A Plant Growth Promoting Rhizospheric Pseudomonas Aeruginosa Strain Inhibits Seed Germination in Triticum Aestivum (L) and Zea Mays

Microbiology Research 2017; volume 8:7233

A plant growth promoting rhi- seeds inoculated with the strain itself, zospheric Pseudomonas aerugi- failed to germinate. The seed turned dor- Correspondence: Jay Shankar Singh, mant and grew a pale brown color show- Department of Environmental Microbiology, nosa strain inhibits seed ing bacterial growth over it. Thus it maybe Babasaheb Bhimrao Ambedkar University, germination in Triticum concluded that the isolated strain of P. Lucknow, Uttar Pradesh, 226025 India. aestivum (L) and Zea mays (L) aeruginosa has abilities to both promote E-mail: [email protected] and at the same time inhibit plant growth (by inhibiting seed germination). Key words: Antagonism; MALDI-TOF MS; Pankaj Tiwari, Jay Shankar Singh PGPR; Phytohormones; Rhizobacteria

Department of Environmental Acknowledgments: The authors thank the Microbiology, Babasaheb Bhimrao Head, Department of Environmental Ambedkar University, Lucknow, Uttar Introduction Microbiology, Babasaheb Bhimrao Ambedkar Pradesh, India P. aeruginosa has long been under University for providing all the necessary human concern. Its plant growth promoting facilities for this work. characteristics have always beendebatable, Contributions: PT, generated the idea, Abstract a major cause being its opportunistic patho- designed the study, performed the experiments genic nature causing a wide array of hospi- and wrote the manuscript. JSS helped in con- 1 Various rhizobacteria strains are isolat- tal born infections in humans. It is ducting the experiments, evaluated data and ed and tested worldwide for their plant amongst the most successful organisms that edited the manuscript. growth promoting activities. A strain is said have been able to modify themselves to sur- to be a plant growth promoter if it has the vive the changing environment; whether be Conflict of interest: the authors declare no ability to influence plant growth directly or it saline or acidic conditions or the antibiot- potential conflict of interest. indirectly. Though the main focus has ic menace. Today P. aeruginosa is one of always been to realize the plant growth pro- the most potent multiple drug resistant bac- Received for publication: 29 May 2017. moting traits of rhizobacteria and use them terium which has been able to resist an onlyAccepted for publication: 5 June 2017. as biofertilizers, but the disadvantages or innumerable antibiotics.2 This is what deleterious effects of such plant growth This work is licensed under a Creative makes it interesting for the researchers to Commons Attribution NonCommercial 4.0 promoting rhizobacteria (PGPR) if any study on this particular unpredictable bac- License (CC BY-NC 4.0). have also been ignored to agreater extent. terium. Plant growth promoting rhizobacte-use The present study deals with the character- ria are a group of bacteria that reside inside ©Copyright P. Tiwari and J.S. Singh, 2017 ization of both promoting as well as delete- the rhizosphere and help in plant develop- Licensee PAGEPress, Italy rious activities of an isolated strain of ment.3 Rhizosphere is that part of the soil Microbiology Research 2017; 8:7233 Pseudomonas aeruginosa on plants. The which is strictly governed by the roots of doi:10.4081/mr.2017.7233 bacterium was isolated from the soil of the the plant.4 These plant growth promoters college campus of Babasaheb Bhimrao may help the plant directly or indirectly. important role in natural as well as artificial Ambedkar University, Lucknow, Uttar Direct methods include phytohormone pro- environment (agricultural fields). Therefore Pradesh and was characterized as P. aerug- duction such as Indole Acetic Acid (IAA), it is necessary to carry out experiments to inosa by various positive biochemical tests Gibberellic Acid (GA) etc., ammonia understand the nature of such bacterial such as oxidase, catalase, citrate tests and production (as N source), atmospheric strains. confirmed by MALDI-TOF-MS. Its growth nitrogen fixation, siderophore production as well as IAA producing ability at different commercial(for iron chelation), zinc and phosphate sol- salt (NaCl) concentrations was tested. Tests ubilization and providing tolerance against examining HCN, Ammonia and unfavorable conditions (high salinity) etc.5 siderophore production, flocculation abili- Indirect methods of plant growth inducing Materials and Methods ty, phosphate and zinc solubilization by the methods involve anti-phytopathogenic abil- Isolation strain were carried out. Its antNoni-microbial ity by producing secondary metabolites The soil sample was serially diluted up property was tested against three phytopath- such as antibiotics or hydrogen cyanide that to 10-4 dilutions and filtered in test tubes. ogenic fungi: Fusarium solani, Aspergillus restrict the growth of various fungal as well 0.25 mL of the filtered solution was spread niger and Curvularia lunata and two bacte- as bacterial species that produce deleterious 5 using an L-shaped spreader on nutrient agar rial strains- Klebsiella pneumoniae and E. effects over plant. media containing per litre of doubled dis- coli.Its effect over seedgermination of Seeds are the most important part of an tilled water, 5.0 gpeptone, 1.5 gyeast Triticum aestivum and Zea mays was angiosperm’s lifecycle. Weather the plant’s extract, 1.5 gbeefextract, 5.0 gNaCl and inspected in plates as well as in pots. The geneline will continue or not depends on 18 g agar, pH was maintained at 7.2 and strain showed a great potential as a plant its seeds’ ability to germinate. There are plates were incubated at 28.5°C for 72 hrs. growth promoter growing up to 10% NaCl various factors that regulate seed germina- concentration and producing considerable tion such as temperature, light, moisture, amount of IAA upto 8% NaCl concentra- pH, etc.6,7 Some rhizobacterial species Selective media for Pseudomonas tion. It showed antagonistic nature against may inhibit seed germination completely aeruginosa the three phytopathogenic fungi, solubilized or reduce its ability to germinate.8 Hence The obtained pure colonies were trans- phosphate and produced siderophore and if a microorganism shows an array of plant ferred to cetrimide agar media containing 9 NH3. Though supernatant of the bacterial growth promoting characters in vitro but per litre of distilled water, 20 genzymatic culture did not show any deleterious hinders seed germination it is contradictory digest of gelatin, 1.4 g MgCl2, 10 g KCl, 0.3 effect over seed (of the test plants T. aes- to consider it as a plant growth promoter. gcetrimide (Cetrimethylammonium tivum and Z. mays) germination but the Also these microorganisms may have an Bromide), 10 mL glycerol and 13.6 g

