MULTI-TRAITS OF NON-PATHOGENIC FLUORESCENT PSEUDOMONAS AND EVALUATION OF THEIR POTENTIEL AS BIOCONTROL AGENTS

In recent years, certain strains of fluorescent pse udomonads called PGPB have drawn attention due to t heir abilities to promote plant growth. Therefore, in th is investigation, we have explored the adhesive pro perties, the phytostimulator effects and the biocontrol acti vities of 40 isolates, with the aim to select poten tial inoculants to improve crop yields. Ten different co lony morphotypes were detected on CRA plates. PsTp172 described as “ST” showed the highest adhere nc ability to abiotic surface (OD 550 = 2.102). 31 isolates were positive for the plant growth-promoti ng hormone (IAA) production and 30 stains solubilis ed tri-calcium phosphate in Pikovskaya’s agar. Further more, the highest pyoverdine concentration was detected with PsTp172 strain (172.50 μM) under iron starvation conditions. This strain also exhibited a coresistance against Zn 2+ and Mn and displayed high values of Minimum Inhibitory Con ce trations (MIC) for each heavy metal. Additionally, among the teste d isolates, eight strains (PsS15, PsTp172, PsS28, PsTp171, PsS31, PsS67, PsS18, PsS39 and PsS93) were found to be efficient antagonists against the 3 pathogenic strains and 6 isolates (PsS15, PsTp156, PsTp172, PsC54, PsTp171 and PsS102) were considered as lactone inhibitors of the 3 tested strains, as s hown by their ability to inhibit the cellular commu nication. The majority of isolates showed various phytobenefi cial traits and the most effective strains are P. putida (PsTp172 and PsS15) and P. mosseli (PsTp171).


INTRODUCTION
In diver's environments, beneficial plant-associated bacteria exhibit a significant role in plant growth and health . The success of Plant Growth Promoting Bacteria (PGPB) is due to their efficient colonization of plant roots by forming microcolonies or biofilms which promotes the plantmicrobe interaction. These bacterial consortiums have the ability to communicate chemically with one another through quorum-sensing (Mishra et al., 2010). This signalling mechanism coordinates bacterial communications with plants, comprising antibiotic production, phytohormone excretion, toxin release and Horizontal Gene Transfer (HGT) (Von Bodman et al., 2003). Though, it has been portrayed that certain PGPB are able to quench phytopathogens quorum-sensing capability by degrading autoinducer signals, thereby blocking expression of virulence genes (Compant et al., 2005).
An efficient PGPB should display more than two plant growth promoting activities which are root colonization competency, biofertilizer, phytostimulator and phytopathogen biocontrol activities (Bloemberg and Lugtenberg, 2001). Of the various rhizospheric bacteria, Pseudomonas species are the most popular bacteria that combine all these characters; enhance plant growth by direct and indirect mechanisms. The direct promotion involves either supplying plant with growth promoting substances (such as auxins, gibberellins) or helping plant nutrients uptake from environments (Husen et al., 2011). The indirect promotion arises when PGPB avoid harmful effects of phytopathogenic microorganisms (bacteria, Science Publications AJES fungi, nematode) by the production of siderophores, HCN, antibiotics, bacteriocins, volatile metabolites (Saharan and Nehra, 2011).
The performance of PGPB is influenced by a wide range of the environmental conditions (soil composition or characteristics: pH, heavy metals contamination, weather conditions, etc.) that may affect the plant growth (Sajani and Muthukkaruppan, 2011). So, application of highly heavy metal tolerant PGPB has been exploited in contaminated soil allowing the vegetables grow under hard conditions (Zhuang et al., 2007).
Therefore to exploit the potential of native strains of non-pathogenic fluorescent Pseudomonas, the present study attempts to characterize, screen and select nonpathogenic Pseudomonas bacteria exhibiting the highest number of traits.

Growth media and culture conditions
All samples were transfered to the laboratory in sterile stomacher containers, stocked at 4°C and analyzed within 24 h. 10 g or 10 mL of sample were suspended in 90 mL sterile distilled water. 50 µL of the appropriate dilution was spread on King's B (King et al., 1954) agar medium. After 48 h of incubation at 25°C, each different colonie showing fluorescent halo was purified (Munsch et al., 2000). Each colonie confirmed as putative Pseudomonas spp. (gram negative, catalase positive and oxidase positive) was utilized for further characterization.
Strains whose designations begin with PsS, PsTp, PsC and PsWw were collected respectively from soil rhizosphere, wastewater treatment plant rhizosphere, compost and wastewater.
The NCBI Accession Numbers for the 16S rDNA gene sequences of 40 isolates determined in this present investigation are listed in Table 1.

