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Current Trends in Biotechnology and PharmacyVol. 10 (2) 161-168 April 2016, ISSN 0973-8916 (Print), 2230-7303 (Online)

161

AbstractThe use of plant growth-promoting

rhizobacteria (PGPR) is steadily increasing inagriculture and offers an attractive way to replacechemical fertilizers, pesticides, and supplements.Genus Pseudomonas is widespread bacteria inagricultural soils and has many traits that makethem well-matched as PGPR. Pseudomonasaeruginosa HMR16 isolated from heavy metalcontaminated soil of Zawar, Udaipur was testedfor direct plant growth promoting activities (IAAproduction, production of ammonia andphosphate solubilization) and indirect growthpromoting activities (HCN production,siderophore production). Effect of heavy metalssuch as zinc, lead, chromium on growth ofPseudomonas aeruginosa HMR16 was alsodetermined on nutrient agar and nutrient brothmedium supplemented with respective heavymetals concentrations. Pseudomonasaeruginosa HMR16 was found to be positive forthe production of plant growth-promotinghormone (IAA), ammonia, HCN, siderophoresand solublize phosphate with solubilizationefficiency of 115.38. Minimum inhibitoryconcentration (MIC) of zinc, lead, and chromiumagainst Pseudomonas aeruginosa HMR16strains were 16.0mM, 1.25 mM and 0.30mMrespectively. Pseudomonas aeruginosa HMR16showed various plant growth promoting activitiesand tolerance to heavy metals thus it can be usedto improve plant growth in heavy metalcontaminated soil.

Keywords: Zawar; PGPR, Heavy metals,Pseudomonas, IAA, Phosphate solubilization

IntroductionHeavy metal pollution in soils is the most

serious environmental problem and hassignificant implications for most of the organisms.In plants, such effects of heavy metalcontamination may include growth inhibition,structural damage, and a decline of physiologicaland biochemical activities. Metal-contaminatedsoils represent one of the most difficultchallenges facing bioremediation.Phytoremediation assisted with the heavy metaltolerant bacteria offers more benefits thanconventional technology in accumulating heavymetals from the soil as it is less expensive andsafer for the environment. The soil bacteria thataggressively colonize the root zone and promoteplant growth are generally termed as PlantGrowth Promoting Rhizobacteria (PGPR).Fluorescent Pseudomonads are the dominantgroup of bacteria that preferably lives in closevicinity to the root or on root surface and play acrucial role in soil health and plant development(1). These bacteria have several mechanismsto survive in heavy metal contaminated soil andalso influence plant growth directly or indirectly(2). Their tolerance to adverse environmentalconditions, capacity to solubilize phosphate,hydrocyanic acid, indole acetic acid, sidero-phores and ability to effectively colonize roots isresponsible for plant growth promotion (3).

Study of Potential Plant Growth-Promoting Activitiesand Heavy Metal Tolerance of Pseudomonas aeruginosa

HMR16 Isolated from Zawar, Udaipur, India

Ali Asger Bhojiya and Harshada Joshi1*

1 Department of Biotechnology, Vigyan Bhawan, Block B, Mohanlal Sukhadia University Udaipur,Rajasthan, India

*For Correspondence - [email protected]

Study of Potential Plant Growth-Promoting Activities


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The aim of this study was to determine thepotential plant growth promoting activities ofindigenous Pseudomonas aeruginosa HMR16strain isolated from heavy metal contaminatedsite of Zawar, Udaipur and to determine theminimum inhibitory concentration of variousheavy metals i.e. zinc, lead and chromium.

Materials and MethodsSource and maintenance of bacteria: Heavymetal tolerant bacterial isolate Pseudomonasaeruginosa HMR16 (Accession No. KU174205)previously isolated from heavy metalcontaminated sites of Zawar, Udaipur (4) onNutrient agar medium supplemented with zincsulphate heptahydrate was used in this study.Isolate Pseudomonas aeruginosa HMR16 wasroutinely grown for 24h at 37oC on Nutrient agarmedium supplemented with 1 mM zinc sulphateheptahydrate concentration and stored at -20oCin glycerol.