[Microbiology Research 2017; 8:7233] [page 73] Article agar.Plates were incubated for 48hrs at hrs. Color of the media changed from green centration. All the vials were marked 28.5°C. Colonies identical to P. aerugi- to blue indicating a positive test and bacter- according to their salt concentrations. The nosa showed bluish green fluorescence ial capability to utilize citrate as carbon vials containing media were autoclaved at under UV light (254 nm). source by producing the enzyme citrate per- 121°C at 15 lbs for 15 minutes. mease.13 Each vial was inoculated with 75 µL Characterization of 24 hrs old bacterial culture prepared in Gram staining and microscopy Urease test nutrient broth and kept in incubator at The protocol was carried out as stat- Christensen’s ureaagarcontaining per 28.5°C. Optical density of each triplicate ed by Bartholomew and Mittwer litre distilled H2O, 20 gurea, 5 g NaCl, 2g sample was noted using UV-visible spec- (1952).10 Pinch of colonies were taken on monopotassium phosphate, 1gpeptone, 1g trophotometer at different time intervals: 0 a slide and a thin smear was formed by dextrose, 18 gagar, and 0.012 g phenol red; hr, 3 hrs, 6 hrs, 9 hrs, 12 hrs, 15 hrs, 18 hrs, adding a drop of distilled water. Smear was prepared and sterilized. The media was 21 hrs and 24 hrs. Growth curves under var- was air dried and heat fixed. Slide was poured in test tubes and slants were pre- ious salt concentrations were monitored. flooded with crystal violet, kept for 1 min pared. The slant was streaked with the bac- and was gently washed in an indirect stream terial isolate and incubated at 28.5°C for 48 Antibiotic sensitivity of tap water. A drop of Gram’s iodine was hrs. Development of bright pink color indi- Antibiotic sensitivity was tested by 14 applied as a mordant for 1min. and the cated urease positive. disk diffusion methodagainst 6 different slide was again gently washed. Alcohol- antibiotics namely Amikacin 10 mcg acetone decolorizer was poured drop by MALDI-TOF mass spectrometry (AK10), Bacitracin (B), Clindamycin 10 drop on the inclined slide until the decol- Matrix Assisted Laser Desorption mcg (CD10), Gentamycin 120 mcg orizing agent ran clear. The slide was Ionization Time-of-Flight Mass (HLG120), Chloramphenicol 30 mcg (C30) counter stained with safranin for 45 sec- Spectrometry (MALDI- TOF MS) is one and Ciprofloxacin 5mcg(CIP5).16 Mueller onds, washed under an indirect streamand of the most famous modern approaches Hinton agar media containing per litre dis- excess water was absorbed on atissue used in bacterial cell identification. The tilled H2O, 2gbeefextract, 17.5 g acid paper. Violet to purple color colony are said procedure was followed as stated by hydrolysateonlyof casein, 1.5 g starch, and 17 g to be gram positive whereas pinkish-red Etienne et al.15 Condensed biopolymer agar was prepared, sterilized and plated. 24 colony show gram negative test. Cell mor- molecules were converted into intact, ion- hrs old bacterial cultureswerespread on the phology was observed under oil immer- ized molecules in the gas phase. Ions were media and antibiotic discs were placed (3 in sion (100×) of abright field compound then separated based on their molecular one plate). The plates were incubated for 48 microscope. mass (m), the charge (z), the useratio hrs at 28.5°C. Zones around the antibiotic mass/charge (m/z), and the relative inten- disk indicated bacterial susceptibility Biochemical assay sity of the signal. Initially crystals between whereas no zone indicated