PGPR Isolates From The Rhizosphere Of Vegetable Crop .
Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1789-1802International Journal of Current Microbiology and Applied SciencesISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 1789-1802Journal homepage: http://www.ijcmas.comOriginal Research 5PGPR Isolates from the Rhizosphere of Vegetable CropMomordica charantia: Characterization and Application as BiofertilizerRitu Singh*, Kapil D. Pandey, Ajay Kumar and Monika SinghCentre of Advance Study in Botany, Banaras Hindu University, Varanasi- 221 005, India*Corresponding authorABSTRACTKeywordsBitter melon,plant growthpromotingrhizobacteria(PGPR), PGPtraits, biofertilizer.Article InfoAccepted:24 February 2017Available Online:10 March 2017Plant growth promoting Rhizobacteria (PGPR) enhance the plant growth and productivitythrough wide variety of mechanism. The strains isolated from rhizosphere of Momordicacharantia, mainly belonged to Bacillus, Azotobacter, Pseudomonas and Acinetobacter.Nearly 50% isolates of Pseudomonas and Acinetobacter and all the isolates of Bacillus andAzotobacter were able to solubilise tricalcium phosphate. All the isolates were positive forCatalase activity and IAA production except Acinetobacter with 75% isolates were IAAproducing. Pseudomonas (100%), Bacillus (80%), Acinetobacter (75%) and Azotobacter(40%) produced ammonia and all the isolates were siderophore producing exceptAzotobacter. The Bacillus (75%) and Pseudomonas (50%) also produce HCN. Bestperformers in above plant growth promotion (PGP) traits, isolate of Bacillus (R7),Azotobacter (RS7), Pseudomonas (RS1) and Acinetobacter (R12) evaluated for their effecton plant vigour. The antibacterial activity and plant growth promotion in Pseudomonas(RS1), salt tolerance in Bacillus (R7), and nitrogenase activity (C2H2-reduction) inAzotobacter (RS7) was highest. Pseudomonas (RS1) was most efficient PGPR andBacillus (R7) most dominant in rhizosphere of bitter melon. Nitrogenase (C2H2reduction), phosphate solubilisation and growth promotion activities in Bacillus (R7),Azotobacter (RS7) and Pseudomonas (RS1) suggested that these isolates may be used as abiofertilizer for bitter melon.IntroductionPlant Growth Promoting Rhizobacteria(PGPR) are a group of naturally occurring soilbacteria that aggressively colonize plant rootsand increase plant growth and yield (Wu etal., 2005). They prevent the colonization ofdeleterious rhizospheric microorganisms(DRMOs) at micro-niches and can promoteplant growth directly or indirectly. It isinteresting to study the diverse bacterial taxafor their PGPR characteristics (Lucy et al.,2004). The numbers of PGPR species hasincreased due to studies on a wide range ofplant species, advancement in bacterialtaxonomy and progress in understanding thedifferent mechanisms of action of PGPR. Theincreased number of PGPR for various cropsand vegetables has been commercialize as abiofertilizer, recently includes Pseudomonas(Loper et al., 2007), Azospirillum (Cassán andGarcía Salamone, 2008), Bacillus (Jacobsonet al., 2004), Stenotrophomonas (Ryan et al.,2009), Rhizobium (Long, 2001), Serratia (DeVleeschauwer and Höfte, 2007), hrobacter, Burkholderia (Joseph et al.,2007) and Streptomyces (Schrey and Tarkka.,1789
Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1789-18022008). Pseudomonas and Bacillus are themost commonly investigated PGPR and oftendominate the rhizosphere (Morgan et al.,2005).The mode of action of PGPR involvescomplex mechanisms to promote plantgrowth, development and protection. Asbioprotectant, they suppress the plant diseasesthrough induction of systemic resistance andproduction of antibiotics and siderophore.PGPR promotes plant growth by improvingnutrient acquisition, as a biofertilizer and byproducing phytohormones such as indoleacetic acid, gibberellic acid, cytokinin andethylene (Glick, 1995) as a biostimulant.Some PGPR may promote plant growthindirectly by stimulating symbiotic N2fixation, nodulation and nodule occupancy(Joseph