Qualitative Detection of Biofilm Formation (Detection of Colony Morphology Variant)
All strains were spread on Congo Red Agar (CRA) containig 0.8 g of Congo red (Sigma) and 36 g of saccharose to 1 l of brain heart infusion agar (Bio-rad). A concentrated aqueous solution of Congo red stain was prepared, autoclaved separately for 15 min at 121°C and added when the agar had cooled to 55°C (Freeman et al., 1989). Plates were incubated at 30°C for 48 h. Morphology, pigmentation and mucosity were observed.

Quantitative Estimation of Biofilm Formation on Polystyrene Surface
All bacteria were cultured overnight in Brain Infusion Broth (BHI-0.25 glucose at 30°C). The culture was diluted 1:20 in fresh BHI plus (0.25%) glucose at 30°C. This suspension (200 µL) was utilized to inoculate sterile 96-well-polystyrene microtiter plates. The plates were incubated at 30°C aerobically for 24h. The cultures were eliminated and the microtiter wells were washed twice with phosphate-buffered saline (7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 and 130 mM NaCl at pH 7.4) to remove non-adherent cells and were dried in an inverted position. Then, bacteria that not adherate to microtiter plates were stained with 1% Crystal violet for 15 min. The wells were washed once more and the Crystal violet was dissolved in 200 µL of ethanol (95%). An automated PR3 100 TSC (Bio-Rad) was used to measure the absorbance at 550 nm (OD 550 ) (O'Toole et al., 1999). Each essay was performed in triplicate. The following values were attributed for biofilm determination: OD 550 ≤0.1 non biofilm forming; 0.1≤OD 550 ≤0.5 weak biofilm forming; 0.5≤OD 550 ≤1 medium biofilm forming; 1≤OD 550 ≤2 strong biofilm forming; OD 550 ≥2 very strong biofilm forming.

Heavy Metal Tolerance
Metal Salts were utilized in the following forms: ZnSO 4 ·7 H 2 O (iron content,<10 ppm) and MnSO 4 · H 2 O (iron content, <0.001%). Stocks of 10mM 1 ZnSO 4 and MnSO 4 salts were prepared and sterilized with 0.22 µm filters under aseptic condition. The stock solutions were incorporated to autoclaved CAA media (CAA+Mn and CAA+Zn) at a final concentration of 60 µM for each metal. In the purpose to establish the Maximum Tolerable Concentration (MTC) of metals at which growth was observed, the 40 strains were monitored in CAA medium as a function of increasing concentrations of Mn (II) and Zn (II) from 0.1 to 475 µM in 5 mL CAA medium (Mehri et al., 2012).

In Vitro Antagonism Against Pathogenic and Phytopathogenic Pseudomonas Bacteria
Culture supernatant was prepared as follows: A centrifugation at 150 rpm of an overnight culture. Resulting supernatant was neutralized, sterilized by filtering and assayed for the presence of an inhibitor in the broth; following the agar well diffusion assay technique (Barefoot et al., 1983). Nutrient agar was first seeded with pathogenic organism (110 µL of overnight culture per 20 mL of agar) in sterile Petri dishes and after solidification, dried for 15 min. Wells were bored in the agar. Aliquots of the supernatant were distributed in holes and plates were incubated 24h at 30°C. Inhibition of growth was determined by an area of inhibition surrounding each agar well.

Quorum-Quenching (QQ) Bioassays
N-Acyl Homoserine Lactone (NAHL) degradation ability of the isolates was detected by cross streaking against Chromobacterium violaceum CV026 as the AHL biosensor (Swift et al., 1999). Supernatants from 7 days old cultures of the isolates were adjusted to pH 7 and 15µl of each supernatant blended with pathogenic or phytopathogenic Pseudomonas supernatant and dispensed in the wells of a bioassay plate overlaid with C. violaceum CV026.

RESULTS
The 40 isolates were screened for functional properties. PGPB traits were represented in Table 1.

Growth Temperature Tolerance
All isolates, except three strains, were able to grow at maximum temperature of 32°C. On the other hand PsS60, PsS91 and PsS73 strains were able to grow at 37°C and 42°C.

Biofilm Formation on Polystyrene Surface
About 26 isolates showing mucoid, highly mucoïd and stronger mucoïd colony morphotypes were nonbiofilm forming on polystyrene surfaces with an OD 550 ≤0.1. While, PsTp172 (morphotype "ST") produced a very large amount of biofilm (OD 550 = 2.102). This type of morphotype was characterized by hyper-adhesion and autoaggregation to abiotic surfaces (Kirisits et al., 2005). The PsS67 strain was strongly adhesive to polystyrene with a value of 1,238 at 550 nm. Strains PsTp171 and PsS46 were medium adhesive to the abiotic surface with OD 550 values respectively of 0.639 and 0.547. On the other hand, Rakhimova et al. (2008) explained that isolates with light rose or colourless colony morphotypes, suggesting that biofilm formation was affected. Hence PsTp169 and PsS103 showed optical densities respectively of 0.069 and 0.041. All these data were summarized in Table 1.

