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Microbiological Research 163 (2008) 173181

Screening of free-living rhizospheric bacteria for

their multiple plant growth promoting activities
Farah Ahmad, Iqbal Ahmad, M.S. Khan

Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh 202002, India

Accepted 10 April 2006

Plant growth promoting rhizobacteria (PGPR) are known to influence plant growth by
Indoleacetic acid;
various direct or indirect mechanisms. In search of efficient PGPR strains with
Siderophores and
multiple activities, a total of 72 bacterial isolates belonging to Azotobacter,
antifungal activity
fluorescent Pseudomonas, Mesorhizobium and Bacillus were isolated from different
rhizospheric soil and plant root nodules in the vicinity of Aligarh. These test isolates
were biochemically characterized. These isolates were screened in vitro for their
plant growth promoting traits like production of indoleacetic acid (IAA), ammonia
(NH3), hydrogen cyanide (HCN), siderophore, phosphate solubilization and antifungal
activity. More than 80% of the isolates of Azotobacter, fluorescent Pseudomonas and
Mesorhizobium ciceri produced IAA, whereas only 20% of Bacillus isolates was IAA
producer. Solubilization of phosphate was commonly detected in the isolates of
Bacillus (80%) followed by Azotobacter (74.47%), Pseudomonas (55.56%) and
Mesorhizobium (16.67%). All test isolates could produce ammonia but none of the
isolates hydrolyzed chitin. Siderophore production and antifungal activity of these
isolates except Mesorhizobium were exhibited by 1012.77% isolates. HCN
production was more common trait of Pseudomonas (88.89%) and Bacillus (50%).
On the basis of multiple plant growth promoting activities, eleven bacterial isolates
(seven Azotobacter, three Pseudomonas and one Bacillus) were evaluated for their
quantitative IAA production, and broad-spectrum (active against X three test fungi)
antifungal activity. Almost at all concentration of tryptophan (50500 mg/ml), IAA
production was highest in the Pseudomonas followed by Azotobacter and Bacillus
isolates. Azotobacter isolates (AZT3, AZT13, AZT23), Pseudomonas (Ps5) and Bacillus
(B1) showed broad-spectrum antifungal activity on Muller-Hinton medium against
Aspergillus, one or more species of Fusarium and Rhizoctonia bataticola. Further
evaluation of the isolates exhibiting multiple plant growth promoting (PGP) traits on
soilplant system is needed to uncover their efficacy as effective PGPR.
& 2006 Elsevier GmbH. All rights reserved.

Corresponding author. Tel.: +91 9412371170; fax: +91 571 2703516.

E-mail address: (I. Ahmad).

0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved.
174 F. Ahmad et al.