et al., 2007) and by asymbiotic N2fixation (Boddey and Dobereiner, 1995). Inaddition to above traits, the plant growthpromoting strain must be rhizosphericcompetent, able to survive and colonize in therhizospheric soil (Cattelan et al., 1999).Unfortunately, the interaction betweenassociative PGPR and plants can be unstableand the good results obtained in vitro(Zhender et al., 1999). The variability in theperformance of PGPR may be due to variousenvironmental factors that affect the plantsgrowth, includes climate, weather conditions,soil characteristics or the composition andactivity of the indigenous microflora of thesoil (Joseph et al., 2007). Inoculation ofornamentals, horticultural and forest treesnurseries, vegetable and agricultural cropseedlings, seeds or soil with PGPR may resultin multiple effect on plant growth, such asenhancement of seed germination, seedlinggrowth, seedling health, plant vigor, plantheight, shoot weight, nutrient content of shoottissues, early bloom, chlorophyll content andincreased nodulation in legumes (Saharan,2011).Isolating native strains adapted to the localenvironment has always been successful tothe formulation of PGPR inoculants to use invegetables crops. Our interest is to isolate andscreen some well known PGPR strains ofBacillus, Pseudomonas, Azotobacter andAcinetobacterfromrhizosphereofnutritionally and pharmaceutically importantvegetable crop, Momordica charantia (bittermelon) and also to evaluate plant growthpromotion potentials of the above PGPR onbitter melon plants.Materials and MethodsSiteThe study conducted in earthen pot filled withamended natural soil kept in Botanicalgarden, Banaras Hindu University (20 18ʹ Nand 83 36ʹ E, elevation 80.71 m).Plants and soil samplingMomordica charantia Linn. (Bitter melon),seeds were sown in natural soil added withmanure and sand (2:1:1) in earthen pots andseedlings appears within 10- 15 days aftersowing (DAS). At flowering stage (45 DAS),rhizospheric soil was collected by gentleshaking the entire root system to removeloosely adhering soil. Physicochemicalanalysis of soil collected from bitter melonpots at the flowering stage was performed andbrief description of methods was as givenbelow:Total soil organic carbon, Total N, AvailableN, P and K determined by method describedby (Jackson, 1967).Soil microbial biomass C and N wereestimated by direct chloroform fumigationand extraction method as described by (Vanceet al., 1987).1790
Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1789-1802Soil pH was measured in the soil solution(1:2; soil: water, w/v) using the combinedglass electrode pH meter. 2.2.4 Soil MoistureContent was determined based on freshweight and soil dried to constant weight at80 CIsolation of Plant Growth PromotingBacteriaRhizospheric soil (fresh weight) wassuspended in 50 ml buffer solution (phosphatebuffer 10mM, pH- 7.0), shaken vigorously 1hon a gravatory shaker and serially diluted upto 10-5. Bacterial strains isolated by platingthe soil dilution on agar plate; Bacillus andAcinetobacter were isolated on LB nutrientagar plates (Carlton and Brown, 1981),Azotobacter on Jensen’s agar plates (Norrisand Chapman,1968) and Pseudomonas onKing’s B agar plates ( King, 1954). The plateswere incubated at 30 C for 2- 7 days. Thebacterial colonies were chosen based oncolony morphology and visual growth rates.Clones picked up and purified by streaking onthe respective plates. Colonies wererestreaked on fresh nutrient agar plates forisolation and maintenance of isolates.Pure isolates of PGPR from rhizospheric soilwas characterized by using the criteriadescribed in Bergey’s Manual of tive Bacteriology 9th, 1994) gey’sManualofDeterminative Bacteriology, 1984). ThePGPR strain was identified on the basis ofcolony behaviour, morphological andbiochemical characteristics.Morphological Characterization of PGPRColony morphology of each isolate wasexamined on LB agar plates. After 3 day ofincubation, different characteristics ofcolonies such as shape, size, tics, pigmentation, recorded.A