Antimicrobial Activity of Potato Rhizospheric Pseudomonas chlororaphis subsp . aureofaciens from Sétif Algeria

Aims: This study was assessed to demonstrate the antimicrobial activity in vitro of an identified fluorescent Pseudomonas strain characterized for its capacity to produce phenazine compounds. Methodology: First Pseudomonas chlororaphis subsp aureofaciens was inoculated on Nutrient Broth supplemented with Yeast Extract (NBY) and with glucose at a final concentration of 2%, after incubation the filtered culture was acidified with HCl to pH 2. The solution was extracted twice with the same volume of ethyl-acetate. The organic supernatants were combined, dried over anhydrous Na2SO4, and evaporated to dryness. The crude extract was resuspended in methanol and tested Original Research Article Mezaache-Aichour et al.; ARRB, 11(5): 1-7, 2016; Article no.ARRB.31651 2 for antimicrobial activity. Antimicrobial activity was determined (i) by disc diffusion technique for bacteria and (ii) using serial dilution technique in soft PDA for fungi. Secondly the antifungal activity of the bacterial strain was tested against several phytopathogenic fungi in dual culture. Results: The studied strain has an important activity against the phytopathogenic bacteria and fungi tested. Among the tested fungi Fusarium oxysporum f. sp. albedinis is the most sensitive to the actions of this Pseudomonas, where the inhibition rate reached 77.78%. The less sensitive one was Pythium ultimum with a rate of 55.56%. While for pathogenic bacteria only Salmonella enteridis was sensitive to the tested strain. Conclusion: Pseudomonas chlororaphis subsp aureofaciens showed appreciable antagonistic activity, in vitro, against special forms of Fusarium oxysporum and the tested phytopathogenic bacteria.


INTRODUCTION
Plant pathogens can cause serious damage to agriculture; they are responsible for the loss of 10 to 20 percent of agricultural production worldwide, despite the several billion dollars spent for their control by synthetic chemicals.Telluric pathogens are recognized to be difficult to manage in narrow rotations.Resistant plant varieties are not available for several soil-borne pathogens and chemical control is often insufficiently effective in soil.There is a growing awareness that integrated pest management (IPM) tactics and strategies provide more environmentally and economically acceptable alternatives for agriculture [1].Evidence exists correlating the efficacy of bacterial strains with control soil-borne pathogen with their ability to competitively colonize and survive in the root system of the plant to be protected.Several bacterial mechanisms have been described for the protection of plants against fungal diseases [2].Several mechanisms, including the production of siderophores, hydrogen cyanide (HCN) and antibiotics have been shown to play roles in disease suppression [3].The production of antifungal metabolites, induction of systemic resistance and the ability to compete efficiently with resident rhizobacteria are considered to be important prerequisites for the optimal performance of biocontrol agents [1].These biocontrol strains belong to different species and/or phylogenetic groups within genus Pseudomonas and can be closely related to strains without apparent biocontrol activities [4][5].The potential for using strains of beneficial Pseudomonas spp.for biological control of soil borne fungal pathogens has been demonstrated for many crops [1].Fluorescent pseudomonads that protect plant from soil borne fungal pathogens are thought, to act in part, through the secretion of antimicrobial substances with antifungal [6][7] and antibacterial activity [7][8].The present study was performed to demonstrate the antibacterial capacity of a Pseudomonas strain already known for its antifungal one.

Materials
The tested strain was already isolated from potato rhizosphere, characterized and identified by Mezaache-Aichour et al. [9]

Extraction and characterization of antimicrobial metabolites
Antimicrobial metabolite production in vitro was assayed in Nutrient Broth Yeast Extract (NBY; [6]) supplemented with glucose to a final concentration of 2%.Cultures were incubated on a rotary shaker (250 rpm) at 28°C for 5 days [10].
After incubation, the culture filtrate was first acidified with HCl to pH 2 before extraction [6].
Then antimicrobial metabolites were extracted twice from the cultures with an equal volume of ethyl-acetate [11].The ethyl-acetate fraction was decanted, filtered through anhydrous sodium sulfate to remove the aqueous fraction [6], and concentrated by evaporation to dryness by rotary vacuum at 55°C [1,11].The crude extract was resuspended in methanol [12].

Antimicrobial activity
To determine the effect of the isolated strain on bacteria, cells of examined strains were plated by an inoculating needle to the surface of Luria Bertani (LB) agar medium [8] in a band approximately 2.5 cm wide [3] and grown at 28º C for two days.The grown colonies were killed with chloroform vapors, plates overlayed with 3 ml of 0.6% agar containing 2x10 7 cells of indicator strains of phytopathogenic and pathogenic bacteria.Incubation of plates proceeded for 18-20 h at 28ºC.The antimicrobial activity was judged from the radius of indicator growth inhibition zones around colonies of tested bacteria [8].
Antibacterial activity of supernatant from the selected strain (ethyl-acetate fraction, dried and resuspended in methanol) was determined using the disc technique.Bacterial suspensions (100 µl) were spread on Tryptone Soya Agar (TSA) medium and discs containing 20 µl supernatant were placed on the inoculated plates.Plates were stored overnight at 4°C and then incubated at 37°C for 24 h [13].
The antifungal activity of the strain was first screened in dual culture as described by Vincent et al. [12] against the phytopathogenic fungi cited earlier.The hyphal inhibition was measured, and the percentage of fungal inhibition was calculated using the following formula: Where Dc: Control diameter (fungi alone); Ds: Sample diameter (fungi in dual culture with tested bacteria).
Secondly the Antimicrobial activity of bacterial extracts was determined using serial dilution technique in soft PDA inoculated with 2 mm plug of a seven days fungal culture (diameter of the plate hole is 9 mm).

