P. aeruginosa has long been under human concern. Its plant growth promoting characteristics have always been debatable, a major cause being its opportunistic pathogenic nature causing a wide array of hospital born infections in humans.1 It is amongst the most successful organisms that have been able to modify themselves to survive the changing environment; whether be it saline or acidic conditions or the antibiotic menace. Today P. aeruginosa is one of the most potent multiple drug resistant bacterium which has been able to resist an innumerable antibiotics.2 This is what makes it interesting for the researchers to study on this particular unpredictable bacterium. Plant growth promoting rhizobacteria are a group of bacteria that reside inside the rhizosphere and help in plant development. 3 Rhizosphere is that part of the soil which is strictly governed by the roots of the plant.4 These plant growth promoters may help the plant directly or indirectly. Direct methods include phytohormone production such as Indole Acetic Acid (IAA), Gibberellic Acid (GA) etc., ammonia production (as N source), atmospheric nitrogen fixation, siderophore production (for iron chelation), zinc and phosphate solubilization and providing tolerance against unfavorable conditions (high salinity) etc.5 Indirect methods of plant growth inducing methods involve anti-phytopathogenic ability by producing secondary metabolites such as antibiotics or hydrogen cyanide that restrict the growth of various fungal as well as bacterial species that produce deleterious effects over plant.5
Seeds are the most important part of an angiosperm’s lifecycle. Weather the plant’s geneline will continue or not depends on its seeds’ ability to germinate. There are various factors that regulate seed germination such as temperature, light, moisture, pH, etc.6,7 Some rhizobacterial species may inhibit seed germination completely or reduce its ability to germinate.8 Hence if a microorganism shows an array of plant growth promoting characters in vitro but hinders seed germination it is contradictory to consider it as a plant growth promoter. Also these microorganisms may have an important role in natural as well as artificial environment (agricultural fields). Therefore it is necessary to carry out experiments to understand the nature of such bacterial strains.
Materials and Methods Isolation
The soil sample was serially diluted up to 10-4 dilutions and filtered in test tubes. 0.25 mL of the filtered solution was spread using an L-shaped spreader on nutrient agar media containing per litre of doubled distilled water, 5.0 g peptone, 1.5 g yeast extract, 1.5 g beef extract, 5.0 g NaCl and 18 g agar, pH was maintained at 7.2 and plates were incubated at 28.5°C for 72 hrs.
Selective media for Pseudomonas Aeruginosa
The obtained pure colonies were transferred to cetrimide agar media containing per litre of distilled water,9 20 g enzymatic digest of gelatin, 1.4 g MgCl2, 10 g KCl, 0.3 g cetrimide (Cetrimethylammonium Bromide), 10 mL glycerol and 13.6 g agar. Plates were incubated for 48hrs at 28.5°C. Colonies identical to P. aeruginosa showed bluish green fluorescence under UV light (254 nm).
Gram staining and microscopy
The protocol was carried out as stated by Bartholomew and Mittwer (1952).10 Pinch of colonies were taken on a slide and a thin smear was formed by adding a drop of distilled water. Smear was air dried and heat fixed. Slide was flooded with crystal violet, kept for 1 min and was gently washed in an indirect stream of tap water. A drop of Gram’s iodine was applied as a mordant for 1 min. and the slide was again gently washed. Alcoholacetone decolorizer was poured drop by drop on the inclined slide until the decolorizing agent ran clear. The slide was counter stained with safranin for 45 seconds, washed under an indirect stream and excess water was absorbed on a tissue paper. Violet to purple color colony are said to be gram positive whereas pinkish-red colony show gram negative test. Cell morphology was observed under oil immersion (100×) of a bright field compound microscope.
A loop full of cells from a 24 hrs old bacterial culture was transferred to a slide and a drop of 3% hydrogen peroxide was added. Occurrence of bubbles indicated a positive test for catalase production by the bacteria.11
The test was performed as stated by Isenberg (2004).12 Bacterial cells were cultivated in tripticase soy agar containing per litre of doubled distilled H2O, 17 g tryptone, 3 g soytone, 2.5 g dextrose, 5 g NaCl, 2.5 g K2HPO2, 15g agar, pH 7.2 and incubated for 24 hrs at 28.5°C. Pinch of colonies were transferred to a sterile filter paper aseptically with a glass rod and 2-3 drops of N, N-dimethyl-pphenylenediamine (DMPD) was added. A color change from violet to purple indicated a positive oxidase test confirming cytochrome oxidase production by the bacteria.
