Bioprospecting Phosphate Solubilisation and PGP Activities of Native Strains of Pseudomonas...

Bioprospecting Phosphate Solubilisation and PGP Activities of Native Strains of Pseudomonas Aeruginasa and Pseudomonas Fluorescens from Bamboo (Bambusa Bamboo) Rhizosphere: Jyotsna Kiran Peter & Neeraj Pandey 702

International Journal of Research (IJR) Vol-1, Issue-4, May 2014 ISSN 2348-6848

Bioprospecting Phosphate Solubilisation and PGP Activities of Native Strains of Pseudomonas Aeruginasa and Pseudomonas Fluorescens from Bamboo (Bambusa Bamboo) Rhizosphere

Jyotsna Kiran Peter1 & Neeraj Pandey2

ABSTRACT

Bamboo rhizosphere soil was considered for isolation and screening of phosphate solubilising Pseudomonas spp. 22 Pseudomonas isolates were obtained among which incidence of Pseudomonas auruginosa was 59%and Pseudomonas fluorescens was 40.9%. All strains were analyzed for abiotic stress tolerance of temperature, pH, NaCl conc. The isolates showed confluent growth at 10˚C, pH 5, 7and 8 ; NaCl conc tolerated were 2, 4 and 6% while at 50˚C, 3, 9 and 11 pH and 8 and 10% did not support growth. Phosphate solubilization was screened on medium and PGP activities were characterized on basis of PQQ, IAA and HCN production by the isolated strains which were found positive. In conclusion, it was revealed in the investigation that the rhizoshere of Bamboo plant is rich in phosphate solubilising –PGP traits showing Pseudomonas auruginosa and Pseudomonas fluorescens that confers antibiosis against plant fungal pathogens like Fusarium oxysporum, Rhizoctonia solani and Alternaria solani.

Key words:

Bamboo rhizosphere, Pseudomonas auruginosa, Pseudomonas fluorescens, Phosphate solubilization, abiotic stress tolerance, antibiosis

1 Department of Microbiology and Fermentation Technology (MBFT), Jacob School of Bioengineering and Biotechnology (JSBB), Sam Higginbottom Institute of Agriculture Technology and Sciences (SHIATS) –Deemed to be University, Naini, India, 211008, email: [email protected]

2 Department of Microbiology and Fermentation Technology (MBFT), Jacob School of Bioengineering and Biotechnology (JSBB), Sam Higginbottom Institute of Agriculture Technology and Sciences (SHIATS) –Deemed to be University, Naini, India, 211008



INTRODUCTION

Soil is the dynamic system and is an ecological niche of constant biological activity. Soil Phosphorus dynamics is characterized by physicochemical (sorption-desorption) and biological (immobilization-mineralization) processes. Phosphorous is one of the major plant nutrients, lack of which limits plant growth. Most agriculture soil contains Phosphorus in the range of 200-500 mg /kg-1 with an average of 600 mg/kg-1 (Fernandez et al., 2007). Phosphorus is an integral part of the cellular activities of living organisms. It has a defined role in plant metabolism such as cell division, development, photosynthesis, break down of sugar, nutrient transport within the plant (Tandon, 1987; Armstrong, 1988; Theodorou and Plaxton,1993). The plants obtain their ‘P’ requirements from the soil pool. Phosphorus is second only to nitrogen among mineral nutrients most commonly limiting the growth of plant development and growth making upto about 0.2% of plant dry weight. Plants acquire Phosphorus from soil solution as Phosphate anions (Keneni et al., 2010).

Pseudomonas species are well known biocontrol agents against many phytopathogenic fungi like Phythium ultimum, Fusarium sp., Alternaria sp., Phytophthora copirci and Rhizoctonia solani, certain strain inhibits some bacteria, Yeasts and Protozoa growth and suppress some plant disease development on Maize, Cotton, Pea, Tomatoes and Pepper (Coenye and Vandamme, 2003).

Bamboo is one of the most important forest resources on earth and contains over 70 genera and 1,200 species. Bamboo is mainly distributed in tropical and subtropical regions, with a few species found in temperate and frigid regions (Gui et al., 2007). Bamboo is a fast growing plant and can come up in water stress (Nagasampige et al., 2008). It is well known that in most cases, there were no grasses growing beneath and near Bamboo plants. Bamboos are an ornamental plant, used as medicine and had a major role constructions and various other purposes (Dhamangaonkar, 2009). The different species of Pseudomonas are isolated from Bamboo rhizosphere soil as Pseudomonas nitroreducens 4CD2 from Bamboo (Abuse chungii) soil (Zhang et al., 2009). Some other species like Pseudomonas extremorientalis, Pseudomonas putida, Pseudomonas reinekei and Pseudomonas paonaceae from moso Bamboo (Phyllostachys edulis) rhizosphere soil (Han et al., 2009).