Introduction and exert their effect on the plant. The environ-

mental factors include climate, weather condi-
Plant growth promoting rhizobacteria (PGPR) are tions, soil characteristics or the composition or
a heterogeneous group of bacteria that can be activity of the indigenous microbial flora of the soil.
found in the rhizosphere, at root surfaces and in To achieve the maximum growth promoting inter-
association with roots, which can improve the action between PGPR and nursery seedlings it is
extent or quality of plant growth directly and or important to discover how the rhizobacteria exert-
indirectly. In last few decades a large array of ing their effects on plant and whether the effects
bacteria including species of Pseudomonas, Azos- are altered by various environmental factors,
pirillum, Azotobacter, Klebsiella, Enterobacter, including the presence of other micro-organisms
Alcaligens, Arthobacter, Burkholderia, Bacillus (Bent et al., 2001).
and Serratia have reported to enhance plant Therefore, it is necessary to develop efficient
growth (Kloepper et al., 1989; Okon and Laban- strains in field conditions. One possible approach is
dera-Gonzalez, 1994; Glick, 1995). The direct to explore soil microbial diversity for PGPR having
promotion by PGPR entails either providing the combination of PGP activities and well adapted to
plant with a plant growth promoting substances particular soil environment. So keeping in view the
that is synthesized by the bacterium or facilitating above constrains, the present study was designed
the uptake of certain plant nutrients from the to screen certain rhizospheric bacterial isolates
environment. The indirect promotion of plant belonging to Azotobacter, Mesorhizobium ciceri,
growth occurs when PGPR lessen or prevent the fluorescent Pseudomonas and Bacillus for their
deleterious effect of one or more phytopathogenic multiple plant growth promoting activities.
The exact mechanisms by which PGPR promote
plant growth are not fully understood, but are
thought to include (i) the ability to produce or Materials and methods
change the concentration of plant growth regula-
tors like indoleacetic acid, gibberellic acid, cyto- Isolation and characterization
kinins and ethylene (Arshad and Frankenberger,
1993; Glick, 1995), (ii) asymbiotic N2 fixation All the isolates of Azotobacter, Pseudomonas and
(Boddey and Dobereiner, 1995), (iii) antagonism Bacillus were isolated from the rhizospheric soil of
against phytopathogenic microorganisms by pro- different crops (mustard, barseem, wheat, sugar-
duction of siderophores (Scher and Baker, 1982), cane, brinjal, onion, cauliflower, cabbage and chick
antibiotics (Shanahan et al., 1992) and cyanide pea) grown in vicinity of Aligarh, UP, India.
(Flaishman et al., 1996), (iv) solubilization of Mesorhizobium was isolated from the nodules of
mineral phosphates and other nutrients (De Freitas chickpea on yeast extract mannitol agar containing
et al., 1997; Gaur, 1990). Most popular bacteria per liter of distilled water: 10 g mannitol, 0.5 g
studied and exploited as biocontrol agent includes K2HPO4, 0.2 g MgSO4  7H2O, 0.1 g NaCl, 1.0 g yeast
the species of fluorescent Pseudomonas and Bacil- extract, 3.0 g CaCO3, 15 ml Congo red (1:400
lus. Some PGPR may promote plant growth indir- aqueous solution), 20 g agar, pH 6.87.0. M. ciceri
ectly by affecting symbiotic N2 fixation, nodulation isolates were confirmed by nodulation assay under
or nodule occupancy (Fuhrmann and Wollum, sterile pot condition as described by Vincent
1989). However, role of cyanide production is (1970).
contradictory as it may be associated with deleter- Whereas other bacteria were isolated on their
ious as well as beneficial rhizobacteria (Bakker and respective media, Azotobacter on Jensens medium
Schippers, 1987; Alstrom and Burns, 1989). containing per liter of distilled water: 20 g sucrose,
In addition to these traits, plant growth promot- 1 g K2HPO4, 0.5 g MgSO4  7H2O, 0.5 g NaCl, 0.1 g
ing bacterial strains must be rhizospheric compe- K2SO4, 0.005 g Na2MoO4, 20 g agar, pH 6.9, Pseudo-
tent, able to survive and colonize in the monas on Kings B medium containing per liter of
rhizospheric soil (Cattelan et al., 1999). Unfortu- distilled water: 10 g proteose peptone, 10 ml
nately, the interaction between associative PGPR glycerol, 1.5 g K2HPO4, 1.5 g MgSO4, 20 g agar, pH
and plants can be unstable. The good results 7.2 and Bacillus on nutrient agar (NA) containing
obtained in vitro cannot always be dependably per liter of distilled water: 5.0 g peptone, 1.5 g
reproduced under field conditions (Chanway and yeast extract, 1.5 g beef extract, 5.0 g NaCl, 20 g
Holl, 1993; Zhender et al., 1999). The variability in agar, pH 7.2. Bacterial cultures were maintained on
the performance of PGPR may be due to various the respective slants. Fluorescence of Pseudomo-
environmental factors that may affect their growth nas colonies was observed on Kings B medium
Screening of free-living rhizospheric bacteria 175