bacterial resist- Catalase test the sample and an organic matrix (co-crys- ance. A loop full of cells from a 24 hrs old tallization) were formed. The sample was bacterial culture was transferred to a slide then spotted on MALDI-TOF sample target Flocculation test and adrop of 3% hydrogen peroxide was with an appropriate matrix (Sinapinic Acid) Nutrient broth was prepared and steril- added. Occurrence of bubbles indicated a and was allowed to air dry at room temper- ized in a conical flask. It was inoculated positive test for catalase production by the ature. Then, the plate was inserted into the with a freshly prepared bacterial culture bacteria.11 MS; the dried matrix was bombarded with a and incubated at 28.5°C in a normal incu- laser to create gas phase ions that were then bator for 72 hrs. It was made sure that the Oxidase test pulsed into a flight tube. Generally only a conical flask is undisturbed. Floating bac- The test was performed as stated by sincommercialgly ionized species having a single terial clumps indicated bacterial ability to Isenberg (2004).12 Bacterial cells were culti- charge is produced. The species of interest flocculate. vated in tripticase soy agar containing per litre were identified by their mass/charge ratio. of doubled distilled H2O, 17 g tryptone, 3 g The m/z value was obtained from the cen- In vitro assays for PGPR abilities soytone, 2.5 g dextrose, 5 g NaCl,Non 2.5 g troid of the peak. Among the compounds Phosphate solubilization K2HPO2, 15g agar, pH 7.2 and incubated for detected in the spectrum, some peaks (mol- The method stated by Pikovskaya was 24 hrs at 28.5°C. Pinch of colonies were trans- ecular masses) are specific to genus, species followed.17 Pikovskaya Agar medium con- ferred to a sterile filter paper aseptically with a and sometime to subspecies. Matrix is sure- taining per litre distilled HO, 10 g glucose, glass rod and 2-3 drops of N, N-dimethyl-p- ly the most important part of this process 5 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.2 g NaCl, phenylenediamine (DMPD) was added. A to acquire areliable result with least vari- 0.1 g MgSO47H2O, 0.1 g KCl, 0.5 g yeast color change from violet to purple indicated a ance. Thus keeping this in mind Sinapinic extract, 0.002 g MnSO4H2O, 0.002 g positive oxidase test confirming cytochrome Acid was used as the matrix for the identifi- FeSO47H2O, 18 g agar was prepared and oxidase production by the bacteria. cation of the isolate. poured in plates after sterilization. The media was spot inoculated in the center and Citrate test Growth at various salt concentra- incubated at 28.5°C for 48 hrs. Atranspar- Simmon’s citrate agar containing per tions ent zone around the inoculums showed litre of distilled H2O, 5 g NaCl, 2 g sodium Nutrient broth containing per litre dis- phosphate solubilizing ability of the strain. citrate, 1 g ammonium dihydrogen phos- tilled H2O, 5 g peptone, 1.5 gyeast extract, phate, 1 g dipotassium phosphate, 0.2 g 1.5 gbeef extractwasprepared and 4 mL Zinc solubilization magnesium sulphate, 0.08 g bromothymol of it was poured in 10 mL sterile vials Nutrient agar medium supplied with blue, 15 g agar; was prepared, autoclaved each. NaCl was added from 1% to 10 % in 0.1% zinc oxide was prepared, sterilized and poured in plates. The media was spot set of 24 vials each (240 vials), another set and poured in plates. It was spot inoculated inoculated and incubated at 28.5°C for 24 of 24 vials lacked NaCl for 0% NaCl con- in the center using an inoculation loop and