Statistical analysis
Data were analyzed by the one way analysis of variance (ANOVA) and the test with P=0.05 was considered as statistically significant.

Ps. chlororaphis subsp. aureofaciens
The strain was screened visually on agar for the ability to produce antimicrobial substances.The antibiotic was produced consistently and could be screened on NBY or PDA plates, on which colonies appeared orange and were highly colored in dual culture with pathogenic fungi when near mycelium [14].However, the presence of antimicrobial substances produced varied greatly with media (the amount was greater on NBY, LB, than on PDA) and the age of the cultures (the amount was greater in 72 h than 24 h cultures).When spread on the medium bacterial cells produces the antimicrobial substances which inhibited other bacterial cells spread above (Fig. 1).Using the disc technique, the crude bacterial extracts inhibited the growth of Bacillus subtilis and Pseudomonas diminutus, but had an intermediate effect against Micrococcus luteus and Paracoccus paratrophus (Fig. 2).While on dual culture there is another strain which is inhibited by the selected strain Salmonella enteritidis (Table 1).
The fluorescent pseudomonads bacteria are used as biocontrol agents of bacteria, fungi, and viral diseases of plants [15][16].They suppress the pathogen and reduce disease incidence by competition, antibiosis or parasitism [15].They act in part, through the secretion of antimicrobial substances with antifungal [6][7] and antibacterial activity [7][8].Our results demonstrate that selection of antagonistic strains among competitive Potato root colonizers yields a low frequency of strains producing well known antibiotics.The isolated bacterial colonies were orange and fluoresced orange under long wave (365 nm) UV irradiation.They were surrounded by a dark halo of UV absorbing material consistent with production of antimicrobial metabolites [17], which were identified as phenazines [14].Our results showed that the crude extract was active against gram positive bacteria.Veselova et al. [8] demonstrated that extracts of Pseudomonas chlororaphis 449 which produces phenazines inhibited the growth of the gram positive bacteria, Bacillus subtilis and Staphylococcus aureus.Pseudomonas spp.produces phenazine antibiotics-nitrogencontaining heterocyclic pigments, which exhibit broad-spectrum activity against numerous bacteria and fungi [7,[18][19][20].
The crude extract inhibited the fungal growth.The diameters of the fungal colonies were 3.99, 3.37, 3.08, 2.0 and 2.25 mm for Pythium ultimum, Rhizoctonia solani, Fusarium solani; Fusarium oxysporum f. sp.albedinis and Fusarium oxysporum f. sp.lycopersici respectively (Fig. 4).The tested strain was more active than the strain 30-84 which produces well known antibiotics [21].Vincent et al. [12] using this bacterium (30-84) also demonstrated inhibition of Gaeummanomyces graminis var.tritici.In other experiments in Algeria other strains isolated in the same time from the same field were only active on media depleted in iron or containing low concentrations of iron, such as King's B [21].The selection of antagonistic strains among competitive Potato root colonizers yields a low frequency of strains producing well known antibiotics, contributes to the isolation of rhizobacterial strains with a higher variety of strains that could be useful in final protection of the plant against the phytopathogens.Ps. chlororaphis subsp.aureofaciens exhibited antagonism towards the test pathogens in dual culture, and showed maximum inhibition on PDA of the mycelial growth of F. oxysporum.However, the antifungal efficacy is less against F. solani, P. ultimum and R. solani [22][23].Earlier, Alabouvette [24], Deacon and Berry [25], demonstrated that the suppressiveness is specific of Fusarium wilts and not effective against diseases caused by non vascular Fusarium species including F. roseum and F. solani, or other soilborne pathogens.On the other hand we have already demonstrated that this bacterium produces bacteriocins [26].These metabolites allow the bacterium to be competitive against the same genera or bacteria from other genera [21,26] if introduced in the field [21].

CONCLUSION
This antagonism was less specific for the studied isolate, whose activity was expressed on the three culture media used, and with all the fungal and phytopathogenic bacteria isolates tested.These results complete previous studies, and show that activity of the indigenous Ps. chlororaphis subsp.aureofaciens isolate is not specific to a kind of bacteria or fungi but could be useful in protection of plant against a large bacterial and fungal phytopathogens.It can constitute an effective mean of biological control in natural soil, to limit the use of Chemical inputs.