under UV exposure. All the microbiological media were sealed with parafilm and incubated at
and media ingredients were purchased from Hi- 2872 1C for 4 days. Development of orange to red
Media Lab. Pvt. Ltd., Mumbai, India. colour indicated HCN production.
The bacterial isolates were characterized by
their cultural conditions, morphological and bio- Siderophore production
chemical characteristics (hydrolysis of starch, lipid Bacterial isolates were assayed for siderophores
and chitin, utilization of glucose, sucrose, lactose, production on the Chrome azurol S agar medium
mannitol, citrate and catalase reactions) using (Sigma, Ltd.) described by Schwyn and Neilands
standard methods (Cappuccino and Sherman, (1987). Chrome azurol S agar plates were prepared
1992). Biotin prototrophy was determined by and divided into equal sectors and spot inoculated
growing the isolates on Bacto biotin assay medium with test organism (10 ml of 106 CFU/ml) and
(Difco Laboratories) for 4872 h at 2872 1C. incubated at 2872 1C for 4872 h. Development of
yelloworange halo around the growth was con-
In vitro screening of bacterial isolates for sidered as positive for siderophore production.
their plant growth promoting (PGP) activities
Phosphate solubilization by test bacteria
Assay for indoleacetic acid (IAA) production All isolates were first screened on Pikovskayas
IAA production was detected by the modified agar plates for phosphate solubilization as de-
method as described by Brick et al. (1991). scribed by Gaur (1990). Quantitative analysis of
Quantitative analysis of IAA was performed using solubilization of tricalcium phosphate in liquid
the method of Loper and Scroth (1986) at different medium was made as described by King (1932).
concentrations of tryptophan (0, 50, 150, 300, 400 Briefly, the test isolates were inoculated in 25 ml
and 500 mg/ml). Bacterial cultures were grown for Pikovskayas broth and incubated for 4 days at
72 h (Azotobacter) and 48 h (Pseudomonas and 2872 1C. The bacterial cultures were centrifuged
Bacillus) on their respective media at 2872 1C. at 15,000 rpm for 30 min. Supernatant (1 ml) was
Fully grown cultures were centrifuged at 3000 rpm mixed with 10 ml of chloromolibidic acid and the
for 30 min. The supernatant (2 ml) was mixed with volume was made up to 45 ml with distilled water.
two drops of orthophosphoric acid and 4 ml of the Cholorostannous acid (0.25 ml) was added and the
Salkowski reagent (50 ml, 35% of perchloric acid, volume was made up to 50 ml with distilled water.
1 ml 0.5 M FeCl3 solution). Development of pink The absorbance of the developing blue colour was
colour indicates IAA production. Optical density read at 600 nm. The amount of soluble phosphorus
was taken at 530 nm with the help of spectro- was detected from the standard curve of KH2PO4.
photometer Spectronic 20 D+. Concentration of IAA
produced by cultures was measured with the help
Antifungal assay
of standard graph of IAA (Hi-media) obtained in the
The agar well diffusion method as adopted
range of 10100 mg/ml.
earlier (Mehmood et al., 1999) was used. The
bacterial isolates tested for their antifungal activ-
NH3 production ity were fully grown in the respective broth media.
Bacterial isolates were tested for the production Test fungi were grown on Sabaroud dextrose agar
of ammonia in peptone water. Freshly grown (SDA), (per liter of distilled water: 40 g dextrose,
cultures were inoculated in 10 ml peptone water 10 g peptone, 20 g agar) slants. The spores were
in each tube and incubated for 4872 h at 2872 1C. scraped and suspended in 10 ml of sterile normal
Nesslers reagent (0.5 ml) was added in each tube. saline solution (NSS). Diluted spore suspension
Development of brown to yellow colour was a (0.1 ml, 105 CFU/ml) of the fungi was spread on
positive test for ammonia production (Cappuccino Muller Hinton agar (per liter of distilled water:
and Sherman, 1992). 300 g beef infusion, 17.5 g casein acid hydrolysate,
1.5 g starch, 20 g agar, pH 7.2), NA and SDA plates.
HCN production Wells of 8 mm diameter were punched into the agar
All the isolates were screened for the production medium and filled with 200 ml (2  107 CFU/ml) of
of hydrogen cyanide by adapting the method of bacterial culture. Nutrient broth was taken as
Lorck (1948). Briefly, nutrient broth was amended negative control and 100 mg/ml antifungal antibio-
with 4.4 g glycine/l and bacteria were streaked on tic, nystatin was used as positive control. The
modified agar plate. A Whatman filter paper no. 1 plates were incubated for 56 days at 2872 1C. The
soaked in 2% sodium carbonate in 0.5% picric acid antifungal activity was evaluated by measuring the
solution was placed in the top of the plate. Plates growth inhibition zone against test fungi.
176 F. Ahmad et al.