Simmon’s citrate agar containing per litre of distilled H2O, 5 g NaCl, 2 g sodium citrate, 1 g ammonium dihydrogen phosphate, 1 g dipotassium phosphate, 0.2 g magnesium sulphate, 0.08 g bromothymol blue, 15 g agar; was prepared, autoclaved and poured in plates. The media was spot inoculated and incubated at 28.5°C for 24 hrs. Color of the media changed from green to blue indicating a positive test and bacterial capability to utilize citrate as carbon source by producing the enzyme citrate permease. 13
Christensen’s urea agar containing per litre distilled H2O, 20 g urea, 5 g NaCl, 2 g monopotassium phosphate, 1 g peptone, 1 g dextrose, 18 g agar, and 0.012 g phenol red; was prepared and sterilized. The media was poured in test tubes and slants were prepared. The slant was streaked with the bacterial isolate and incubated at 28.5°C for 48 hrs. Development of bright pink color indicated urease positive.14
MALDI-TOF mass spectrometry
Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI- TOF MS) is one of the most famous modern approaches used in bacterial cell identification. The procedure was followed as stated by Etienne et al.15 Condensed biopolymer molecules were converted into intact, ionized molecules in the gas phase. Ions were then separated based on their molecular mass (m), the charge (z), the ratio mass/charge (m/z), and the relative intensity of the signal. Initially crystals between the sample and an organic matrix (co-crystallization) were formed. The sample was then spotted on MALDI-TOF sample target with an appropriate matrix (Sinapinic Acid) and was allowed to air dry at room temperature. Then, the plate was inserted into the MS; the dried matrix was bombarded with a laser to create gas phase ions that were then pulsed into a flight tube. Generally only a singly ionized species having a single charge is produced. The species of interest were identified by their mass/charge ratio. The m/z value was obtained from the centroid of the peak. Among the compounds detected in the spectrum, some peaks (molecular masses) are specific to genus, species and sometime to subspecies. Matrix is surely the most important part of this process to acquire a reliable result with least variance. Thus keeping this in mind Sinapinic Acid was used as the matrix for the identification of the isolate.
Growth at various salt concentrations
Nutrient broth containing per litre distilled H2O, 5 g peptone, 1.5 g yeast extract, 1.5 g beef extract was prepared and 4 mL of it was poured in 10 mL sterile vials each. NaCl was added from 1% to 10 % in set of 24 vials each (240 vials), another set of 24 vials lacked NaCl for 0% NaCl concentration. All the vials were marked according to their salt concentrations. The vials containing media were autoclaved at 121°C at 15 lbs for 15 minutes.
Each vial was inoculated with 75 μL of 24 hrs old bacterial culture prepared in nutrient broth and kept in incubator at 28.5°C. Optical density of each triplicate sample was noted using UV-visible spectrophotometer at different time intervals: 0 hr, 3 hrs, 6 hrs, 9 hrs, 12 hrs, 15 hrs, 18 hrs, 21 hrs and 24 hrs. Growth curves under various salt concentrations were monitored.
Antibiotic sensitivity was tested by disk diffusion method against 6 different antibiotics namely Amikacin 10 mcg (AK10), Bacitracin (B), Clindamycin 10 mcg (CD10), Gentamycin 120 mcg (HLG120), Chloramphenicol 30 mcg (C30) and Ciprofloxacin 5 mcg (CIP5).16 Mueller Hinton agar media containing per litre distilled H2O, 2 g beef extract, 17.5 g acid hydrolysate of casein, 1.5 g starch, and 17 g agar was prepared, sterilized and plated. 24 hrs old bacterial cultures were spread on the media and antibiotic discs were placed (3 in one plate). The plates were incubated for 48 hrs at 28.5°C. Zones around the antibiotic disk indicated bacterial susceptibility whereas no zone indicated bacterial resistance.
Nutrient broth was prepared and sterilized in a conical flask. It was inoculated with a freshly prepared bacterial culture and incubated at 28.5°C in a normal incubator for 72 hrs. It was made sure that the conical flask is undisturbed. Floating bacterial clumps indicated bacterial ability to flocculate.