[page 74] [Microbiology Research 2017; 8:72233] Article incubated for 28.5°C for 24 hrs. A transpar- centration and autoclaved. Each vial was inoc- fungal culture) was placed at the extreme ent zone around the inoculum showed zinc ulated with 0.1% 24 hrs old bacterial cultures opposite corner. solubilizing ability of the strain.18 and incubated at 28.5°C for 72 hrs. The culture was centrifuged at 8,000 rpm for 10 min Double streak method NH3 production and the supernatant was recollected in the Two straight line streak of bacterial 0.1 mL bacterial culture was added to respective vials. 2 mL Salkowski reagent con- strain were made at extreme opposite cor- 10 mL sterilized peptone water in aconical taining per litre distilled H2O, 600 mL H2SO4, ners of the plate and 8 mm fungal colony flask, and incubated for 28.5°C for 48 hrs. 20 mL FeCl3 was added to each supernatant. was placed in the center. 0.5 mL Nessler’s reagent was added to it. Color change to reddish pink indicated posi- Development of yellowish brown color tive test for IAA production. Spread method indicated ammonia production.19 The plates were spread with 0.1 mL Quantitative test bacterial culture and 8 mm fungal colony HCN production Optical density of the above reddish was placedat the center of the plate. Production of HCN was tested as stated pink solution was taken at 530 nm with the The plates were then incubated at by Castric (1975).20 HCN induction media help of UV-VIS Spectrophotometer.23 28.5°C for 7days.Control plates were not – Kings media Bbase containing per litre Concentration of IAA produced by the cul- inoculated with bacterial culture.Inhibition distilled H2O, 20 gpeptone, 1.5 g dipotassi- ture at various salt concentrations was of fungal growth was calculated using the um hydrogen phosphate, 1.5 g magnesium measured using standard graph of IAA formula: growth inhibition = (control − test sulphate heptahydrate, 10 g NaCl, 15 mL obtained in the range of 10-100 µg/mL. / control) × 100 as proposed by Skidmore glycerol, 20 g agar; supplemented with 4.4 and Dickinson (1976).25 Here control and gglycine was prepared, sterilized and plat- Confirmatory test test signifies the fungal growth (diameter) ed. The media was streaked with bacterial 72 hrs grown cultures(inLB-Try in control and test plates respectively. The culture and aWhatmanfilter paper no. 1 media supplemented with various NaCl above set up was tested with 3 different soaked in 2% sodium carbonate in 0.5% concentrations) were centrifuged at 8,000 sp. of fungi namely Fusarium solani, picric acid solution was placed inside the rpm for 10 minutes. The supernatants were Aspergillusonly niger and Curvularia lunata. top of the plate. Plates were sealed with collected and acidified with HCl (pH 2.8). It parafilm and incubated at 28.5°C for 72 was extracted adding ethyl acetate of equal Bacterial effect on seed germination hrs. The change in color of the filter volume. The extracts were air dried, recol- In vitro assay paper from light yellow to reddish brown lected in ethanol and analyzed using thin Seeds were prepared according to the indicated HCN production. The filter paper layer chromatography (TLC) to confiuserm the procedure stated by Lubna et al.26 Seeds of 22 is soaked in 10 mL doubled distilled H2O presence of IAA. Z. mays and T. aestivum were surface and optical density of this solution is taken sterilized with 0.1% HgCl2 for 2minutes at 540 nm. The control