Results Pseudomonas (11.11%) and Bacillus (10%). All

isolates were negative for chitin hydrolysis whereas
Isolation and biochemical characterization positive for ammonia production.

On the basis of cultural, morphological and Quantitative assay of IAA production by

biochemical characteristics a total of 66 soil selected isolates
isolates were grouped into Azotobacter, Bacillus,
fluorescent Pseudomonas as described in Bergeys A total of 11 selected isolates of Azotobacter
Manual of Determinative Bacteriology (Holt et al., (seven), fluorescent Pseudomonas (three) and
1994). Six nodule isolates of chickpea (Cicer Bacillus (one) were tested for the quantitative
aretinum) were characterized as M. ciceri (Nour estimation of IAA in the presence of different
et al., 1994). General features of the test isolates concentrations of tryptophan. With no addition of
are illustrated in Table 1. All the isolates of M. tryptophan, production of IAA was not observed.
ciceri were prototrophic for biotin whereas 91.49% With the addition of tryptophan from 50 to 500 mg/
isolates of Azotobacter, 83.33% isolates of fluor- ml the production of IAA was increased. The
escent Pseudomonas and 80% isolates of Bacillus production of IAA was highest in isolates of
were prototrophic for biotin. fluorescent Pseudomonas, followed by Azotobacter
and Bacillus, respectively. Amongst the Azotobac-
Plant growth promoting traits of test isolates ter, isolates AZT26 produced highest amount of IAA
followed by AZT34AZT134AZT234AZT14AZT20 as
Screening results of PGP traits are depicted in depicted in Table 2.
Figs. 1 and 2. IAA production was shown in all the
isolates of fluorescent Pseudomonas followed by Antifungal activity of the test isolates
Azotobacter (83.3%), M. ciceri (83.3%) and Bacillus
(20%). Phosphate solubilization was detected in 80% Antifungal activity of AZT1, AZT3, AZT9, AZT13,
of isolates of Bacillus followed by Azotobacter AZT20, AZT23, Ps5 and B1 was checked against
(74.47%), fluorescent Pseudomonas (55.56%) and M. Aspergillus sp., Fusarium solani, F. ciceri, F.
ciceri (16.67%). Production of siderophore and oxysporum, Rhizoctonia bataticola using three
antifungal activity was simultaneously exhibited different media, MH, NA and SDA (Table 3). The
by isolates of Azotobacter (16.22%), fluorescent antifungal activity of strains tested varied with

Table 1. Biochemical characterization of the test isolates

Biochemical characters Azotobacter species Mesorhizobium Fluorescent Bacillus species

ciceri Pseudomonas

Number of isolates 47 6 9 10
Pigmentation Transparent, milky, Fluorescent
some becomes blackish green
brown on aging
Colony morphology Watery, mucilaginous Pin head, Button shaped Serrated
shrink, serrated mucilaginous margins
margins white
Polysaccharide production + +  
Gram reaction, cell shape  rods  rods  rods + rods
Growth on N2 free medium +   
Catalase, citrate test 100 100 100 100
Starch 68.09 55.56 80
Lipid 48.94 50 77.78 80
Biotin prototrophy 91.49 100 83.33 80
Carbohydrate utilization
Glucose 63.83 83.33 55.56 80
Lactose 70.21 16.67 11.11 20
Sucrose 78.72 83.33 33.33 80
Mannitol 36.17 16.67 70
Screening of free-living rhizospheric bacteria 177

IAA production

Phosphate solubilization

NH3 production

Percent isolates

Azotobacter spp. Mesorhizobium Fluorescent Bacillus spp.
ciceri Pseudomonas

Figure 1. Direct PGP activities of test isolates.

Siderophore production
Antifungal activity
80 HCN production

Percent isolates

Azotobacter Mesorhizobium Fluorescent Bacillus spp.
spp. ciceri Pseudomonas

Figure 2. Indirect PGP activities of test isolates.