phenotypic characteristic of bacterial cell ofeach isolates were examined by preparing asmear of diluted a loopful of bacterial cultureon slide and fixed by heating on a flame. Theslide was flooded with crystal violet solutionfor 3min, washed gently in flow of tap waterand air-dried. The slide was observed undermicroscope and recorded the shape.The smear prepared as above flooded withcrystal violet solution (1min), washed gentlyin flow of tap water and then flooded withiodine solution, for 1min. Iodine complex wasdrained out followed by washing with 95%ethanol. The smear incubated with safraninsolution for 1 min. Then, the slide observedunder microscope and Gram-reactionrecorded.Motility of bacteria was determined byhanging drop method. A drop of loopful 2day-old bacteria in 1 ml of nigrosin solutiontook on a cover slip that hanged on a hollowslide with Vaseline. The slide was thenobserved under microscope to test motility ofbacteria.Biochemical characterization of PGPRIsolates of Bacillus (15), Pseudomonas (2),Azotobacter (5) and Acinetobacter (4) werebiochemically characterized by Oxidase test,H2S production, Citrate test, Urease test,Carbohydrates production, Starch hydrolysisand Nitrate reduction by the followingmethod.Oxidase Test- The loop full culture fromsingle colony was just touched on theStandard Oxidase disc and immediatedevelopment of blue colour considered aspositive.1791
Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1789-1802Hydrogen Sulphide Test- SIM medium withlead acetate as indicator used for testing theability of strains to produce H2S.Citrate Test- Simmon’s Citrate medium greenin colour, if the inoculated bacteria utilizecitrate the pH increases gradually andmedium becomes blue in alkaline pH.Urease Test- Few drop of indicator phenolred added to the medium, a colour changefrom yellow to bright pinkish-red is positive;lack of colour change is a negative result forurease.Nitrate Reduction- Addition of three drops ofeach solution A (Sulphanilic acid) andsolution B (dimethyl-alpha-naphthyalmine) totwo days old culture of an isolate in nitratebroth and appearance of pink colourconsidered as positive to nitrate reductionability.Carbohydrates fermentation Test- An isolategrown in Hugh- Leifson’s test mediumcontaining bromothymol blue at 37 C for 24hours to 4 days. Development of yellowcolour due to change in colour of indicator,shows acid production from carbohydratefermentation.Starch hydrolysis Test- Few drops of Gram’siodine solution were added to the two daysold culture of an isolate grown on starch agarplate. The dark blue colour developed due toformation of starch iodine complex. Cleararea around streaked culture indicates thedegradation of starch due to production ofamylase.CharacterizationofPlantgrowthpromoting bacteria for PGP traitsPhosphate solubilisation: A loopful (20 µl)of each bacterial isolate was placed on thecentre of modified Pikovskaya agar platecontaining insoluble tricalcium phosphate(TCP) and incubated at 30 0.1 C for 5 days.The formation of clear zone indicated thephosphate solubilising potential of bacteria(Gupta et al., 1994).Quantitative estimation of phosphate: ThePhosphate solubilisation activity of theselected PGPR isolates was estimated as permethodology described by authors [Nautiyaland Mehta, 2001; Jackson, 1973). Bacterialisolates grown in National Botanical ng 0.5% tricalcium phosphate. Theflask containing 100 ml medium inoculatedwith 1 ml 48 h old bacterial culture intriplicate and incubated at 28 C for 7 days ona shaker at 180 rpm. Simultaneously, undersimilar conditions the uninoculated controlalso kept. The test cultures centrifuged at10,000 rpm for 10 min. To a 0.5 ml aliquot ofthe supernatant, 2.5 ml Barton's reagent addedand made the volume to 50 ml with deionized water. The absorbance of the resultantcolour read after 10 min at 430 nm inUV/Visible Spectrophotometer. The totalsoluble phosphorus calculated from theregression equation of standard curveprepared from