Effect of PGPR Studies on Some Angiospermic Plants
Kaviyarasi K.*, Cholarajan A., Bharathidasan R., Madhanraj P. and Panneerselvam A .
P.G. and Research Department of Botany and
Microbiology, A.V.V.M Sri Pushpam College (Autonomous),
Poondi -613503, Thanjavur, Tamilnadu.
ABSTRACT:
PGPR
offers an environmentally sustainable approach to crop production and health. Many
members of genera pseudomonas sp. perform
beneficial effects on plants. Among them P.
putida and P.
fluorescens are fluorescent pseudomonas that
represent Significant part of these Members. The rhizosphere
soil was collected from cultivated plots. P.
fluorescence was isolated and identified through morphologicaland
hemical tests and it was treated against some plant
pathogens. The biocontrol potential of P. fluorescens
against plant pathogen such as A. terreus sp, Circinella sp, Verticillium sp,
was assessed by dual plate technique. The seed treatment was made on four
plants namely Hibiscus esculentus, Oryza sativa,
Capsicum annum, Spinacia olerace.
Pot culture techniques was applied for the growth of plants along with p. fluorescence inoculums (100ml) and
chemical fertilizer (10g).The plant height, root length, root biomass, shoot biomass
were also estimated. The results were discussed.
KEYWORDS: PGPR, Pseudomonas
fluorescence.
INTRODUCTION:
The
thin layer of soil about 1 to 2 mm thick Surrounding crop roots and Volume of
soil occupied by roots is known as volume of soil occupied by roots is known as
the Rhizosphere. Root Colonizing bacteria that exert
Beneficial effects on plant development via direct or indirect mechanisms have been defined as plant growth promoting rhizobacteria Although Significant control of plant
pathogens or direct enhancement of plant development has been less consistent
.Recent Progress in our an Understanding of their Diversity colonization
ability, mechanism of action Formulation ,and Application Should facilitate their development as
reliable components in the Management of Sustainable agricultural systems
[Louis Nelson et al, 2004 ). In search of efficient PGPR strains With Multiple
activities a total of 150 bacterial isolates belonging to Bacillus, pseudomonas, Azotobactes and Rhizobium were isolates could Exhibit More than two or
three PGPR traits which may promote plant growth, directly or indirectly or
synergistically. Further, rhizobacteria tolerant to multiple
heavy metals exhibited a couple of PGPR Activities
(Joseph et al, 2007 )PGPR isolates were
Screened against TMV disease on tomato, out of these, 17 isolates were highly effective
and completely controlled the disease. Based on visual plant growth and disease
Control (Kirankuman et al, 2008 ) this study was conducted to assess the
possible role of the integrated use of seed inoculation with plant growth
promoting Rhizobacteria (PGPR ).
Compost
and mineral fertilizers for improving growth and yield of wheat sown at
different plant Spacing PGPR were Isolated Form Rhizosphere
Soil of Wheat Plants. Maximum grain yield and 1000 grain weight were observed where
PGPR inoculated seeds were used in combination with commended chemical
fertilizer [Javed Akhtar et
al, 2009 ]
MATERIALS AND
METHODS:
Isolation of Bacteria from Rhizosphere:
Rhizosphere
soil was collected from Peikkarumbankottai village of
Thanjavur District and it was separated from 5-6
roots with the help of a brush in a petridish 10 gram
of rhizosphere soil was added in 100ml sterile water
blank and shaken it for 15 minutes on a magnetic shaker. Serial dilutions were prepared and
transferred 1ml each of dilution 10-4 to 10-6 to sterile petriplates. Then,
the melted and cooked nutrient agar medium was poured to plates. The plates were incubated at 35°C for 24-48
hours.
Identification and Characterization of
Bacterial strain:
The
tests were undertaken to identify and characterize bacterial isolates as per
standard procedures described (Holt et al, 1994) and the biochemical test were analyzed.
Morphological Analysis:
Cultural Characterization:
The
colony morphology was studied based on the colour,
shape, size and margin of the colonies. Gram staining, motility, Biochemical
test were used to identify the colonics.