In vitro screening of bacterial isolate for theirplant growth promoting (PGP) activitiesProduction of IAA: IAA production wasquantitatively measured by the method asdescribed by Gordon and Weber (5). Bacterialculture was grown for 48 h on LB broth and LBbroth amended with 100 μg/mL tryptophan. Fullygrown culture was centrifuged at 5000 rpm for15 min. The supernatant (2 mL) was mixed withtwo drops of orthophosphoric acid and 4 mL ofthe Salkowski reagent (50 mL, 35% of perchloricacid, 1 mL 0.5 M FeCl

3 solution). Optical density

was taken at 530 nm with the help ofspectrophotometer. Concentration of IAAproduced by culture was measured with the helpof standard graph of IAA obtained in the range of10-100 μg/ mL.

Production of NH3: Bacterial isolate was testedfor the production of ammonia as described byCappuccino and Sherman (6). Overnight grownbacterial culture was inoculated in 10 mL peptonebroth and incubated at 35±0.1 °C for 48 h inIncubator Shaker. After incubation 0.5 mL ofNessler’s reagent was added.

Production of HCN: Bacterial isolate wasscreened for the production of hydrogen cyanideby adapting the method of Castric (7). Briefly,bacterial culture was streaked on nutrient agarmedium containing 4.4 g per liter of glycine. AWhatman filter paper No. 1 soaked in 0.5% picricacid solution (in 2% sodium carbonate) wasplaced inside the lid of a plate. Plates were sealedwith parafilm and incubated at 25 ± 2°C for 4days.

Production of Siderophore: Bacterial isolatewere assayed for siderophores production on theChrome azurol S agar medium described bySchwyn and Neilands (8). Chrome azurol S agarplates were prepared and divided into equalsectors and spot inoculated with test organism(10μL) and incubated at 25±2ºC for 48-72 h.

Detection of phosphate solubilizing activity:Phosphate solubilizing activity was tested on themodified Sperber’s medium by spot assay. A totalof 10µL bacterial culture was spotted on themedium. Petriplates were incubated at 37 ºC for24 h. The potential to solubilize insolublephosphates on the modified Sperber’s mediumwas determined by measuring clear zone aroundthe colony. The colony and zone diameter wasmeasured. The solubilization efficiency (SE) iscalculated by formula given below.

Determination of MIC (Minimum InhibitoryConcentration) : Minimum inhibitoryconcentrations (MICs) of the metals weredetermined by the plate-dilution and brothmethod. The metal salts used were zinc sulphate[ZnSO4.7H2O], lead nitrate [Pb(NO3)2] andpotassium dichromate [K2Cr2O7]. Heavy metaltolerant isolate Pseudomonas aeruginosaHMR16 was inoculated aseptically on Nutrientagar plates, supplemented with differentconcentrations (mM) of the following heavymetals: Zn (0.0- 16), Pb (0.0-1.5) and Cr (0.0-

Ali Asger Bhojiya and Harshada Joshi


163

0.35). The plates were incubated at 37 ºC for 48h. The concentration of metal where there wasno growth observed is considered as the MIC ofthe metal against the strain tested. The brothmethod was accomplished using tubes of nutrientbroth spiked with different concentrations of filter-sterilized divalent metal ions. Bacterial culturewas pre-incubated in 100 mL metal-deficientnutrient broth to a mid-log phase and 0.1 mLsample was transferred into 10 mL nutrient brothcontaining metals in test tubes. Positive controlsconsisted of metal-deficient medium inoculatedwith bacterial cultures. The tubes were incubatedon rotary shake incubator at 37oC for 48 h. Theconcentration of heavy metals at which noturbidity was observed by spectrophotometer at620nm was considered as the MIC of bacterialisolates against heavy metals.

Results and DiscussionPseudomonas is a well-known beneficial

plant growth promoting bacteria. Underunfavourable conditions, rhizospheric bacteriamay enhance the plant growth by optimizing thesupply of nutrients, stimulating plant growth bythe synthesis of phytohormones IAA,solubilization of inorganic phosphorus andinhibiting the activity of pathogens (9). Previouslycharacterized Pseudomonad strains namelyPseudomonas aeruginosa HMR16 was screenedfor their plant growth promoting activity such asIAA, ammonia, HCN, siderophore, productionand P-solubilization. Effect of heavy metals suchas zinc, lead, chromium on growth of P.aeruginosa HMR16 was also studied. AmongPGP activites, the phytohormone auxin (IAA)release by PGPR has various direct effects onplant growth under stress conditions (10). IAA isone of the most important phytohormone andfunction as important signal molecule in theregulation of plant development. IAA is a commonproduct of L-tryptophan metabolism by variousPGPR strains. Our results suggest thatPseudomonas aeruginosa HMR16 strainproduce IAA. For estimation of IAA production,development of pink color in the tube afteraddition of orthophosphoric acid and Salkowski