Thus, in view of above aspects, the present study entitled was undertaken with the following objectives:-

1. To isolate phosphate solubilising Pseudomonas species from rhizosphere soil of Bamboo plant.

2. To characterize the Phosphate solubilizing Pseudomonas strains on the basis of abiotic stress tolerance and plant growth promoting traits.

3. To determine biocontrol activity of Pseudomonas spp. against selected fungal plant pathogens.



MATERIALS AND METHODS

The rhizospheric soil of Bamboo plant (n=30) was collected from different sites of Sam Higginbottom Institute of Agriculture, Technology & Sciences, campus. Sample was collected from rhizosphere of plant after digging 5 to 15 cm from the stem base and the soil was collected in sterilized polythene bags then carried to the laboratory under aseptic condition for further processing.

Isolation of Pseudomonas species from Bamboo rhizosphere

The adhering soil was removed from root segments. 1 g was added in 9.0 ml Phosphate Buffered Saline and vortexed. The resulting suspension was serially diluted up to 10-5 and 100μl of aliquots from 10-4 and 10-5 dilution was plated onto Nutrient agar and King’s B agar medium. The plates were incubated in dark for 3 days at 28°C. (Mendes et al., 2007). The isolates were maintained by regularly subculturing in Nutrient agar slants and incubated at 28°C for 3 days and stored at 4°C in refrigerator.

Identification of isolates

The isolates were identified on the basis of cultural, morphological and biochemical tests as per Bergey’s Manual of Systemic Bacteriology (Holt et al., 1984). Cultural characterization was done on the basis of different colony characteristics like color, texture, size, shape etc of the colony on culture plates. Morphological characterization included Gram-staining and the slides were observed under 100 X objective lens. For Sugar utilization test the isolates were

examined for carbohydrate fermentation test using a basal medium supplemented with 10% sugar solution in distilled water of Mannitol, Sucrose, Glucose, Lactose, and Maltose and with phenol red as an indicator. The medium was inoculated with the loopful of test culture and then incubated at 28+2°C for 24 to 48 h. Change in colour of the medium from red to yellow indicated acid production which confirmed the fermentation of that particular sugar and the presence of gas in the Durham’s tubes indicated gas production hence, suggested positive result for sugar fermentation. For biochemical characterization of the isolated colonies catalase test, nitrate reduction, urease production, indole production, MR-VP test, citrate utilization, gelatin liquefaction, Ammonification assay were performed.

Screening of Pseudomonas spp. for Phosphate solubilization ability

The Pseudomonas spp. were screened for their Phosphate solubilizing ability by inoculating the cultures in National Botanical Research Institute’s Phosphate Growth Bromo-Phenol Blue medium (Mehta and Nautiyal, 2001). The tubes were incubated at 30°C for 10 days on a rotary shaker along with control and were observed visually. The decolourization of Bromo phenol blue dye indicated solubilization of phosphate by the organism.

Characterization of phosphate solubilizing Pseudomonas spp.

PQQ (Pyrolequnioline Quinone) activity (Groen et al., 1983)



The ability of PQQ independent/dependent activity of Glucose dehydrogenase (GDH) of Pseudomonas spp. was examined using MacConkey Agar (Appendix 1.3) plate containing 1% Glucose. The Pseudomonas spp. were spot inoculated on media plate with one uninoculated plate as control and was kept at 30°C in BOD incubator. The appearance of red colour zone around the colony was indication of PQQ independent activity.

HCN Production

100 µl of freshly grown culture was inoculated on Nutrient Agar (Appendix 1.7) plates using spread plate technique except one, which served as control. A Whatman’s filter paper No.1 was impregnated with alkaline Picric acid solution and placed on the upper lid of inoculated petriplate under aseptic conditions. The plates were incubated at 30°C for 24 h. A change in colour from yellow to orange of Whatman’s filter paper was indication of cyanogenic activity. (Castric and Castric, 1993).

Auxin production

24 hours old culture of the isolates were spot inoculated on the Nutrient Agar (Appendix 1.7) Tryptophan medium plates and then overlaid by Whatman no.1 filter paper and left overnight for incubation at 30°C for Auxin production. After incubation the filter paper was treated with Salkowski’s reagent. The change in colour of filter paper to pink indicated the positive result.