plate’s filter paper Antimicrobial assay and washed with sterile water. Seeds were (not inoculated, hence no change in color) Antibacterial assay constantly shaken with 24 hrs old bacterial is also soaked in 10 mL distilled H2O and Well diffusion method was used in culture (in nutrient broth) until a thin layer the solution is used as blank. which 24 hrs old cultures of Klebsiella sp. of bacteria developed around seeds. and E. coli (in nutrient broth) was spread Carboxymethyl cellulose (CMC) was used Siderophore production separately on plates containing nutrient agar as a binding agent. Seeds were air dried and Chrome azurol Sagarwaspreparedby media.24 8 mm wells were made in the placed in plates containing soft agar media mixing per 990 mL distilled H2O, 60.5 mg center of the plates and were filled with (75 g agar per litre distilled H2O). Control chrome azurol S, 72.9 mg hexade- 200 µL of P. aeruginosa strain. It was plates were prepared with sterilized seeds cyltrimethyl ammonium bromide, 10 mL of commercialincubated for 72 hrs at 28.5°C. Nutrient without bacterial inoculation. The plates of 1mMFeCl3.6 H2O solution prepared in 10 broth was taken as a negative control Zea mays and T. aestivum were kept at mM HCl, 42.23 g Kings media Bbase; whereas the antibiotic Ceftazidime (100 20±2°C and 28±2°C respectively. Media was autoclaved and poured in plates µg/L) was used as a positive control. for solidification. It was spotNon inoculated Development of clear zones around wells Effect of antibiotics over seed germina- with 24 hrs old culture of test organism (10 indicated positive antagonistic activity of tion inoculated with P. aeruginosa µL) and incubated at 28.5°C for 72 hrs. the bacterial strain. Six antibiotics namely Amikacin (AK), Development of yellow orange halo around Bacitracin (B), Clindamycin (CD), the bacterial growth was considered posi- Antifungal assay Gentamycin (HLG), Chloramphenicol (C) 21 tive for siderophore production. Potato dextrose agar (PDA) media and Ciprofloxacin (CIP) in concentration of containing per litre distilled water, 200 g 2000 µg/L were added separately to plates Production of indole 3-acetic acid at potato infusion, 20 g dextrose, and 20 g containing soft agar media. Seeds were various salt concentrations agar powder was prepared, autoclaved and sterilized and inoculated with the isolated Qualitative test poured in plates. Further 3 different modi- bacterial culture as mentioned above. Protocol stated by Ehmann (1977) was fied dual culture methods were used to Control plates were prepared with sterilized followed.22 Luria-Bertani broth containing per examine the antifungal property of the iso- seeds without bacterial inoculation. The litre distilled H2O, 10 g tryptone, 5 g yeast lated strain. plates of Z. mays and T. aestivum are kept extract; supplied with 5 mg/mL tryptophan at 20±2°C and 28±2°C respectively. was prepared and poured into vials (2 mL Single streak method each). Salt (NaCl) was added from 1% to 10% A straight line streak of bacterial strain Pot experiment in different vials in triplicates. Another set of was made at one corner of the plate con- Soil was collected and autoclaved 3 times 3 vials were left for 0% salt concentration. taining PDA media using inoculation loop. to kill any possible microbial life. Sterilized Vials were marked according to their salt con- An 8 mm fungal colony (cut from a pure pots were filled with the soil. Set of test pots