In vitro assays for PGPR abilities
The method stated by Pikovskaya was followed.17 Pikovskaya Agar medium containing per litre distilled HO, 10 g glucose, 5 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.2 g NaCl, 0.1 g MgSO47H2O, 0.1 g KCl, 0.5 g yeast extract, 0.002 g MnSO4H2O, 0.002 g FeSO47H2O, 18 g agar was prepared and poured in plates after sterilization. The media was spot inoculated in the center and incubated at 28.5°C for 48 hrs. A transparent zone around the inoculums showed phosphate solubilizing ability of the strain.
Nutrient agar medium supplied with 0.1% zinc oxide was prepared, sterilized and poured in plates. It was spot inoculated in the center using an inoculation loop and incubated for 28.5°C for 24 hrs. A transparent zone around the inoculum showed zinc solubilizing ability of the strain.18
0.1 mL bacterial culture was added to 10 mL sterilized peptone water in a conical flask, and incubated for 28.5°C for 48 hrs. 0.5 mL Nessler’s reagent was added to it. Development of yellowish brown color indicated ammonia production.19
Production of HCN was tested as stated by Castric (1975).20 HCN induction media – Kings media B base containing per litre distilled H2O, 20 g peptone, 1.5 g dipotassium hydrogen phosphate, 1.5 g magnesium sulphate heptahydrate, 10 g NaCl, 15 mL glycerol, 20 g agar; supplemented with 4.4 g glycine was prepared, sterilized and plated. The media was streaked with bacterial culture and a Whatman filter paper no. 1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed inside the top of the plate. Plates were sealed with parafilm and incubated at 28.5°C for 72 hrs. The change in color of the filter paper from light yellow to reddish brown indicated HCN production. The filter paper is soaked in 10 mL doubled distilled H2O and optical density of this solution is taken at 540 nm. The control plate’s filter paper (not inoculated, hence no change in color) is also soaked in 10 mL distilled H2O and the solution is used as blank.
Chrome azurol S agar was prepared by mixing per 990 mL distilled H2O, 60.5 mg chrome azurol S, 72.9 mg hexadecyltrimethyl ammonium bromide, 10 mL of 1 mMFeCl3.6 H2O solution prepared in 10 mM HCl, 42.23 g Kings media B base; Media was autoclaved and poured in plates for solidification. It was spot inoculated with 24 hrs old culture of test organism (10 μL) and incubated at 28.5°C for 72 hrs. Development of yellow orange halo around the bacterial growth was considered positive for siderophore production.21
Production of indole 3-acetic acid at various salt concentrations
Protocol stated by Ehmann (1977) was followed.22 Luria-Bertani broth containing per litre distilled H2O, 10 g tryptone, 5 g yeast extract; supplied with 5 mg/mL tryptophan was prepared and poured into vials (2 mL each). Salt (NaCl) was added from 1% to 10% in different vials in triplicates. Another set of 3 vials were left for 0% salt concentration. Vials were marked according to their salt concentration and autoclaved. Each vial was inoculated with 0.1% 24 hrs old bacterial cultures and incubated at 28.5°C for 72 hrs. The culture was centrifuged at 8,000 rpm for 10 min and the supernatant was recollected in the respective vials. 2 mL Salkowski reagent containing per litre distilled H2O, 600 mL H2SO4, 20 mL FeCl3 was added to each supernatant. Color change to reddish pink indicated positive test for IAA production.
Optical density of the above reddish pink solution was taken at 530 nm with the help of UV-VIS Spectrophotometer.23 Concentration of IAA produced by the culture at various salt concentrations was measured using standard graph of IAA obtained in the range of 10-100 μg/mL.
72 hrs grown cultures (in LB-Try media supplemented with various NaCl concentrations) were centrifuged at 8,000 rpm for 10 minutes. The supernatants were collected and acidified with HCl (pH 2.8). It was extracted adding ethyl acetate of equal volume. The extracts were air dried, recollected in ethanol and analyzed using thin layer chromatography (TLC) to confirm the presence of IAA.22
Well diffusion method was used in which 24 hrs old cultures of Klebsiella sp. and E. coli (in nutrient broth) was spread separately on plates containing nutrient agar media.24 8 mm wells were made in the center of the plates and were filled with 200 μL of P. aeruginosa strain. It was incubated for 72 hrs at 28.5°C. Nutrient broth was taken as a negative control whereas the antibiotic Ceftazidime (100 μg/L) was used as a positive control. Development of clear zones around wells indicated positive antagonistic activity of the bacterial strain.