Table 2. Production of indoleacetic acid by selected bacterial isolates grown on their respective medium

Isolate designation IAA production (mg/ml7SD) at different tryptophan concentrations (mg/ml)

0 50 150 300 400 500

AZT1 ND 1.4770.25 3.5370.32 6.5770.31 9.3770.15 11.8370.21

AZT3 ND 1.7770.21 5.0070.20 8.9070.30 11.2770.12 13.4770.45
AZT13 ND 1.6070.30 3.7770.25 7.0770.15 9.7370.25 12.8070.20
AZT9 ND 1.2770.31 3.5370.25 6.6370.40 8.5770.25 10.1770.25
AZT20 ND 1.2770.25 3.4070.20 6.4070.20 8.3370.25 10.4770.15
AZT23 ND 1.5070.20 3.6070.30 6.4770.31 8.9370.25 11.9070.20
AZT26 ND 2.1370.15 4.6770.15 7.6770.15 10.4370.47 15.0070.26
Ps5 ND 3.0070.20 7.4770.25 13.3770.35 16.8070.20 18.7070.30
Ps7 ND 2.6070.20 5.9070.20 11.3070.36 15.1770.25 18.0770.25
Ps9 ND 3.6070.20 6.1070.20 11.4770.15 14.9370.15 22.0270.20
B1 ND ND ND 3.4070.20 5.0370.15 7.0370.15

NDnot detectable.
 For Azotobacter Jensens, Pseudomonas Kings B, Bacillus nutrient media was used.
Table 3. Antifungal activity of the test isolates on different media

Test isolates Media used Zone size (mm 8 SD)

Aspergillus sp. Fusarium solani Fusarium ciceri Fusarium oxysporum Rhizoctonia bataticola

AZT1 MH 22.6770.58 ND ND 15.6770.58 ND

NA 18.3370.58 ND ND 14.3370.58 ND
SDA 13.0071.41 ND ND 16.0071.00 ND
AZT3 MH 30.5070.50 23.5370.50 17.6770.58 30.8370.76 15.0071.00
NA 25.6770.58 18.6771.15 15.3370.58 22.3370.58 ND
SDA 19.0071.00 25.8370.76 21.3371.15 24.1770.29 ND
AZT9 MH 25.0071.00 ND ND 13.3371.15 ND

NA 21.0071.00 ND ND ND ND
SDA 16.6770.58 ND ND ND ND
AZT13 MH 31.5070.50 21.0071.00 16.6770.58 17.6770.58 16.5070.50
NA 26.3370.58 16.00 14.5070.71 16.00 13.8370.76
SDA 22.0072.00 17.00 21.0071.00 17.5070.50 ND
AZT20 MH 19.0071.00 ND ND 15.6770.58 ND
NA 16.00 ND ND ND ND
AZT23 MH 17.5070.50 16.3370.58 ND 18.6770.58 ND
NA 14.00 ND ND 15.3370.58 ND
SDA ND ND ND 19.0071.00 ND
Ps5 MH 15.5070.50 15.3370.58 15.3370.58 19.0071.00 ND
NA 13.8370.7 12.0071.00 12.0071.00 14.8370.76 ND
SDA ND ND ND 12.0071.00 ND
B1 MH 15.5070.50 16.5070.50 16.5070.50 20.1770.76 ND
NA 12.8370.29 13.3370.58 13.3370.58 15.5070.50 ND
SDA 11.5070.71 14.2570.35 14.2570.35 12.6770.58 ND