[Microbiology Research 2017; 8:7233] [page 75] Article were added with 1 mL 24 hrs old bacterial cul- aeruginosa (Table 1). Further MALDI-TOF Plant growth promoting characteristics ture (in nutrient broth) and control pots were MS (Figure 1) confirmed it to be P. aerug- Awell-developed zone around the left un-inoculated. The sterilized seeds were inosa at a confidence value of 89.2. inoculum in Pikovskaya medium confirmed sown in both test pots and control pots. The The isolated strain was resistant to 5 of the bacterial ability to solubilize phosphate pots were kept at favorable temperature (men- the 6 tested antibiotics, being sensitive only (Figure 3A). Diameter of the transparent tioned above) and mentored for a weak to to Ciprofloxacin (Table 2). zone (indicating P solubilization) after 24 observe germination. The bacterial strain was found to hrs, 48 hrs and 72 hrs was measured to be thrive and even grow up to 1.71 mol 1.1±0.1 cm, 1.9±0.1 cm, and 3.3±0.2 cm (10%) salt (NaCl) concentrations. Mean of respectively. An orange brown color devel- the triplicates was calculated and growth oped on addition of Nessler’s reagent to the curves were drawn (Figure2). The growth Results 24 hrs old bacterial culture in peptone curves showed a gradual decline in bacter- water which indicated the bacterial ability Characterization and identification ial growth rate as the salt concentration All the biochemical and morphological to produce ammonia. After 72 hrs incuba- rose. Also the log phase became dilatory characteristics of the isolated strain strongly tion the filter paper soaked with picric acid with increase in salt concentration. indicated its similarity with that of P. solution turned brownish red confirming the production of HCN by the isolated strain. The opticaldensity 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 ayelloworange halo in chrome azurol S agar medium(Figure 3B).

