International Journal of Chemical Studies 2019; 7(1): 1785-1788

P-ISSN: 2349–8528
E-ISSN: 2321–4902
IJCS 2019; 7(1): 1785-1788 Biochemical and physiological characterizations
© 2019 IJCS
Received: 03-11-2018 of Pseudomonas fluorescens
Accepted: 07-12-2018

Labhasetwar AA Labhasetwar AA, SB Bramhankar, TS Pillai, SS Isokar, GT Dinkwar, SS

Plant Pathology Section, College
of Agriculture, Nagpur,
Bhure and VM Kharat
Maharashtra, India
Abstract
SB Bramhankar Seven Pseudomonas isolates obtained from rhizosphere of different crops were designed as PF-1 to PF-7
Plant Pathology Section, College which showed the characteristics of Pseudomonas. All the isolates were gram negative, rod shaped and
of Agriculture, Nagpur, produced yellow and greenish yellow pigment. Biochemical characterizations were studied viz., KOH
Maharashtra, India test, catalase test, starch hydrolysis, gelatin liquefaction, H2S production, acid and gas production, some
of the isolates show both positive and negative to starch hydrolysis, H2S production, IAA production and
TS Pillai
phosphate solubilization. The bacterium failed to produce hydrogen cyanide (HCN). Physiological
Plant Pathology Section, College
of Agriculture, Nagpur, studies revealed that all isolates show maximum growth at 25 and 30°C temperature with OD value ≥ 60.
Maharashtra, India All the isolates show maximum growth at pH 7 and 8 with OD vale ≥ 60. Antagonistic activity of
Pseudomonas fluorescens isolates were studied against Xanthomonas axonopodis pv. citri. There were
SS Isokar significant differences on inhibition zone. Isolate PF-7 recorded maximum zone of inhibition (29.1 mm)
Plant Pathology Section, College followed by PF-4 (28.7 mm), whereas no inhibition showed by isolate PF-6.
of Agriculture, Nagpur,
Maharashtra, India Keywords: Pseudomonads fluorescens, Isolation, Characterizations, Xanthomonas axonopodis pv. citri

GT Dinkwar
Introduction
Plant Pathology Section, College
of Agriculture, Nagpur, The Triclosan genus Pseudomonas is the most heterogeneous and ecologically significant
Maharashtra, India group of known bacteria, and includes Gram-negative motile aerobic rods that are widespread
throughout nature and characterized by elevated metabolic versatility. The nutritional
SS Bhure requirements of Pseudomonas spp. are very simple, and the genus is foundin natural habitats
Plant Pathology Section, College
like soil, fresh water, marine environments etc., but it has also been isolated from clinical
of Agriculture, Nagpur,
Maharashtra, India instruments, aseptic solutions, cosmetics and medical products. The use of chemical fertilizer
and pesticides has caused an incredible harm to environment. These agents are both hazardous
VM Kharat to animal and humans and may persist and problem is replacing chemical with biological
Plant Pathology Section, College approaches, which are consider more environment friendly in the long term. One of the
of Agriculture, Nagpur,
emerging research areas for the control of different phytopathogenic agents is the use of plant
Maharashtra, India
growth promoting rhizobacteria (PGPR), which are capable of suppressing or preventing the
Phyto pathogen damage. In present study P. fluorescensiso late from different rhizosphere
were characterized for different biochemical and physiological test.

Materials and Methods

Isolation of Pseudomonas fluorescens: The bio agent involve in the study i.e. Pseudomonas
fluorescens were isolated from rhizosphere of different crops on King’s B media by serial
dilution and pour plate technique (King et al., 1954) [9]. possible to perform on a small sample
aliquot. The limit of quantification value in fish tissue was 0.083 mg g-1 and the limit of
detection was 0.016 mg g-1.

Morphological character of Pseudomonas fluorescens

Morphological character of Pseudomonas fluorescens such as shape, pigmentation and Gram
reaction were studied as per Migula (1894) [12].