Determination of Abiotic stress tolerance (Zhang et al., 2009)

The Pseudomonas spp. isolates were tested for abiotic stress conditions as temperature, pH and salt (NaCl) tolerance (Plate 4.6, 4.7, 4.8, 4.9, 4.10, 4.11 and 4.12).

Temperature tolerance

The Pseudomonas spp. were spot inoculated on Nutrient Agar (Appendix 1.7) plates at 10°C and 50°C. One set was incubated at 10°C and other set was incubated at 50°C in BOD incubator and growth was observed visually for 10 days.

pH tolerance

The ability of pH tolerance of isolated strains were studied by growing them in Nutrient Agar plates with pH 3, 5, 7, 9 and 11 incubated at 30°C using BOD incubator and growth was observed visually for 10 days.

Salt (NaCl) tolerance

The Pseudomonas spp. were spot inoculated on Nutrient Agar plates containing 2%, 4%, 6%, 8% and 10% NaCl concentration. The strains were also spot inoculated on Nutrient Agar plates to be used as organism control. The plates were incubated at 30°C in BOD incubator and growth was observed visually for 10 days.

Biocontrol activity

Biocontrol activity of Pseudomonas species was determined by dual culture method as per the procedure of Topps and Wain, 1957. The antagonistic property of Pseudomonas species against F. oxysporum, Rhizoctoniasolani and Alternariasolani was examined by streak- inoculation of Pseudomonas spp to four sides of F. oxysporum, Rhizoctoniasolaniand



Alternariasolani placed at the centre on Potato Dextrose Agar (PDA) medium. Then plate was incubated at 30°C for 6 days. Diameters of colonies were measured in every 24 hours. When growth of fungal isolate just covered the untreated (control) plate, percent inhibition (I %) was calculated using the formula

I% = [(A-B) /A] x 100 Where, I% = percentage of inhibition A = Mean diameter growth in control, B = Mean diameter growth in a given treatment.

RESULTS AND DISCUSSION

Incidence of Pseudomonas spp among 30 rhizospheric soil samples of Bamboo plant studied was 73.34% i.e 22 Pseudomonas isolates were obtained (Fig:1; Fig: 2). Upon morphological, biochemical, physiological and sugar fermentation tests the identified strains were Pseudomonas aeruginosa whose occurrence in those samples was 59% and Pseudomonas fluorescens with 40.9% incidence. All the 22 isolates obtained from rhizospheric soil of Bamboo plant were screened for phosphate solubilizing ability on NBRIP-BPB medium. Out of 22 isolates 19 were able to solubilize phosphate among which 12 (54.5%) were Pseudomonas aeruginosa and 7 (32%)were Pseudomonas fluorescens. Data was analysed statistically using (P< 0.05).

Fig:1 Incidence of Pseudomonas spp. from rhizospheric soil of Bamboo plant

Fig:2 Incidence of phosphate solubilizing Pseudomonas spp

59%40.90%

Incidence of Pseudomonas spp. from rhizospheric soil of Bamboo

Pseudomonas aeruginosaPseudomonas fluorescens

54.50%

32%

Incidence of phosphate solubilizing Pseudomonas spp.Pseudomonas aeruginosa

Pseudomonas fluorescens



Plate: 1.1

Incidence of Phosphate solubilizing

Pseudomonas aeruginosa

Plate: 1.2 Incidence of Phosphate

solubilizing Pseudomonas fluorescence

Plate: 1.3 cultural characteristic of Pseudomonas fluorescens and Pseudomonas aeruginosa on Nutrient agar medium Plant growth promoting traits of Phosphate solubilizing Pseudomonas spp.

Phosphate solubilising strains of Pseudomonas aeruginosa and Pseudomonas fluorescens were examined for plant growth promoting traits. All



isolates showed positive PQQ activity, HCN production and auxin production.

Plate:1.4 PQQ (Pyrole qunioline Quinone) activity of Pseudomonas spp.

PGP traits Pseudomonas aeruginosa Pseudomonas fluorescens PQQ activity + +

HCN production + + Auxin production + +



Plate 1.5 HCN production of Pseudomonas spp.

Plate 1.6 Auxin production of Pseudomonas spp.

Abiotic stress tolerance activity of Phosphate solubilizing Pseudomonas spp.