K2HPO4. The values of solublephosphate liberated expressed as µg ml-1 overcontrol.Ammonia production: Bacterial isolates weretested for the production of ammonia inpeptone water. Freshly grown cultures wereinoculated in 10 ml peptone water andincubated for 48 to 78 h at 36 2 C.Nessler’s reagent (0.5 ml) was added in eachtube. Development of brown to yellow colourwas a positive test for ammonia production(Cappuccino and Sherman, 1992)Detection of indole acetic acid production:IAA production was detected by the modifiedmethod described by Brick et al., 1991.Quantitative analysis of IAA was performed1792
Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1789-1802using the method (Loper and Scroth, 1986) at50 μg/ml concentrations of tryptophan.Bacterial cultures were grown for 48 h in LBBroth medium at 28 2 C. Bacterial cultureswere centrifuged at 3000 rpm for 30 min. Thesupernatant (2 ml) was mixed with two dropsof orthophosphoric acid and 4 ml of theSalkowski reagent (50 ml, 35% of perchloricacid,1 ml0.5 MFeCl3solution).Development of pink colour indicates IAAproduction. Absorbance was read at 535 nmwiththehelpofUV/Visiblespectrophotometer. Concentration of IAAproduced by cultures was measured with thehelp of standard graph of IAA (Hi-media)obtained in the range of 10–100 μg/ml.Hydrogen cyanide production: Screening ofbacterial isolates for hydrogen cyanide (HCN)production was done by adapted method(Castric, 1975). Bacterial cultures werestreaked on nutrient agar medium containing4.4 g per litre of glycine. A Whatman filterpaper no.1 soaked in 0.5% picric acid solution(in 0.2% sodium carbonate) was placed insidethe lid of a plate. Plates were sealed withparaffin and incubated at 30 0.1 C for 4days. Development of light brown to darkbrown colour indicated HCN production.free medium (48 h at 30 C) were transferred(1 ml) to each vial sealed with airtight rubberstopper and the headspace was injected with10% (v/v) acetylene after removing equalamount of air. Gas samples (50 µl) removedafter 1h and assayed for ethylene productionin a gas chromatograph equipped withPoropak-R column and a flame ionizationdetector (Varian, USA). Ethylene producedwas determined using standard ethylene(Scott Specialty Gases, Deerfield IL 60015).Values expressed as nmol m-1 h-1.Antimicrobial activity: The antimicrobialactivity of selected isolates of PGPR strainswas done by agar well diffusion method(Mehmood et al., 1999). The PGPR isolatestested for their antibacterial activity weregrown in LB broth medium. Test bacteriasuch as human pathogenic strains ofEscherichiacoliandPseudomonasaeruginosa were grown in LB broth medium.LB broth was taken as negative control and30µg/ml Chlorempenicol was used as positivecontrol. After 5-6 days of incubation at 30 C,the antibacterial activity was evaluated bymeasuring inhibition zone against testorganisms.Salt stressCatalase activity: Immediate gas bubblesformation was considered Catalase positivewhen single colony was mixed 2-3 drops of3% H2O2 on clean grease free glass slide.Siderophore production: Bacterial strainsgrown on blue agar plates containing the dyechromazurol S (CAS), the formation oforange halos around the colonies wereindicative for siderophore production(Schwyn and Neilands, 1987)N2 -fixation (C2H2 -reduction) assay: TheNitrogenase activity (C2H2- reduction assay)(Schollhorn and Burris, 1967) was performedin 10 ml vacuotainer tubes containing 2.5 mlof the medium. Isolates grown in Burk’s N-The selected bacterial strains were tested fortheir tolerance to salt stress by inoculatingthem in LB broth medium containing variousconcentration of NaCl (1% to12%) and 0.1 mlof culture (1.0 x 107 cell ml-1) was inoculatedin 10 ml LB broth. The culture tubesincubated at 30 C for 48 hours and analyzedfor influence of salt by taking OD at 600 nmby UV/Visible spectrophotometer just afterinoculation and after incubation for 24 h.Experimental DesignThe effect of PGPR isolates Bacillus (R7),Azotobacter (RS7), Pseudomonas (RS10) and1793
Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1789-1802Acinetobacter (R12) on bitter melon wasdetermined by conducting a pot experiment.The experiment was designed in five sets withthree earthen pots in each set and soil frombotanical garden added with manure and sandin proportion 2:1:1 ratio. Set 1 served ascontrol without treatment of bacterium and inSet. 2, 3, 4 and 5, (one set for each PGPRstrain) sterile soil seeded with three bittermelon seeds. Seeds were inoculated withBacillus(R7),Azotobacter(RS7),Pseudomonas (RS10) or Acinetobacter (R12)culture (109cell ml-1) and pelleted with CaCO3powder.All five sets of pots kept under naturalenvironment in botanical garden and wateredon each alternate day. Different traits of plantwere studied such as seed germination,seedling length, plant height, number of fruitsper plant, fruits size and number of seeds perfruit.Results and DiscussionThe physicochemical characteristics of therhizospheric soil such as soil organic carbon(0.85%), total nitrogen (0.085%), moisturecontent (17.10%), soil pH (7.1) and otherparameters were at a level, which could befavorable for the growth of PGPR(data notpresented).The Momordica charantia rhizosphere wasassociated with a diversity of bacterialpopulation. Among the selected bacterialstrains population (CFU per gram dry soil)Bacillus sps (4.7x 108) was dominant in therhizosphere followed by, Acinetobacter (0.24x 108), Pseudomonas sps (0.1 x 108), andAzotobacter sps (0.51 x 107)(Fig- 1). On thebasis of cultural, morphological andbiochemical characteristics out of total 42 soilisolates, 26 isolates grouped as Bacillus,Azotobacter,PseudomonasandAcinetobacter. The cultural (3-4 days colony),morphologicalandbiochemicalcharacteristics of all the test isolates are givenin Table 1.All the 26 isolates of four bacterial generawere characterized for their PGP traits in vitroare depicted in Table 2. Catalase activity wasdetected by all the bacterial strains that maybe potentially very advantageous. OnlyBacillus and Pseudomonas exhibited HCNproduction. The IAA production, ammoniaproduction and phosphate solubilisationactivity was present in all the isolates ofBacillus, Azotobacter, Pseudomonas andAcinetobacter. Except Azotobacter, all thebacterial isolates produced siderophore.We had selected four isolates, one each ofBacillus, Azotobacter, Pseudomonas andAcinetobacter based on high PGP activity andcoded as R7, RS7, RS1 and R12, respectively.These isolates formed high phosphatesolubilisation zone (16- 24 mm) and liberatedhigh quantity of soluble phosphate (210418µg ml-1). All the selected isolates such asBacillus(R7),Azotobacter(RS7),Pseudomonas (RS1) and Acinetobacter (R12)produced IAA (ranges from 0.2 - 3.1 µg ml-1)at 50µg Tryptophan concentration. ExceptAcinetobacter(R12),Bacillus(R7),Azotobacter (RS7) and Pseudomonas (RS1)isolates exhibited high nitrogenase (C2H2reduction) activity which was maximum inAzotobacter (RS7) (72 nmol. C2H4 ml-1 h-1).All the three later strains also ic strains of E. Coli with inhibitionzone (12 to 23 mm) whereas only Bacillus(R7) and Pseudomonas (RS1) exhibitedantibacterial activity against P. aeruginosawith inhibition zone of 25 and 11 mm.Pseudomonas (RS1) has maximum Psolubilisation (418µg ml-1), IAA production(3.1 µg ml-1) and antibacterial activities (23mm in E. coli and 25 mm in P. aeruginosa)(Table 3). These strains further selected for1794
Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 1789-1802the salinity tolerance and traits of growthpromotion on bitter melon.Among all the four selected strains of plantgrowth promoting Rhizobacteria, Bacillus(R7) tolerated high concentration of salt(NaCl) and could surv
Jun 03, 2017 · Starch hydrolysis Test-Few drops of Gram’s iodine solution were added to the two days old culture of an isolate grown on starch agar plate. The dark blue colour developed due to formation of starch iodine complex. Clear area around streaked culture indicates the degradation
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