IAA production and Phosphorus solubilisation
About 31 strains (76%) of fluorescent Pseudomonas produced the IAA hormone in various levels, while 9 strains showed no producing activity. The isolate PsS93 produced the highest level of IAA (7.4 µg mL −1 ), whereas PsS4 and PsS31 were found the weak producer isolates (respectively 0.11-0.15 µg mL −1 ). On the other hand, 30 Pseudomonas spp. (75%) were capable to solubilise, in Pikovskaya's agar, tri-calcium phosphate by the formation of clear halo zone (Fig. 2).

Pyoverdine Quantification
When the cells grown under iron starvation Conditions (CAA medium) different level of siderophore was detected in culture supernatant. The highest metabolite concentration was obtained with PsTp172 strain (172.50 µM). The least pyoverdine production was detected in PsS73 isolate (2.82 µM).

Heavy metals Resistance
The 40 isolates were screened for their metal tolerance on CAA media containing different metal concentrations of each Zn 2+ and Mn 2+ . The MTC for each metal was determined to select isolates able to grow and resist high level of metal toxicity. Generally, the bacterial isolates showed the maximum resistance against Zn 2+ (MTC: 100-300 µM) and Mn 2+ (MTC: 300-475 µM). PsTp172 strain was the most tolerant Science Publications AJES bacteria to tested metals. This isolate resisted to Zn 2+ up to 300 µM and to Mn 2+ up to 475 µM.

