas fluorescens DS17R chloroform extract against Phytophthora colocasiae, the causal agent of the TLB. They correlated the observed activities with the presence of some secondary metabolites like phenols and flavonoids. For successful use of Pseudomonas fluorescens DS17R as biocontrol agent, we need to understand which and how environmental factors affect the production of antimicrobial components. The identification of conditions that control secondary metabolite production by P. fluorescens DS17R could lead to a better understanding of their regulation and their exploitation. The present study aimed to investigate the effects of various abiotic conditions on phenazine production by Pseudomonas fluorescens isolate DS17R and their respective antimicrobial potential against Phytophthora colocasiae.

Materials and Methods

Microbial strains

The microbial strain used in this study (Pseudomonas fluorescens DS17R and Phytophthora colocasiae) came from the culture collection of the laboratory of Biochemistry, University of Douala (Cameroon). The data regarding the characterization of these isolates were given by Ntyam et al. and Sameza et al. [20]. Bacterium was routinely cultivated on King B medium and preserved in King B Broth 20% (v/v) glycerol at -70°C for long-term maintenance whereas Phytophthora colocasiae was maintained on Potato Dextrose Agar (PDA) slant at 4°C.

Various growth conditions of Pseudomonas fluorescens DS17R

Carbon and nitrogen source: For carbon source, basic medium (KMP) consisted of 1.5 g/l KH2PO4, 1,5 g/l K2HPO4, 2 g/l MgSO4, peptone 20 g/l. Various carbon sources (Glycerol, Glucose, Mannitol and Sucrose) were added at a rate of 10g /l. The basic medium of nitrogen was made of GKM (10 ml Glycerol, 1.5 g/l KH2PO4, 1,5 g/l K2HPO4, 2 g/l MgSO4).This medium was supplemented respectively with nitrogen sources at a rate of 20 g/l each. They were: Peptone, NH4Cl, (NH4)2SO4, (NH3)2SO4 and yeast extract. A volume of 200 ml of each medium was introduced in 250 ml conical flasks and the pH adjusted to 6.7 after sterilization. Two microliters of two days preculture of the bacteria suspension (1.5. 108 UFC/ml) were introduced into each flask. After 3 days of incubation, at 26°C, samples were collected and the cell growth monitored by measuring the optical density at 600 nm using UV spectroscopy (Biotech Ultrospec®3000).

Osmotic stress

The study of osmotic stress was made only with the best media conditions of C and N sources. Each medium was supplemented with different salt (NaCl, KCl and Na2SO4) at concentrations of 2.5; 5; 7.5 and 10% (w/v). Pseudomonas fluorescens DS17R inoculum was introduced in the media and growth monitoring was made as previously described.

pH and temperature

The effect of the pH and the temperature on the growth of P. fluorescens DS17R was performed as described by Petatán-Sagahón [21] with some modifications. Preculture of the rhizobacterium was grown at different pH conditions (4, 6, 8 and 10) and temperatures (20, 25, 28, 30, 35 and 40°C). The growth was monitored as previously described.

Secondary metabolites extraction

The crude extracts were prepared according to Liu et al. [22] and Ntyam et al. with some modifications. Briefly, the bacterium was grown in 200 ml of King B broth at 26±2°C for 72 hours and the pH value of this broth adjusted to 2.0 with 1 M HCl. After centrifugation at 28,000 rpm for 30 min, the supernatant was collected, and extracted four times with total of 120 ml of chloroform. Finally, the organic phase was evaporated under vacuum and residue dissolved in 500 μl of sterilized distilled water.

In vitro antimicrobial test

The antimicrobial assay of extracts against P. colocasiae was performed on Potato Dextrose Agar (PDA) plate using well diffusion method. A 5 mm agar plug of P. colocasiae was inoculated in the center of the plate and two wells (5 mm diameter) were created at the border of the plate equidistant from the center by punching the plate using sterile cork borer. One hundred microliter (100 μl) of chloroform extracts were added in each well while 100 μl of sterile distilled water were added in the control plates. The plates were then incubated at 26±2°C for 7 days and the radial growth of the pathogen measured. The inhibition of the radial growth was calculated according to the formula: %I=(Do-Dx)/Do X 100. Do is the growth of the pathogen on the control plate and Dx the growth in the test plate. All the tests were done in triplicate and the experiment repeated twice [21].

Quantification of Phenazines

Preliminary screening of the crude extracts for phenazines content was done by the reduction MnO2-based assay. Briefly, aqueous chloroform extracts (200 μl) obtained directly after extraction was mixed with 100 μl of MnO2 (3mM) in Eppendorf tube. The reaction of phenazine in the extract with MnO2 was detected after incubation at 30°C. The reduction of brown colored insoluble Mn (IV) to colorless Mn (II) was observed daily for 7 day. Phenazines were quantified on extracts from various nutritional conditions of growth (C and N source, pH and osmotic stress) by measuring the optical density at 367 nm using UV spectroscopy [23,24]. The total amount of phenazine in each extract was estimated using standard curve obtained by a range of concentrations (0.5, 1, 2, 4 and 16 μg/ mL) from a synthetic phenazine (Figure 1).