reagent to supernatant of Pseudomonas culturein LB broth supplemented with and without L-tryptophan confirmed that Pseudomonasaeruginosa HMR16 strain produce indole aceticacid (Fig 1a).Colorimetric estimation of IAA by P.aeruginosa HMR16 showed 1.50 μg/mL and6.832 μg/mL IAA production in absence andpresence of L-tryptophan respectively. Theseresults were found to be much better than that ofKarnwal (11) where P.aeruginosa AK2 produced0.8 μg/mL and 3.9 μg/mL IAA in absence andpresence of 100 μg/mL L-tryptophan respectively.Our findings of IAA in Pseudomonas isolate is inagreement with other worker where in thepresence of 100 μg/mL L-tryptophan,Pseudomonas spp. produced IAA in the rangeof 6.00-7.93 μg/mL (12 ). Ammonia productionby rhizobacteria also plays an important role inbiocontrol activity of PGPRs. For estimation ofammonia, the development of faint yellow to darkbrown color after addition of Nessler’s reagentto overnight grown bacterial culture in peptonebroth indicated the production of ammonia by P.aeruginosa HMR16 (Fig 1b). These results arein close agreement with those of Chacko et al.,(13) who isolated the Pseudomonas putida fromthe rhizosphere. This bacterium shows differentPGPR characteristics and was also foundpositive for the production of ammonia. HCNproduction by rhizobacteria has been postulatedto play an important role in the biological controlof pathogens (14). Genus Pseudomonas is oneof the leading bacteria which inhibit growth ofpathogenic fungus in agriculture fields. Forestimation of HCN production, the changes incolor of the filter paper from yellow (0.5% picricacid in 1% Na

2CO

3) to brown after 24 h incubation

showed that P. aeruginosa HMR16 strain produceHCN (Fig 1c) which acts as an inducer of plantresistance. Lanteigne et al., (15) isolated HCNproducing Pseudomonas and observed biologicalcontrol activity of Pseudomonas. Bakker andSchippers (16) also observed that nearly 50% ofthe pseudomonads from potato and wheatrhizospheres produce HCN which has a primarymechanism in suppression of root fungalpathogens. Siderophores are known to chelates



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with iron and other metals and contribute todisease resistance by limiting the supply ofessential trace minerals in natural habitats.Siderophore producing PGPR help to preventplants from becoming chlorotic due to theavailability of Fe to plants when they were grownin heavy metal polluted soil (3, 17). For estimationof siderophore production, formation of yellow-orange halo around the colony on CAS agarmedium showed that P. aeruginosa HMR16produce siderophore (Fig 1d). These findings arein close corroboration with those of Islam et al.(18) which shows that the highest orange halozone was produced by P. aeruginosa bacterialisolate. Phosphate solubilising microorganismssolubilize insoluble phosphates mainly bysecreting acids (19) which can be observed as asolubilization zone on the medium. Efficiency ofphosphate solubilization by P. aeruginosa HMR16was determined. The Pseudomonas aeruginosaHMR16 was found to be potent phosphatesolubilizers showing clear halo zone around theircolonies. The zone size was gradually increasedwhen incubation was extended beyond 24 h.These results are similar to Kumar et al., (20)who isolated phosphate solubilisingPseudomonas sp and showed highest phosphatesolubilization zone (20 mm) in PVK agar. TheSolubilization efficiency of 115.38 and maximumzone size of 1.5 cm was recorded after incubationperiod of 24 h (Fig 1e). Reena et al., (21) foundthe phosphate-solubilizing capacity of P.aeruginosa as 126.11 on 6th day which iscomparatively low than that of our present study.Genus Pseudomonas is well-studied and is ofgreat interest because of their high resistance toheavy metals and other toxic substances. In thepresent study efforts were made to determinethe minimum inhibitory concentration (MIC) ofzinc, lead, chromium for this bacterial isolate. Thepresent results showed P. aeruginosa HMR16to be tolerant to all the three heavy metals tested.Fairly high tolerance was observed towardselevated concentration of zinc followed by leadand chromium. On determining MIC by agardilution method, the well defined colonies wereobserved after 48 h of incubation in the medium