F. Ahmad et al.
NDnot detected.
Screening of free-living rhizospheric bacteria 179

inhibition zones in diameter from 11.50 to production. While antifungal activity was shown
35.00 mm. Strains AZT3 and AZT13 induced larger by 12.77% of Azotobacter isolates, followed by
inhibition zones compared to the other strains. Pseudomonas (11.11%) and Bacillus isolates (10%).
AZT1 showed activity against Aspergillus sp. and F. Some of the above-tested isolates could exhibit
oxysporum on all three media. AZT3 and AZT13 more than two or three PGP traits, which may
showed good antifungal activity against all the promote plant growth directly or indirectly or
fungi but no activity was detected on SDA and or NA synergistically. Similar to our findings of multiple
against R. bataticola. AZT9 and AZT20 isolate PGP activities among PGPR have been reported by
showed activity against Aspergillus sp. on all three some other workers while such findings on indigen-
media whereas antifungal activity against F. oxy- ous isolates of India are less commonly explored
sporum was observed on MH medium. AZT23 was (Gupta et al., 1998). On the basis of preliminary
effective against Aspergillus sp., F. oxysporum and screening, quantitative analysis of IAA production
F. solani but results varied with different media. was made on seven Azotobacter isolates, three
PS5 and B1 could also exhibit broad-spectrum fluorescent Pseudomonas and one Bacillus isolate.
activities against test fungi. MH medium was most There was an increase in the level of IAA with the
appropriate medium for screening of antifungal increasing concentration of tryptophan (50500 mg/
activity and Aspergillus sp. was most susceptible ml). Similar trend of IAA production with the
organism (Table 3). increasing concentration of tryptophan was also
reported by Barazani and Friedman (2000). Isolates
AZT26, AZT3, AZT13 could produce relatively high
concentration of IAA compared to other Azotobac-
Discussion ter isolates. Production of high levels of IAA by
fluorescent Pseudomonas is a general characteristic
Plant rhizosphere is known to be preferred of our test isolates. Similar high level of IAA
ecological niche for various types of soil micro- production was recorded by other workers (Xie et
organisms due to rich nutrient availability. It has al., 1996). The production of IAA was found
been assumed that inoculation with diazotrophic dependant upon bacterial isolates and concentra-
bacteria like Rhizobium, Azotobacter and Azospir- tion of tryptophan. Such findings may have direct
illum enhanced the plant growth as a result of their practical application, although intrinsic ability of
ability to fix nitrogen. However, despite of exten- bacteria to produce IAA in the rhizosphere depends
sive research efforts only rhizobia have been shown on the availability of precursors and uptake of
to increase yields from dinitrogen fixation. Growth microbial IAA by plant (Arshad and Frankenberger,
promotion may be attributed to other mechanisms 1993).
such as production of plant growth promoting Another important trait of PGPR, that may
hormones in the rhizosphere and other PGP indirectly influence the plant growth, is the
activities (Arshad and Frankenberger, 1993; Glick, production of siderophores. They bind to the
1995). In the present investigation 47 isolates of available form of iron Fe3+ in the rhizosphere, thus
Azotobacter and 25 isolates belonging to M. ciceri, making it unavailable to the phytopathogens and
fluorescent Pseudomonas and Bacillus species were protecting the plant health. In the present inves-
screened in vitro for PGP activities. IAA production tigation six isolates of Azotobacter and the fluor-
was detected in all the test isolates of fluorescent escent Pseudomonas strain Ps5 showed multiple
Pseudomonas, 83.3%, of both Azotobacter and M. PGP activities including siderophore production and
ciceri isolates. Our findings of IAA production in antifungal activities against one or more test fungi.
Azotobacter isolates are in agreement with other On the basis of data obtained it could be stated
workers (Gonzalez-Lopez et al., 1986; Jagnow, that: (i) sensitivity of test fungi was in order of
1987; Nieto and Frankenberger, 1989). Aspergillus sp.4F. oxysporum4F. solani4Rhizoc-
Phosphate solubilization was most frequently tonia bataticola; (ii) MullerHinton medium was
encountered by Bacillus isolates (80%), followed best out of the three tested media to detect the in
by Azotobacter, Pseudomonas and least by Mesor- vitro antifungal activity which is probably due to
hizobium isolates. However, production of ammo- the non-interfering composition of this medium
nia was a common trait in all selected group of with the assay system; and (iii) isolate AZT3 and
bacteria. Siderophore production was detected AZT13 demonstrated broad spectrum antifungal
among some isolates of Azotobacter (12.77%), activity against the five tested fungi. The anti-
followed by Pseudomonas and Bacillus isolates. fungal activity of the test isolates indicated a close
However, 50% and 80% isolates of Bacillus and relationship between production of HCN and side-
Pseudomonas were detected positive for HCN rophores. The antifungal activity of the test
180 F. Ahmad et al.