IAAonly producing ability at various salt concentrations Appearance of pink color on addition of Salkowski reagent up to 8% NaCl con- usecentr ation indicated the production of IAA

Table 1. General characteristics of the isolated strain. Characters studied Observations Colony morphology Pigmentation Fluorescent green Appearance Sticky, mucilaginous Odor Fruity Exopolysaccharide secretion Present commercial Ability to flocculate Present Cell morphology Shape Rods Figure 1. MALDI-TOF analysis report. Size Small Gram staining Negative Non Motility Positive Biochemical tests Catalase Positive Oxidase Positive Citrate Positive Cetrimide Positive

Table 2. Antibiotic resistance. Antibiotic resistance Results Ciprofloxacin 5 mcg (CIP5) Negative Amikacin 10 mcg (AK10) Positive Bacitracin (B) Positive Clindamycin 10 mcg (CD10) Positive Gentamycin 120 mcg (HLG120) Positive Figure 2. Growth curves showing bacterial growth (Optical density) at different salt con- Chloramphenicol 30 mcg (C30) Positive centrations at different time intervals.

[page 76] [Microbiology Research 2017; 8:7233] Article by the bacteria when supplemented with dard IAA and the compound produced by Antifungal nature tryptophan (Figure 4). Furthermore it was the bacterial strain was similar (0.92) con- The bacterial strain was able to inhibit observed that the Rf value for both stan- firming their homology. It was also record- fungal growth up to 100% when spread ed that with increase in salt concentration onto the culture media. The effectiveness up to 6%, there was no major fall in IAA increased in the order: single streak method production by the bacteria but it abruptly < double streak method < spread method A descended above that making it negligible (Figures 5 and 6). The average percentage above 8% salt concentration. The bacterial growth inhibition by the bacterial strain strain was able to produce 13.2±0.20 µg/mL was calculated from the triplicate (Tables 4 of IAA when supplemented with 5 mg/mL and 5). Tryptophan as determined from the standard IAA graph. Deleterious effects over seeds Though the control seeds of T. aestivum Antagonistic effect and Zea mays that germinated within 24 The isolated strain showed antagonistic and 72 hrs respectively in soft agar media, effect against the 5 tested microbial cultures it was observed that the test seeds (inocu- B namely Klebsiella sp., E. coli, Fusarium lated with the bacterial strain) of both the solani, Aspergillus niger and Curvularia plant species did not germinate at all lunata.Itwasobserved that as the microbes (Figure 7). The seeds became soft, covered came in contact with the isolate they with a thick, light browncolored bio-film of stopped growing. the bacterial strain (Figure 7B,C) The pot experiment gave the same result with no seedgermination in the test pots at all Antibacterial nature (Figure 7D). Hence it can be concluded that The isolated bacterial strain inhibited theonly seedgermination was hinderedby the the growth of both the test bacteria. The bacterial strain of P. aeruginosa. Also when diameter of growth inhibition the zones media was supplemented with the 6 antibi- were calculated and mean values were otics namely Amikacin (AK), Bacitracin consideredfrom the triplicate set. The (B), Clindamycin (CD), Gentamycin Figure 3. A) P. solubilization; B) diameter of inhibition zone useagainst (HLG), Chloramphenicol (C) and Siderophore production. Klebsiella sp. was noted to be greater Ciprofloxacin (CIP), seeds failed to germi- (27±0.75) in comparison to E. coli (19±1.0 nate in all except in CIP to which the bacte- mm) (Table 3). ria was tested sensitive.

Table 3. Antibacterial activity of isolated bacterial strain against test bacterial agents. Test bacterial agents Diameter of inhibition commercial zones (mm) Klebsiella sp. 27±0.75 E. coli 19±1.0 Figure 4. IAA production up to 8% NaCl concentration. Non A B C

Figure 5. Bacterial antagonism against Curvularia lunata: A) single streak method, B) double streak method, C) spread method.

[Microbiology Research 2017; 8:7233] [page 77] Article

The seedwerenot affected by the supernatant (24 hrs old bacterial culture A B 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 influ- encing plant growth. Growth of ahealthy 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 knowl- edge and less awareness leads to reduced Figure 6. Bacterial antagonism against Fusarium solani: A) single streak method, B) dou- crop productivity every yearasthe farmers ble streak method. keep on supplementing the soil with unnec- essary formulations including biofertilizers. The present study focused on exploring the characteristic features (both positive and negative) of the isolated bacterial strain of P. aeruginosa.Itwasobserved that the bac- only terial strain survive at NaCl concentration A B of 10% and produce a considerable amount of IAA up to 8% NaCl concentration. It is able in producing siderophores, HCN and use 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 commercial germinate.27 It occurred that the cellular products of the strain did not have anega- C D tive impactover the seedgermination from which it can be concluded that the deleterious nature of the strain is only viableNon up till the cells are able to infect and proliferate over seeds interfering with the seedgermination mechanism. Its ability to sustain its growth under saline conditions, HCN and siderophore production and antibiotic resistance makes it ahighly 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.

References Figure 7. A) Inability of wheat seeds to germinate, B) seed showing bacterial growth, C) and D) showing maize grains after 12 days of sowing. 1. Emori TG, Gaynes RP. An overview of nosocomial infections, including the role of the microbiology laboratory. Clin Microbio Rev 1993;6:428-42. 2. Aloush V, Navon-Venezia S, Seigman-

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Table 4. Percentage growth inhibition in target microbial agents. Fungi tested against Single streak method Double streak method Spread method Aspergillus niger 36.21% 68.35% 100% Fusarium solani 57.12% 74.72% 100% Curvularia lunata 24.37% 72.13% 100%

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