STENOTROPHOMONAS MALTOPHILIA: A NOVEL PLANT GROWTH PROMOTER AND BIOCONTROL AGENT FROM MARINE ENVIRONMENT

1. Research Scholar, Research and Development Centre, Bharathiar University, Coimbatore, India. 2. Professor & Head, Department of Microbiology, Sree Narayana Guru College, Coimbatore, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


In-vitro screening of marine bacterial strains for Plant Growth Promoting potentials:-Phosphate Solubilization Assay:-
Phosphate solubilizing activity was screened on Pikovskays Agar medium [Pikovaskya (1948)], for the clear zone formation around the bacterial colony after incubation at 30°C for 3-5 days and it was expressed as phosphate solubilizing index (PSI) [Katznelson and Bose, (1959)]. Solubilization Index (PSI) = Colony diameter + halo zone diameter Colony diameter

Zinc Solubilization Assay:-
All the isolates were inoculated on to the modified Pikovskaya medium containing 1% insoluble zinc compound (ZNO). The halo zone formation around the colony after incubation at 30°C for 48 hrs indicates zinc solubilization [Pikovaskya (1948)].

Production of Indole 3-Acetic Acid (Auxin):-
In this method, tryptophan rich Luria Bertani (LB) broth was inoculated with the bacterial isolates. After 24hr incubation, 2ml culture supernatant was mixed with 2 drops of ortho phosphoric acid and 4ml of Salkowsky reagent (50 ml, of 35% sulphuric acid, 1 ml of 0.5 M FeCl3) and incubated for 25 min at room temperature; development of pink colour indicates IAA production. The intensity of pink color developed was read at 535 nm using a UV-VIS spectrophotometer [Gordon and Weber, (1951)].

Nitrogen Fixation Test:-
The marine isolates were cultured onto nitrogen free Jensen's media and incubated for 2 or 3 days at 30ºC. Growth after incubation indicates that the ability of organisms to fix atmospheric nitrogen [Noori and Saud, (2012)].
Organic Acid Production:-Bacterial cultures were grown in M9 agar medium with methyl red as pH indicator. The color of the media surrounding the organic acid producing strains changed from yellow to pink when pH changed below 5 due to acid production.

Production of Extracellular Enzymes:-
The marine bacterial isolates were subjected for the screening of extracellular enzymes production, namely protease, lipase, amylase, chitinase and DNAase using simple qualitative plate assay. The basal nutrient agar medium was prepared with different substrates of the tested enzymes at 1% concentration such as skim milk for protease, tween-80 for lipase, starch for amylase, colloidal chitin for chitinase and DNA for DNAase. After 48 hrs incubation at room temperature, different visualization tests were performed to observe enzyme activity. A zone of clearance around the bacterial colony indicates positive protease, lipase and chitinase activity. For amylase test, iodine was used as indicator. Iodine reacts with the unhydrolyzed starch area to give a blue-black color. A clear zone around the colony at the site of hydrolyzed starch shows amylase production. For DNAase assay, plates were flooded with dilute HCI solution. HCL precipitates unhydrolyzed DNA and a clear zone of hydrolyzed DNA around the colony indicates DNAase activity [Ramesh and Mathivanan, (2009)]. Gelatinase test was performed by using the method described by [Blazevic and Ederer, (1975)]. It was done by inoculating the bacteria into gelatin tubes and incubated for 24-48hrs. If gelatinase was produced, the liquid medium fails to solidify upon refrigeration at 4 ºC for about an hour after incubation at 24-48hrs at room temperature.

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Biocontrol Assay:-Production of Siderophore:-The marine bacterial isolates were subjected for the production of siderophore by using modified Chrome Azurol Sulfonate (CAS) assay [Alexander and Zuberer, (1991)]. To prepare blue or green agar 60.5mg of CAS is dissolved in 50ml of distilled water and mixed with 10ml of Iron solution(1Mm FeCl.6H2O in 10Mm HCl). While constantly stirring, this solution is slowly added to 72.9 mg of hexadecyltrimethyl ammonium bromide (HDTMA) dissolved in 40ml of water. The resultant dark blue/green liquid mixture was sterilized and added to sterile nutrient agar to make Chrome Azural S (CAS) agar. Spot inoculated and incubated at 30°C for 48-72 hrs. Development of yellow-orange halo around the growth is considered as positive for siderophore production.

Production of Hydrogen Cyanide (HCN):-
The bacteria were streaked on to nutrient agar amended with glycine (4.4 g/l) plates. Whatman no.1 filter paper soaked in 2% sodium carbonate in 0.5% picric acid was placed at the inner surface of the lid of the Petri plate. Plates were sealed with parafilm and incubated at 30°C for 4 days. Development of orange to red color in the filter paper indicates HCN production [Lorck (1948)].