Potato dextrose agar (PDA) media containing per litre distilled water, 200 g potato infusion, 20 g dextrose, and 20 g agar powder was prepared, autoclaved and poured in plates. Further 3 different modified dual culture methods were used to examine the antifungal property of the isolated strain.
Single streak method
A straight line streak of bacterial strain was made at one corner of the plate containing PDA media using inoculation loop. An 8 mm fungal colony (cut from a pure fungal culture) was placed at the extreme opposite corner.
Double streak method
Two straight line streak of bacterial strain were made at extreme opposite corners of the plate and 8 mm fungal colony was placed in the center.
The plates were spread with 0.1 mL bacterial culture and 8 mm fungal colony was placed at the center of the plate.
The plates were then incubated at 28.5°C for 7 days. Control plates were not inoculated with bacterial culture. Inhibition of fungal growth was calculated using the formula: growth inhibition = (control − test / control) × 100 as proposed by Skidmore and Dickinson (1976).25 Here control and test signifies the fungal growth (diameter) in control and test plates respectively. The above set up was tested with 3 different sp. of fungi namely Fusarium solani, Aspergillus niger and Curvularia lunata.
Bacterial effect on seed germination
In vitro assay
Seeds were prepared according to the procedure stated by Lubna et al.26 Seeds of Z. mays and T. aestivum were surface sterilized with 0.1% HgCl2 for 2 minutes and washed with sterile water. Seeds were constantly shaken with 24 hrs old bacterial culture (in nutrient broth) until a thin layer of bacteria developed around seeds. Carboxymethyl cellulose (CMC) was used as a binding agent. Seeds were air dried and placed in plates containing soft agar media (75 g agar per litre distilled H2O). Control plates were prepared with sterilized seeds without bacterial inoculation. The plates of Zea mays and T. aestivum were kept at 20±2°C and 28±2°C respectively.
Effect of antibiotics over seed germination inoculated with P. aeruginosa
Six antibiotics namely Amikacin (AK), Bacitracin (B), Clindamycin (CD), Gentamycin (HLG), Chloramphenicol (C) and Ciprofloxacin (CIP) in concentration of 2000 μg/L were added separately to plates containing soft agar media. Seeds were sterilized and inoculated with the isolated bacterial culture as mentioned above. Control plates were prepared with sterilized seeds without bacterial inoculation. The plates of Z. mays and T. aestivum are kept at 20±2°C and 28±2°C respectively.
Soil was collected and autoclaved 3 times to kill any possible microbial life. Sterilized pots were filled with the soil. Set of test pots were added with 1 mL 24 hrs old bacterial culture (in nutrient broth) and control pots were left un-inoculated. The sterilized seeds were sown in both test pots and control pots. The pots were kept at favorable temperature (mentioned above) and mentored for a weak to observe germination.
Characterization and identification
All the biochemical and morphological characteristics of the isolated strain strongly indicated its similarity with that of P. aeruginosa (Table 1). Further MALDI-TOF MS (Figure 1) confirmed it to be P. aeruginosa at a confidence value of 89.2.
The isolated strain was resistant to 5 of the 6 tested antibiotics, being sensitive only to Ciprofloxacin (Table 2).
The bacterial strain was found to thrive and even grow up to 1.71 mol (10%) salt (NaCl) concentrations. Mean of the triplicates was calculated and growth curves were drawn (Figure 2). The growth curves showed a gradual decline in bacterial growth rate as the salt concentration rose. Also the log phase became dilatory with increase in salt concentration.
Plant growth promoting characteristics
A well-developed zone around the inoculum in Pikovskaya medium confirmed the bacterial ability to solubilize phosphate (Figure 3A). Diameter of the transparent zone (indicating P solubilization) after 24 hrs, 48 hrs and 72 hrs was measured to be 1.1±0.1 cm, 1.9±0.1 cm, and 3.3±0.2 cm respectively. An orange brown color developed on addition of Nessler’s reagent to the 24 hrs old bacterial culture in peptone water which indicated the bacterial ability to produce ammonia. After 72 hrs incubation the filter paper soaked with picric acid solution turned brownish red confirming the production of HCN by the isolated strain. The optical density for the solution (test filter paper soaked in 10 mL distilled H2O) was found to be 0.743 ± 0.01. The isolated strain of P. aeruginosa was highly able to produce siderophore as it developed around a yellow orange halo in chrome azurol S agar medium (Figure 3B).