isolates might be due to the production of side- Cattelan, A.J., Hartel, P.G., Fuhrmann, J.J., 1999.
rophore and HCN or synergistic interaction of these Screening of plant growthpromoting rhizobacteria
two or with other metabolites. However, role of to promote early soybean growth. Soil Sci. Soc. Am. J.
chitinase was not expected as these isolates were 63, 16701680.
negative for chitin hydrolysis. Several studies have Chanway, C.P., Holl, F.B., 1993. First year yield perfor-
demonstrated that production of siderophores, mance of spruce seedlings inoculated with plant
other secondary metabolites and lytic enzymes by growth promoting rhizobacteria. Can. J. Microbiol.
Pseudomonas strains was most effective in control- 39, 10841088.
ling the plant root pathogens including F. oxyspor- De Freitas, J.R., Banerjee, M.R., Germida, J.J., 1997.
um and R. solani (OSullivan and OGara, 1992; Phosphate solubilizing rhizobacteria enhance the
Nagrajkumar et al., 2004). Further studies on the growth and yield but not phosphorus uptake of canola
(Brassica napus L.). Biol. Fertil. Soil. 24, 358364.
performance of these isolates and their mutants on
Flaishman, M.A., Eyal, Z.A., Zilberstein, A., Voisard, C.,
the growth of plant will uncover the mechanism
Hass, D., 1996. Suppression of Septoria tritci blotch
and potential of these PGPR exhibiting multiple
and leaf rust of wheat by recombinant cyanide
PGP traits.
producing strains of Pseudomonas putida. Mol. Plant
Microbe Interact. 9, 642645.
Fuhrmann, J.J., Wollum II, A.G., 1989. Nodulation
competition among Bradyrhizobium japonicum strains
as influenced by rhizosphere bacteria and iron avail-
ability. Biol. Fertil. Soil. 7, 108112.
We thank the Chairman, Department of Agricul-
Gaur, A.C., 1990. Physiological functions of phosphate
tural Microbiology for providing necessary facilities
solubilizing micro-organisms. In: Gaur, A.C. (Ed.),
for this work, and Mr. Farrukh Aqil for technical
Phosphate Solubilizing Micro-organisms as Biofertili-
help. zers. Omega Scientific Publishers, New Delhi,
pp. 1672.
Glick, B.R., 1995. The enhancement of plant growth by
free living bacteria. Can. J. Microbiol. 41, 109114.
Gonzalez-Lopez, J., Salmeron, V., Martinez-Toledo, M.V.,
Ballesteros, F., Ramos-Cormenzana, A., 1986. Produc-
Alstrom, S., Burns, R.G., 1989. Cyanide production by
tion of auxins, Gibberellins and cytokinins by Azoto-
rhizobacteria as a possible mechanism of plant growth
inhibition. Biol. Fertil. Soil. 7, 232238. bacter vinelandii ATCC 12837 in chemically defined
Arshad, M., Frankenberger Jr., W.T., 1993. Microbial media and dialyzed soil media. Soil Biol. Biochem. 18,
production of plant growth regulators. In: Blaine, F., 119120.
Metting, Jr. (Eds.), Soil Microbial Ecology. Marcel and Gupta, A., Saxena, A.K., Murali, G., Tilak, K.V.B.R.,
Dekker, Inc., New York, pp. 307347. 1998. Effect of plant growth promoting rhizobacteria
Bakker, A.W., Schippers, B., 1987. Microbial cyanide on competitive ability of introduced Bradyrhizobium
production in the rhizosphere in relation to potato sp. (Vigna) for nodulation. J. Sci. Ind. Res. 57,
yield reduction and Pseudomanas sp. mediated plant 720725.
growth stimulation. Soil. Biol. Biochem. 19, 451457. Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T.,
Barazani, O., Friedman, J., 2000. Effect of exogeneously Williams, S.T., 1994. In: Bergys Manual of Determi-
applied L-tryptophan on allelochemical activity of native Bacteriology, ninth ed. Williams and Wilkins
plant growth promoting rhizobacteria (PGPR). J. Pub., MD, USA.
Chem. Ecol. 26, 343349. Jagnow, G., 1987. Inoculation of cereal crops and forage
Bent, E., Tuzun, S., Chanway, C.P., Enebak, S., 2001. grasses with nitrogen fixing rhizosphere bacteria:
Alterations in plant growth and in root hormone levels possible causes of success and failure with regard to
of lodgepole pines inoculated with rhizobacteria. Can.
yield response A review. Z. Pflanzenernnaehr.
J. Microbiol. 47, 793800.
Bodenkd. 150, 361368.
Boddey, R.M., Dobereiner, J., 1995. Nitrogen fixation
King, J.E., 1932. The colorimetric determination of
associated with grasses and cereals: recent progress
phosphorus. Biochem. J. 26, 292.
and perspectives for the future. Fert. Res. 42,
241250. Kloepper, J.W., Lifshitz, R., Zablotowicz, R.M., 1989.
Brick, J.M., Bostock, R.M., Silverstone, S.E., 1991. Rapid Free-living bacterial inocula for enhancing crop
in situ assay for indoleacetic acid production by productivity. Trends Biotechnol. 7, 3943.
bacteria immobilized on nitrocellulose membrane. Loper, J.E., Scroth, M.N., 1986. Influence of bacterial
Appl. Environ. Microbiol. 57, 535538. sources on indole-3 acetic acid on root elongation of
Cappuccino, J.C., Sherman, N., 1992. In: Microbiology: A sugarbeet. Phytopathology 76, 386389.
Laboratory Manual, third ed. Benjamin/cummings Lorck, H., 1948. Production of hydrocyanic acid by
Pub. Co., New York, pp. 125179. bacteria. Physiol. Plant. 1, 142146.
Screening of free-living rhizospheric bacteria 181