up to 14mM concentration of zinc, 1mMconcentration of lead and 0.25 mM concentrationof chromium. Pseudomonas aeruginosa HMR16didn’t show any growth on high concentration ofzinc, lead and chromium i.e.16mM, 1.25mM and0.30mM respectively (Table 1). On determiningMIC by broth method, the bacterial culture wasgrown in nutrient broth supplemented withdifferent concentrations of divalent metal ions.Growth in terms of turbidity was observed at allconcentrations up to 10 mM concentration ofZnSO

4.7H

2O, 0.75 mM concentration of Pb(NO

3)

2

and 0.2mM concentration of K2Cr

2O

7. However,

growth was inhibited at 11 mM, 1.0 mM and0.25mM concentration of zinc, lead andchromium respectively (Fig. 2, 3 and 4). Thisindicates that zinc is less toxic than lead andchromium to the P. aeruginosa HMR16. In thepresent study, Pseudomonas aeruginosa HMR16showed high MIC of 16mM for zinc ions. Thisrange of MIC is quite higher to Pseudomonasaeruginosa isolated from polluted sites in Assiutcity, Egypt for which MIC of 9.2mM was observed(22). A maximum MTC of 5 mM for Zn wasobserved for Pseudomonas (23) which is low ascompared to the MIC of Zn for Pseudomonasaeruginosa HMR16 in our study. Pseudomonasaeruginosa HMR16 shows MIC of 1mM for leadions which is comparable with that ofPseudomonas aeruginosa AD4 isolated by Durveet al., (24). Sheng et al., (25) isolatedPseudomonas sp. from heavy metalcontaminated site and showed MIC for zinc andlead as 0.34mM and 0.60mM respectively. TheseMIC values for Zn and Pb are low as comparedto the MIC of Zn and Pb for Pseudomonasaeruginosa HMR16 in the present study.Pseudomonas aeruginosa HMR16 shows MICof 0.25 mM for Cr ions. Our results arecomparable with previous study by Singh et al.,(26). It was observed in this study that high MICvalue for heavy metals was obtained on agarmedium as compared to broth medium. Hassenet al., (22) also tested the levels of tolerance ofenvironmental bacteria to the different divalentmetal ions including Zn2+ in nutrient broth andreported that the test in liquid media was sensitive



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Table 1. Heavy metal tolerance of Pseudomonas aeruginosa HMR16.

S.No Zinc ions P. Lead ions P. aeruginosa Chromium P.concentration aeruginosa concentration concentration ions aeruginosa

(mM) HMR16 (mM) HMR16 (mM) HMR16

1. 0 +++ 0 +++ 0 +++

2. 2.0 ++ 0.25 ++ 0.10 ++

3. 4.0 + 0.5 + 0.15 +

4. 6.0 + 0.75 + 0.20 +

5. 8.0 + 1.0 + 0.25 +

6. 10.0 + 1.25 - 0.30 -

7. 12.0 + 1.5 - 0.35 -

8. 14.0 +

9. 16.0 -

+++ luxurious growth, ++ good growth, + less growth, - No growth

Fig. 1. Various Plant growth promoting activity of Pseudomonas aeruginosa HMR16



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Fig. 4. Minimum inhibitory concentration of chromium for the strain Pseudomonas aeruginosa HMR16

Fig. 2. Minimum inhibitory concentration of zinc for the strain Pseudomonas aeruginosa HMR16

Fig. 3. Minimum inhibitory concentration of lead for the strain Pseudomonas aeruginosa HMR16



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at concentrations 10 to 1000 times lower thanthose obtained in solid media.

ConclusionsThis study showed Pseudomonas

aeruginosa HMR16 with multiple PGP activitiesIAA, HCN, siderophore, ammonia production andP-solubilization and tolerance to various heavymetals. Thus metal tolerant Pseudomonasaeruginosa HMR16 can be further explored forits application in promoting plant growth in heavymetal contaminated soil.

AcknowledgmentThe first author gratefully acknowledges

the financial support received from Maulana AzadNational Fellowship, University GrantCommission, New Delhi.

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