Correspondence Biochemical characterization of Pseudomonas fluorescens isolates

Labhasetwar AA Biochemical tests viz., KOH test, starch hydrolysis, gelatin liquefaction, H 2S production, acid
Plant Pathology Section, College and gas production, catalase test, was carried out for biochemical confirmation of
of Agriculture, Nagpur, Pseudomonas fluorescens according to Aneja, 2003.
Maharashtra, India

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International Journal of Chemical Studies

Also all the isolates of Pseudomonas fluorescens were isolates were determined by measuring inhibition zone of
evaluated for plant growth promoting properties viz. indole pathogenic bacteria in petriplates.
production, phosphate solubilization and HCN production.
Result and Discussion
Physiological studies Six isolates of Pseudomonas fluorescens were obtained from
Effect of temperature rhizosphere of different crops and one from Plant Pathology
The study was initiated to find the optimum temperature Section, College of Agriculture, Nagpur were designed as PF-
requirement for growth of Pseudomonas fluorescens using 1 to PF-7. Isolate PF-1 was procured from Pathology Section,
King’s B medium. A loop full of 48 hrs old bacterial culture College of Agriculture, Nagpur, PF-2 from groundnut
was inoculated in 100 ml conical flask containing 30 ml of rhizosphere and PF-3 from chickpea rhizosphere. Two
King’s B broth. The inoculated flasks were incubated at isolates namely PF-4 and PF-5 were isolated from soybean
different temperature level viz., 5, 10, 15, 20, 25, 30, 35 and and maize rhizosphere.
40°C respectively for 72 hours. Observations were recorded Isolates namely PF-6 and PF-7 isolated from jowar and citrus
for the optical density of the broth culture turbidiometrically rhizosphere, respectively. The association of bacterial
by using spectrophotometer at 600 nm after 72 hr. bioagent i.e. Pseudomonas fluorescens has also been reported
by (Dave and Dube, 2000, Yeole and Dube, 2001, Gholve et
pH requirement al., 2006 and Kaur et al., 2007) [4, 17, 5, 7] from rhizosphere of
Effect of pH on the growth of Pseudomonas fluorescens was various crop.
studied by adjusting pH of the King’s B medium to various
levels viz., 4, 5, 6, 7, 8, 9 and 10 using appropriate phosphate Morphological and biochemical characterizations
Result presented in Table 1 showed that all isolates were rod
buffer. A loop full of 48 hour old bacterial culture was mixed
and Gram negative in reaction. All the isolates were able to
in 100 ml conical flask containing 30 ml King’s B broth.
liquefy the gelatin and were capable of H2S production.
Inoculated flasks were incubated at room temperature for 72
Among these all the isolates shows positive for KOH test,
hours. After the incubation period observations were recorded
catalase test, acid and gas production. Out of seven isolates of
for the growth of bacterium turbidiometrically by using
Pseudomonas fluorescens only the four isolates were able to
spectrophotometer at 600 nm.
hydrolyse the starch.
Different biochemical tests were carried out for confirmation
Antagonism of Pseudomonas fluorescens against of Pseudomonas fluorescens. Among them test gelatin
Xanthomonas axonopodis pv. citri liquefaction, H2S production, starch hydrolysis, catalase test,
Xanthomonas axonopodis pv. citri isolatewas collected from KOH test, acid and gas production test gave positive reaction
Plant Pathology Section, COA, Nagpur and antagonistic and confirmed the isolate as Gram negative, similar studies
effect of Pseudomonas fluorescens was tested. Different have also reported by several workers.
isolates of Pseudomonas fluorescence were evaluated for their Meera and Balabaskar (2012) [10] studied seven isolates of P.
efficacy against the growth of Xanthomonas axonopodis pv. fluorescens in detail for colony, colour, growth type,
citri by inhibition zone assay method. A suspension (3 day fluorescence and cell shape. P. fluorescens showed that all the
old) of Xanthomonas axonopodis pv. citri multiplied in isolates produced similar results with regard to Gram staining
nutrient broth was mixed with lukewarm nutrient agar (negative), starch hydrolysis (negative), gelatin liquefaction
medium. Fifteen to twenty ml of seeded medium poured into (positive), catalase test (positive), oxidase test (positive) and
the sterilized petriplates and allowed to solidify. A loop full fluorescent pigmentation (positive).
culture of each Pseudomonas fluorescens isolate placed in the Saravanan et al. (2013) [14] reported biochemical
centre of petriplates containing the seeded medium. The characteristics of fluorescent pseudomonas showed that all ten
inoculated plates then incubated at 28°C for 72 hours. isolates were positive to catalase, amylase, gelatinase and
Antagonistic activity of different Pseudomonas fluorescens Siderophores production.