The abiotic stress tolerance activity of Pseudomonas aeruginosa and Pseudomonas fluorescens were tested at different temperature, pH and salt concentrations. The

isolates showed confluent growth at low temperature i.e 10°C while failed to grow at 50°C. growth of isolates were subjected to grow at pH range of 3, 5, 7, 9, and 11 among



which pH 5, 7 and 9 supported the growth of both isolates and at pH 3 and 11 no growth was observed. Salt tolerance was examined using NaCl concentrations of 2, 4, 6, 8, 10% and it

was revealed that the isolates ofPseudomonas spp were able to tolerate NaCl concentrations of 2, 4 and 6% while could not tolerated 8 and 10% NaCl.

Table: Abiotic stress tolerance activity of Phosphate solubilizing Pseudomonas spp.

Abiotic stress tolerance Pseudomonas aeruginosa

Pseudomonas fluorescens

Temperature 10°C + + 50°C - -

pH

3 - - 5 + + 7 + + 9 + + 11 - -

Salt tolerance (NaCl)

2% + + 4% + + 6% + + 8% - - 10% - -

Plate 1.7Growth activity of Pseudomonas spp. at 10°C

Plate 1.8Growth activity of Pseudomonas spp. at 5 pH

Plate 1.9 Growth activity of Pseudomonas spp.

at 7 pH



Plate 1.10 Growth activity of Pseudomonas spp. at 9 pH

Plate 1.11 Growth activity of Pseudomonas

spp. at 9 pH

Plate 1.12 Growth activity of Pseudomonas spp. on 2% salt (NaCl) concentration

Plate 1.13 Growth activity of Pseudomonas

spp. on 4% salt (NaCl) concentration

Biocontrol activity of Pseudomonas auruginosa against different fungal species

Time of observations

Inhibition pattern (mm) of plant fungal pathoges by Pseudomonas auruginosa

Fusarium oxysporum

Rhizoctonia solani

Alternaria solani

2nd Day Control Radial growth 12 12 12

Treatment Radial inhibition 10 12 12

4th Day Control Radial growth 26 26 28






Percent inhibition 59.53 45.95 50

Biocontrol activity of Pseudomonas fluorescens against different fungal species

Time of observations

Inhibition pattern of plant fungal pathoges by Pseudomonas fluorescens

Fusarium oxysporum

Rhizoctonia solani

Alternaria solani

2nd Day Control Radial growth 12 12 12





Treatment Radial inhibition 15 20 19 Percent inhibition 64.29 45.95 52.5

Plate 1.14 Inhibition pattern (mm) of Rhizoctonia solani with Pseudomonas spp. at (6thdays) interval



Plate 1.15 Inhibition pattern (mm) of Fusarium oxysporum with Pseudomonas spp. at (6thdays)

interval

Plate 1.16 Inhibition pattern (mm) of Alternaria solani with Pseudomonas spp. at (6thdays) interval

DISCUSSION

Study conducted by Ahmed and Shahab, (2010) are reliable for current investigation that determined a total of 2212 bases which showed 95% homology with pqq BC gene of Pseudomonas aeruginosa LESB58 and 97% homology to pqq AB gene of Pseudomonas aeruginosa PAO1 and 77% of pqq A-D gene of Pseudomonas fluorescence strain B 16. Study conducted by Suresh et al., (2010) are in accordance to the present investigation in

which 10 fluorescent Pseudomonas were found positive for IAA and HCN. Patten and Glick, (1996) reported Bactria belonging to the genera Pseudomonas to produce Auxin which helps in stimulating plant growth. Production of HCN, IAA and Phosphate solubilization has been implicated in the plant growth promotion by PGPR (O’ Sullivan and O’ Gara, 1992). Similar result was accomplished by Ahmed and Shahab, 2010 a remarkable change in colour from yellow to Reddish-Brown indicates HCN production and which could be used for the