IAA producing ability at various salt Concentrations
Appearance of pink color on addition of Salkowski reagent up to 8% NaCl concentration indicated the production of IAA by the bacteria when supplemented with tryptophan (Figure 4). Furthermore it was observed that the Rf value for both standard IAA and the compound produced by the bacterial strain was similar (0.92) confirming their homology. It was also recorded that with increase in salt concentration up to 6%, there was no major fall in IAA production by the bacteria but it abruptly descended above that making it negligible above 8% salt concentration. The bacterial strain was able to produce 13.2±0.20 μg/mL of IAA when supplemented with 5 mg/mL Tryptophan as determined from the standard IAA graph.
The isolated strain showed antagonistic effect against the 5 tested microbial cultures namely Klebsiella sp., E. coli, Fusarium solani, Aspergillus niger and Curvularia lunata. It was observed that as the microbes came in contact with the isolate they stopped growing.
The isolated bacterial strain inhibited the growth of both the test bacteria. The diameter of growth inhibition the zones were calculated and mean values were considered from the triplicate set. The diameter of inhibition zone against Klebsiella sp. was noted to be greater (27±0.75) in comparison to E. coli (19±1.0 mm) (Table 3).
The bacterial strain was able to inhibit fungal growth up to 100% when spread onto the culture media. The effectiveness increased in the order: single streak method < double streak method < spread method (Figures 5 and 6). The average percentage growth inhibition by the bacterial strain was calculated from the triplicate (Tables 4 and 5).
Deleterious effects over seeds
Though the control seeds of T. aestivum and Zea mays that germinated within 24 and 72 hrs respectively in soft agar media, it was observed that the test seeds (inoculated with the bacterial strain) of both the plant species did not germinate at all (Figure 7). The seeds became soft, covered with a thick, light brown colored bio-film of the bacterial strain (Figure 7B,C) The pot experiment gave the same result with no seed germination in the test pots at all (Figure 7D). Hence it can be concluded that the seed germination was hindered by the bacterial strain of P. aeruginosa. Also when media was supplemented with the 6 antibiotics namely Amikacin (AK), Bacitracin (B), Clindamycin (CD), Gentamycin (HLG), Chloramphenicol (C) and Ciprofloxacin (CIP), seeds failed to germinate in all except in CIP to which the bacteria was tested sensitive.
The seed were not affected by the supernatant (24 hrs old bacterial culture centrifuged at 8000 rpm for 10 minutes) and their germination time showed no prominent difference from the control seeds.
Discussion and Conclusions
The farmers are still unaware of the soil micro-biota that plays a major role in influencing plant growth. Growth of a healthy crop is often credited to the compounds (manure and fertilizers) added to the soil, whereas growth of an unhealthy crop is linked to nutrient deficiency or some kind of insect related diseases. Lack of knowledge and less awareness leads to reduced crop productivity every year as the farmers keep on supplementing the soil with unnecessary formulations including biofertilizers. The present study focused on exploring the characteristic features (both positive and negative) of the isolated bacterial strain of P. aeruginosa. It was observed that the bacterial strain survive at NaCl concentration of 10% and produce a considerable amount of IAA up to 8% NaCl concentration. It is able in producing siderophores, HCN and ammonia and solubilizing phosphate. It has a recommendable antagonistic activity against the three phytopathogenic fungi and two bacterial strains. At the same time it also has its negative effect over seeds of maize and wheat. The seeds treated with isolated strain failed to germinate which indicates that under normal conditions in nature this particular strain inhibits plants to continue their gene line. Also it may be the major reason behind the loss of crop productivity due to inability of seeds to germinate.27 It occurred that the cellular products of the strain did not have a negative impact over the seed germination from which it can be concluded that the deleterious nature of the strain is only viable up till the cells are able to infect and proliferate over seeds interfering with the seed germination mechanism. Its ability to sustain its growth under saline conditions, HCN and siderophore production and antibiotic resistance makes it a highly resistant strain and a potent inhibitor of other microbial agents. From this study we introduce a new bacterial strain of P. aeruginosa that possesses both deleterious as well as plant growth promoting characteristics.