Table 1: Biochemical characterizations of Pseudomonas fluorescens isolated from rhizospheric soil of field crop.
Reaction of isolates
Sr. No. Characters
PF-1 PF-2 PF-3 PF-4 PF-5 PF-6 PF-7
Morphological characterization
1 Gram staining -ve -ve -ve -ve -ve -ve -ve
2 Cell shape Rod Rod Rod Rod Rod Rod Rod
3 Colony colour WY WY WY WY GY WY WY
Biochemical characterization
4 KOH test + + + + + + +
5 Catalase test + + + + + + +
6 Gelatin liquefaction + + + + + + +
7 H2S production + + + + + + +
8 Starch hydrolysis + - + - - + +
9 Acid and gas production + + + + + + +
+: positive reaction -: negative reaction WY: whitish yellow GY: greenish yellow

Plant growth promoting studies of Pseudomonas Pseudomonas fluorescens strain confirmed IAA production.
fluorescens All the isolates were positive for production of IAA except
Table 2 indicate plant growth promoting characteristics of (PF-3). Shinde (2003) studied the plant growth promoting
Pseudomonas fluorescens. Development of cherry red colour activities of rhizobacteria and noted IAA production of nine
upon addition of Kovac’s reagent to culture supernatant of out of ten Pseudomonas fluorescens. Phosphate solubilization
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International Journal of Chemical Studies

by bacterial strain was found positive as they formed clear colour from yellow to reddish brown of filter paper confirmed
zone on Pikovskaya’s agar medium. All the isolates were production of strong HCN. All the isolates of Pseudomonas
shows positive to solubilize the phosphate. Similarly 19 fluorescens showed negative reaction for HCN production.
isolates of phosphate solubilizing bacteria showed the highest Mahesh (2007) [11] also studied ten isolates of Pseudomonas
halo colony ratios, the halo colony ratio indicate that these fluorescens and found that all tested isolates showed negative
isolates were the putative bacteria with phosphate solubilizing for HCN production.
activities reported by Ruangsanka (2014) [13]. Change in

Table 2: Plant growth promoting properties of Pseudomonas fluorescens

Reaction of isolates
SN. Characters
PF-1 PF-2 PF-3 PF-4 PF-5 PF-6 PF-7
1 IAA + + - + + + +
2 Phosphate solubilization + + + + + + +
3 HCN - - - - - - -
‘+’ positive test ‘–’ negative test

Physiological Characterization between 0.01 to 0.30 indicating the physiological properties

Temperature of Pseudomonas fluorescens. The present result are in
Table 3 indicates all the isolates shows the maximum growth conformity with the result obtained by Waghunde et al.
at temperature ranges between 25 and 30°C with an OD value (2013) [16] they isolated seven Pseudomonas isolates from
≥60 followed by 20 and 35°C with an OD value ranging different locations of South Gujarat, all the isolates were able
between 0.31 to 0.60 and minimum growth of bacteria to grow at 10 to 42°C temperature and fail to grow at 4°C.
observed at 5, 10, 15 and 40°C with an OD value ranging