DISCUSSION
Among PGPB, fluorescent pseudomonads have proved a boon in sustainable agriculture for use as biofertilizer (plant growth promoting capacity), biocontrol agent (antagonistic activity) and in bioremediation (degradation of pollutants) (Husen et al., 2011). A total of 40 isolates recovered from diver's origin were subsequently screened for plant growth promoting characteristics and biological control of plant deleterious microbes.
IAA, a member of phytohormone group, acts as important signal molecule in the regulation of plant growth (Kumar et al., 2012). In our study most of the Pseudomonas isolates were positive for IAA production (76%). Among them, P. fluorescens strains isolated from soil rhizosphere are found to be good producers of the phytohormone (PsS93, PsS89, PsS49, PsS90). Saharan and Nehra (2011) study revealed that isolates of the Pseudomonas putida-fluorescens group, which produce auxin, are able to induce statistically important yield increases up to 144% in field tests.
In the soil, most of phosphorus is present in the insoluble form and cannot be used by plants (Pradhan and Sukla, 2006). The study of Rodriguez and Fraga (1999) revealed that the availability of phosphor in soil was increased by strains of Bacillus, Pseudomonas and Rhizobium. In our experiments, all P. putida isolates are able to produce the phosphatase enzyme. Villegas and Fortin (2002) selected P. putida strains as effective agents able to solubilise soil phosphate. The aptitude of various strains to transform the insoluble phosphate suggested their application in agriculture .
Apart the PGPB biofertilizer actions, it is prominent to note that the conformation of adherent microbial population affects significantly the duration and intensity of the plant-microbe interactions (Danhorn and Fuqua, 2007). In general, there should be a correlation between the colony morphotype and the capacity of adhesion to different surfaces. Therefore, PsTp172 strain with rough, wrinkled and Sticky (ST) colony morphotype showed a higher adherence ability (OD 550 = 2.102). "ST" variant is so called because of the hyperadherence to abiotic surfaces and the strong autoaggregative phenotype in liquid culture (Kirisits et al., 2005). On the other hand, some isolated strains showed hypermucoid colony (PsWw128, PsS60, PsS29). In this context, Bianciotto et al. (2001) described that the hypermucoid mutant of P. fluorescens CHA0 demonstrated an important adherence to roots and to the mycelium of arbuscular mycorrhizal fungi. Exopolysaccharide production (alginate) contributes to surface colonising of plants (Danhorn and Fuqua, 2007). Study of Stoodley et al. (2002) portrayed that structure and adherent nature of cells in biofilms modulate the biocontrol activities and antimicrobial tolerance. P. putida and P. fluorescens bacteria are able to coat roots of plant by forming a biofilm, which may save roots against phytopathogens (Espinosa-Urgel et al., 2002;Walker et al., 2004;Ude et al., 2006). Additionally, it has been shown that besides biocontrol activities, biofilmed Pseudomonas spp. inocula possess N 2 -fixing properties and nutrient uptake (Seneviratne et al., 2010).
Siderophores such as pyoverdins are iron chelators molecule. Privated from iron, telluric harmful flora slows its growth and its density is decreased in the rhizosphere. This feature can enhance fluorescent Pseudomonas (PGPR) in the process of colonization and competition for the substrate better than other microbes in the rhizosphere (Lemanceau, 1992). A significant pyoverdin production was detected with isolates PsTp172, PsTp169, PsS46, PsTp171 (>80 µM). Loper and Buyer (1991) explain that the pyoverdine concentration produced by Pseudomonas spp. in the rhizosphere, is important to influence the microbial interactions. Many authors found that mutants, who lost the synthesizing siderophore ability (Sid − ), are not able to determine the beneficial effects recorded with the wild strain (Becker and Cook, 1988). Unfortunately, the involvement of pyoverdin in animals and humans virulence is very well studied in pathogenic isolates (P. aeruginosa), but it is unlikely exposed in saprophytic strains (mainly P. fluorescens) grown at or above 37°C (Donnarumma et al., 2010). These siderophores acquire iron from lactoferrin and transferrin and regulate the production of virulence factors (exotoxin A and endoprotease) (Takase et al., 2000).
Besides iron, several siderophores can bind other essential metals (Mg, K) serving as micronutrients (Shinozaki-Tajiri et al., 2004). Furthermore, microorganisms producing siderophores are able to bind a variety of toxic metals such as Al, Pb, Cd (Mureseanu et al., 2003). While essential metals have important biological role, at high levels they can alter cell membranes, disturb cellular functions, damage the DNA structure and can reduce crop yields (Teitzel et al., 2006). Pseudomonas strains are widely utilized in bioremediation processes as they are able to survive and adapt in extreme environments. PsTp172 isolate is the most tolerant bacteria to tested metals. This metal resistance could be maintained by active efflux, uptake reduction, sequestration, detoxification and binding Science Publications AJES proteins synthesis (Zhuang et al., 2007). In previous investigation, it has been demonstrated that PsTp171 strain was able to bind Zn and to tolerate high concentration of the metal. The result shows that pyoverdins may have the capacity to complex zinc instead of iron. This strain might be able to sequester one of essential element and make it unattainable to phytopathogens (competition phenomenon) (Mehri et al., 2012).
In addition to these traits, PGPB have to be competent in the rhizospheric soil (Sajani and Muthukkaruppan, 2011). So, growth inhibition of pathogenic and phytopathogenic bacteria by antimicrobials released mainly by PsS15, PsTp172, PsS28, PsTp171, PsS31, PsS67, PsS18, PsS39 and PsS93 reflect their rhizospheric competitiveness that can be beneficially combined with plant protection. On the other hand, the discovery of QS enzymes did not only offered a promising means to control bacterial infections, but also provided new challenges to study their roles in host organisms and their potential impacts on environments (Dong and Zhang, 2005). Molina et al. (2003) showed that QS signal of Erwinia carotovora and Agrobacterium tumefaciens can be disturbed by lactonase of P. fluorescens which significantly reduce respectively potato soft rot and crown gall of tomato. Our results revealed that PsS15, PsTp156, PsTp172, PsC54, PsTp171 and PsS102 isolates are capable to disturb and inhibit the QS signal of the 3 pathogenic and phytopathogenic bacteria. Thus, the quorum-quenching mechanism has emerged as novel biocontrol strategy that could substitute pesticides.

CONCLUSION
On the basis of biofertilizer, plant growth promoter and biocontrol capacities we propose three strains (PsTp172, PsTp171 and PsS15) as potential bioinoculants for crop plants to increase seedling emergence, vigor and yield. Selected isolates are metal tolerant and could be used in polluted soils. Further siderophore production and antagonistic activity, which helps in establishing and resisting against deleterious microorganisms, reflect their rhizospheric competitiveness and plant protection that can be beneficially combined with PGPR traits. Additionally, in vitro production of hyper-adherent and/or hyper-mucoïd PGPB inocula could be utilized to increase the crop yields by improving N2 fixation, nutrient uptake and to protect plant against soil pathogens. Moreover, the concept of biological control of plant diseases, using non-pathogenic bacteria as bio-pesticide could substitute chemical fertilizers and pesticides, since they cause an accumulation of toxic compounds in soil. Thus, the use of microorganisms as biological control agent (antimicrobial secretion or NAHL degradation) is a promising strategy to ensure effective plant protection in agricultural ecosystems.
Therefore, most effective Pseudomonas isolates could well be commercialized but we should not overlook the involvement of some species of P. fluorescens, able to grow at or above 37°C that can behave as opportunistic agent in human pathogenicity (endotoxin release). Inoculation of these species (PsS60, PsS73 and PsS91) in raw consumption plants (carrots, cabbage) can cause severe effects in patients with weakened immune systems (Chapalain et al., 2008).