quantitative estimation of IAA in the presence of different concentration of Tryptophan, with addition of Tryptophan from 50 to 500 mg/ml the production of IAA was increased upto 15.3 µg/ml. Nautiyal et al. (2000) investigated the effects of pH, temperature and salt on four PSB isolated from pea rhizosphere in India. Plant growth promoting bacteria may promote growth directly e.g. Phosphate solubilization, Production of plant growth regulates- Auxin and indirectly by improving growth restricting condition e.g. HCN production. In the present investigation both the isolates had shown the plant growth promoting traits viz PQQ activity, HCN production, Auxin production and Phosphate solubilizing activity. PQQ i.e. Pyrroloquinoline [ 4, 5 – dihydro – 4, 5 - dioxa 1 H – pyrrolo- [ 2, 3 – 4] quinoline – 2, 7, 9 – Tricarboxylic acid is an aromatic, Tricyclic orthoquinine that serve as redox cofactor for several bacterial Dehydrogenase. The redox cofactor PQQ links the oxidation of many different compounds to the respiratory chain in gram negative bacteria and acts as reactive oxygen scavenger (ROS) to neutralize free radicals. It also functions as potent growth factor by enhancing DNA synthesis. Thus it has been regarded as plant growth promoting factor (Ahmad and Shahab, 2010). The rhizospheric bacteria are thought to produce HCN towards soil borne pathogens and Pseudomonads are found to predominantly acquire the rhizosphere of plant. Pseudomonas has been found as most Phosphate solubilizing microorganism amongst bacteria in rhizosphere soil. Phosphorus is a major essential macro nutrient for biological growth and development. Phosphorous in soil is

immobilized or become less soluble either by absorption and chemical precipitation or both. Application of chemical Phosphatic fertilizer is practiced though a majority of soil Phosphorus reaction products are only sparingly soluble. Under such condition microorganisms offer a biological rescue system capable of solubilizing the Phosphorus of soil and make it available to plants. However, Phosphate solubilizing activity is influenced under stressed condition of nutritients, temperature and pH. As one of the very important group of PGPR for sustainable agriculture, PSB tolerances to extreme climates are of special interests for bacteria to be used as biofertilizer in arid and semi-arid regions. Thus, the isolates were intensively examined for tolerance toward high temperature, high concentration of NaCl and a wide range of pH. All PSB strains survived at high temperature very well. Decreased GI and PSI at 42˚C for the isolated PSB are acceptable as in natural situation these bacteria survive hot noon of summer time and continue growing on the rest of the days. Givskov et al. (1994) also showed that P. putida survives several hours of incubation at 45˚C. All PSB could tolerate added NaCl to as much as 2.5% which indicate that these isolates would be functionally active in most cultivated lands where salinity of soil is well below 2.5%. It is noteworthy that high salt concentrations increase buffering capacity of medium that may cause reductions in PSI to some degrees. Enhanced PSI of P13 strain at 35˚C and in salt concentrations between 0.5 and 1% may suggest induced acid production in moderately stressed cells. Illemer and Schinner (1995) speculated that the release of protons



accompanying respiration or NH4 assimilation may play the major role in Pi solubilization mechanism, at least for the studied microorganism. Phosphate solubilizing microorganisms produce a variety of organic acids from simple carbohydrates (Bajpai and Rao, 1971) by virtue of which they solubilize insoluble inorganic phosphates (Barik and Dey 1982 and Vazques et al., 2008). Acidification of the eriplasmic space by direct oxidation pathway of glucose to gluconic acid as the major cause of mineral phosphate solubilization in gram-ve bacteria has also been demonstrated (Illmer and Schinner, 1995). Rath and Jena, 2013 analyzed the effect of NaCl on phosphatase activity was studied by growing the isolates on PA medium, supplemented with different concentration of NaCl (5%, 10% & 15%) separately. Only two strains S5-1 (B. magaterium) and S7-1(Pseudomonas sp.) retained phosphatase activities at 10% NaCl concentration. The tritrable acidity was expressed as ml.

CONCLUSION

The present investigation revealed the efficient indigenous phosphate solubilizing species of P. aeruginosa and P. fluorescence isolated from Bamboo plant rhizosphere and the use of the NBRIP-BPB medium was salutary for the quick screening and grouping of phosphate solubilizing bacteria. Furthermore, it was recognized that phosphate solubilizing Pseudomonas spp. possessed tolerance to various abiotic stress viz. pH, temperature and salt concentration. The isolates were observed to grow at wide range of pH, temperature and NaCl concentration. P. aeruginosa and P. fluorescence also had independent PQQ activity as well as HCN and Auxin production as characteristics of plant growth promoting traits. Therefore, the isolates P. aeruginosa and P. fluorescence could be used for commercial preparation of phosphate solubilizing cultures to improve the growth of plants.

ACKNOWLEDGEMENTS

The authors offer gratuitous thanks to Hon’ble Vice Chancellor, Most Rev. Prof. R.B. Lal, SHIATS, Naini, Uttar Pradesh, India for provision of research conductance. Heartfelt thanks to HoD, Department of Microbiology and Fermentation Technology, JSBB, SHIATS, Allahabad for the kind cooperation towards the research.



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