Table 3: Effect of temperature regimes on growth of Pseudomonas fluorescens

SN. Temperature PF-1 PF-2 PF-3 PF-4 PF-5 PF-6 PF-7
1 5°C + + + + + + +
2 10°C + + + + + + +
3 15°C + + + + + + +
4 20°C ++ ++ ++ ++ ++ ++ ++
5 25°C +++ +++ +++ +++ +++ +++ +++
6 30°C +++ +++ +++ +++ +++ +++ +++
7 35°C ++ ++ ++ ++ ++ ++ ++
8 40°C + + + + + + +
+: 0.01 to 0.30 ++: 0.31 to 0.60 +++: ≥60

pH 0.01 to 0.30 (Table 4). The results corroborate with the

All the isolates showed maximum growth at 6 and 7 pH range finding of Stolp and Gadkari, (1970) [15] indicated that
with an OD value ≥60 followed by 8 pH with an OD value optimum temperature and pH for Pseudomonas spp., were
ranging between 0.31 to 0.60. The least growth was observed 30°C and pH 7-8.5, respectively.
only at 4, 5, 9 and 10 pH with an OD value ranging between

Table 4: pH requirement for growth of Pseudomonas fluorescens

SN. pH requirement PF-1 PF-2 PF-3 PF-4 PF-5 PF-6 PF-7
1 4 + + + + + + +
2 5 + + + + + + +
3 6 +++ +++ +++ +++ +++ +++ +++
4 7 +++ +++ +++ +++ +++ +++ +++
5 8 ++ ++ ++ ++ ++ ++ ++
6 9 + + + + + + +
7 10 + + + + + + +
+: 0.01 to 0.30 ++: 0.31 to 0.60 +++: ≥60

Table 5: Antagonistic activity of Pseudomonas fluorescens against Xanthomonas axonopodis pv. Citri
SN. Bacterial strain Inhibition zone (mm)
1 Pseudomonas fluorescens (PF-1) 13.2
2 Pseudomonas fluorescens (PF-2) 20.1
3 Pseudomonas fluorescens (PF-3) 15.3
4 Pseudomonas fluorescens (PF-4) 28.7
5 Pseudomonas fluorescens (PF-5) 18.2
6 Pseudomonas fluorescens (PF-6) –
7 Pseudomonas fluorescens (PF-7) 29.1
8 Control 00

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International Journal of Chemical Studies

Antagonism of Pseudomonas fluorescens against challenged with phytopathogens. African J Crop Sci.
Xanthomonas axonopodis pv. citri 2013; 21(1):29 -36.
The result of antagonistic activity of Pseudomonas 15. Stolp H, Gadkari D. Nonpathogenic Members of the
fluorescens isolates against Xanthomonas axonopodis pv. Genus Pseudomonas. The prokaryotes, 1970, 719-741.
Citri in vitro condition are shown in table 5. It shows 16. Waghunde RR, Sabalpara AN, Deshmukh AJ, Pandya
inhibition zone induced by antagonistic bacteria Pseudomonas JR. Characterization and evaluation of native
fluorescens isolates. The highest inhibition zone observed in Pseudomonas spp. of south Gujarat against leaf blast in
strain PF-7 (29.1 mm) followed by strain PF-4 (28.7 mm). No finger millet. J Biol. Control. 2013; 27(4):312-318.
inhibition zone was induced by strain PF-6. The results 17. Yeole RD, Dube HC. Rhizobacterial Fluorescent
corroborate with the finding of several workers (Kalita et al., Pseudomonads isolates from four crop plants and their
1996; Khodakaramian et al., 2008; Abhang et al., 2015; Apet rhizosphere competence. J Mycol. Pl. Pathol. 2001;
et al., 2018) [6, 8, 2, 3]. 31(3):273-276.

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