Biochemical characterization of bacterial strain:-
The predominant and most potent bacterial strain MB9 was identified by using VITEK 2 compact system-Biomerieus, France automatic system. VITEK-2 system provides an automated, computer based method of species identifications, relies on advanced colorimetric technology, the measurement of light attenuation associated with each biochemical reactions in VITEK cards containing numerous wells to ensure test accuracy.

In-vitro Antifungal Assay:-Cross streak Method:-
The antagonistic property of selected plant growth promoting marine bacterium was tested against different phytopathogens like Colleotrichum sp., Rhizoctonia solani., Curvularia sp., Diploidia sp., Fusarium sp., and Aspergillus sp., by using modified method of [Gupta et al., (2001)]. Agar blocks of 5 day old culture of test pathogens were placed in the centre of Tryptic Soy Agar (TSA). A loopful of 24 hr old culture of bacterial strain was then streaked on either sides of pathogen disc at a distance of 2 cm apart. Control plates with the fungal pathogens alone were inoculated centrally on TSA plates. After 5 days incubation at 25±1ºC, inhibition of the fungal growth was measured. Percentage inhibition of mycelial growth was calculated using the following formula % of inhibition = (C -T)/C × 100, Where, C and T represent the growth of fungus in control plate and treatment plate respectively.

Production of Secondary Metabolite:-
Tryptic digest broth was prepared with 2%Nacl in conical flasks. The selected bacterial strain was inoculated into the sterile broth. Then the flasks were kept in the shaking incubator for 3 days at 180rpm. After the incubation secondary metabolite was extracted.

Extraction of Secondary Metabolite by Solvent Extraction Method:-
After the production of secondary metabolites, the broth was aseptically transferred to sterile centrifuge tubes and centrifuged at 10,000 rpm for 15mins. The supernatant was collected in sterile conical flask and the pellet was discarded. The supernatant was transferred to sterile separating funnel and mixed with ethyl acetate in the ratio 1:1 (supernatant: ethyl acetate). Then the mixture in the separating funnel was shaked continuously for 15mins. After 15mins, keep the separating funnel undisturbed for 10-15mins. Three layers were formed and the middle layer containing metabolite was removed in to a sterile petri plate. The structure of antifungal metabolite was established by the study of Gas Chromatography-mass spectrometry, (MS) [Uzair et al (2006)]. 19 Morphologically distinct bacterial strains were selected (designated as MB1-MB19) and screened for its plant growth promoting potentials. Among the 19 isolates, 15 shows phosphate solubilization, auxin and organic acid 210 production, 10 shows nitrogen fixation ability, 6 shows zinc solubilization, ACC Deaminase and hydrogen cyanide production, 13 shows siderophore production, 12 shows potassium solubilization ( Table 1). The extracellular enzyme production was expressed in (Table 2). The most potent and predominant strain MB9 shows highest activity in all plant growth promoting traits. This potent strain was identified as Stenotrophomonas maltophilia by automated VITEK 2 compact system (Table 3). This bacterium was selected for biocontrol assay against fungal phytopathogens and further characterization, and it shows good antifungal activity (Table 4). Based on the result obtained from Gas Chromatography Mass Spectrometric method (GCMS), the active metabolite that mediates the broad-spectrum antifungal potential of Stenotrophomonas maltophilia has been identified as Dodecanoic acid.    Amylase  Protease  Lipase  Gelatinase  Chitinase  DNAase  MB1  -+  -+  --MB2  +  +  +  +  -+  MB3  +  ---+  -MB4  +  +  +  +  +  +  MB5  +  +  -+  --MB6  +  -+  -+  +  MB7  -+  +  +  --MB8  +  +  -+  --MB9 -    The presence of major plant growth promoting traits such as Indole Acetic Acid, phosphate solubilization, nitrogen fixation, siderophores and chitinase activities in the potent strain Stenotrophomonas maltophilia, may enhance its biofertilizer potential as well as biocontrol efficiency against different fungal phytopathogens. Previous study reported that the solubilization of iron by microbial siderophores and solubilization of phosphorous because of phosphatase were found to significantly increase crop yield [Brown (1974), Kloepper et al., (1988), Glick (1995)]. It has been reported that the production of Indole Acetic Acid by plant beneficial bacteria enhances the development of host plant root system and thus helps in the growth of crop plants [Patten and Glick, (2002), Suresh et al (2010)].

Conclusion:-
The results obtained through this study suggested that the potential use of Stenotrophomonas maltophilia as an effective biocontrol agent that promoting plant growth with reduced incidence of diseases. Further investigation on the type of antimicrobial components and field experiments on Stenotrophomonas maltophilia needed to introduce eco-friendly plant growth promoting and antifungal agents instead of hazardous chemicals.