Mehmood, Z., Ahmad, I., Mohammad, F., Ahmad, S., soil suppressiveness to Fusarium wilt pathogens.
1999. Indian medicinal plants: a potential source of Phytopathology 72, 15671573.
anticandidal drug. Pharmaceut. Biol. 37, 237242. Schwyn, B., Neilands, J.B., 1987. Universal chemical
Nagrajkumar, M., Bhaaskaran, R., Velazhahan, R., 2004. assay for the detection and determination of side-
Involvement of secondary metabolites and extracel- rophores. Anal. Biochem. 160, 4756.
lular lytic enzymes produced by Pseudomonas fluor- Shanahan, P., OSullivan, D.J., Simpson, P., Glennon,
escens in inhibition of Rhizoctonia solani, the rice J.D., OGara, F., 1992. Isolation of 2,4-diacetylphlor-
sheath of blight pathogen. Microbiol. Res. 159, 7381. ogucinol from a fluoroscent pseudomonad and inves-
Nieto, K.F., Frankenberger, W.T., 1989. Biosynthesis of tigation of physiological parameters influencing its
cytokinins produced by Azotobacter chroococcum. Soil production. Appl. Environ. Microbiol. 58, 353358.
Biol. Biochem. 21, 967972. Vincent, J.M., 1970. In: Vincent, J.M. (Ed.), A Manual of
Nour, S.M., Fernandez, M.P., Normand, P., Cleyet-Marel, Practical Study of Root Nodule Bacteria. Blackwell,
J.C., 1994. Rhizobium ciceri sp. nov. consisting of Oxford.
strains that nodulate chickpeas (Cicer aretinum L.). Xie, H., Pasternak, J.J., Glick, B.R., 1996. Isolation and
Int. J. Syst. Bacteriol. 44, 511522. characterization of mutants of plant growth promot-
Okon, Y., Labandera-Gonzalez, C.A., 1994. Agronomic ing rhizobacterium Pseudomonas putida GR 12-2 that
applications of Azospirillum. In: Ryder, M.H., Ste- over produce indoleacetic acid. Curr. Microbiol. 32,
phens, P.M., BOWen, G.D. (Eds.), Improving Plant 6771.
Productivity with Rhizosphere Bacteria. Common- Zhender, G.W., Yao, C., Murphy, J.F., Sikora, E.R.,
wealth Scientific and Industrial Research Organiza- Kloepper, J.W., Schuster, D.J., Polston, J.E., 1999.
tion, Adelaide, Australia, pp. 274278. Microbe-induced resistance against pathogens and
OSullivan, D.J., OGara, F., 1992. Traits of fluorescent herbivores: evidence of effectiveness in agriculture.
Pseudomonas spp. involved in the suppression of plant In: Agarwal, A.A., Tuzun, S., Bent., E. (Eds.), Induced
roots pathogens. Microbiol. Rev. 56, 662676. Plant Defenses Against Pathogens and Herbivores:
Scher, F.M., Baker, R., 1982. Effect of Pseudomonas Biochemistry, Ecology and Agriculture. APS Press, St
putida and a synthetic iron chealator on induction of Paul, MN, p. 33.