Dual Plate Technique:
The
pure plant pathogenic fungi such as Aspergillus terreus, Circinella, Verticillum broth culture were mixed with potato
dextrose agar media. Agar diffusion method was used to bacteria is P. fluorescens
culture were placed centrally on seeded medium. The plates were incubated at
room temperature for 2-3 days and zone of incubation was evaluated by measuring
the diameter of the bacterial growth as compared to control that continued in the
test bacteria.The percentage of inhibition was calculated by applying Vincent
formula.
r-r1
Percentage inhibition of growth = r X100
r=
growth of the fungus was measured from the centre of the colony towards the
centre of the plate in the absence if antagonistic fungus
r1=growth
of the fungus was measured from the centre of the towards the antagonistic
fungus
Inoculation
Test:
The sterile filter papers were taken. Then the seeds
were mixed with 1ml of P. fluorescens. The inoculated seeds were placed on the
plates and incubated. The uninoculated seeds served
as control. The vigour index of the seeds was
estimated by multiplying the height of the plant.
Pot Culture
technique for Hibiscus esculentus, Oryza sativa,
Capsicum annum, Spinacia oleracea:
A pot culture experiment was conducted in garden soil
with the following treatment.
T0 uninoculated
(without P. fluorescens
and without fertilizer)
T 1 fertilizer alone (NPK complex 17:17:17
10g / pot
Fertilizer Basal application)
T2 P. fluorescens + Fertilizer (seeds mixed with P. fluorescens suspension)
Fertilizer Basal application
P. fluorescens was
grown in nutrient broth for 24 hours and centrifuged. The pellets were resuspended
in sterile distilled water to obtain a population density of 106 cfu m-1. This suspension was mixed with 1% carboxymethyl cellulose (CMC) and the slurry coated onto
seeds. Seeds were allowed to air dry
overnight under aseptic conditions and sown in the pots with 3 replicates. The
pots were watered with tap water when required. There are three types of pot
treatments maintained in pot experiments labeled as T0, T1,
T2, which were uninoculated control,
fertilizer control, and fertilizer with Pseudomonas.
Percent germination, fresh shoot weight, fresh root weight were recorded.
Biometric
observations:
Plant samples were collected for biometric observations
at 3 stages. The biometric observations
such as plant height, root length, shoot biomass and root biomass were
recorded.
RESULTS AND
DISCUSSION:
The
following results were obtained from the present study effect of PGPR P. fluorescens
on Oryzae sativa, Capsicum annum, Hibiscus esculentus, Spinacia
oleracea.
Table 1: Cultural and Biochemical
characterization of Pseudomonas fluorescens
|
Characteristics
|
Results
|
|
Colony morphology
|
Round rod
|
|
Cell Shape
|
Short rod
|
|
Motility
|
Motile
|
|
Gram staining
|
Negative
|
|
Biochemical Test
Indole
|
-
|
|
Carbohydrate fermentation
Test
(i)
Glucose
(ii)
Lactose
|
-
-
|
|
Catalase
|
+
|
|
Oxidase
|
+
|
|
Starch hydrolysis
|
-
|
|
Nitrate Reduction
|
+
|
|
Gelatin liquefaction
|
+
|
|
Identified as
|
P. fluorescens
|
Table II: Effect of P. fluorescens on the growth inhibition
of plant pathogenic fungi
|
Pathogen
|
Control (mm)
|
Growth of inhibition (mm)
|
Percent Inhibition
|
|
A. terreus
|
10
|
12.3
|
14.8
|
|
Verticillium
|
-
|
-
|
-
|
|
Cirunella
|
8.9
|
9.5
|
11.4
|
Table III Effect of Pseudomonas fluorescens on plant height
of Capsicum annum, Hibiscus esculentus, Oryza sativa, Spinacia oleracea
|
Treatments
|
Plant Height (cm)
|
|
R1
|
R2
|
R3
|
R4
|
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
|
Control
|
7
|
14.7
|
29.7
|
6.3
|
10.2
|
25.9
|
5.3
|
11
|
23.1
|
7.7
|
14.5
|
28.4
|
|
T1 Fertilizer
|
8.5
|
16.5
|
35.3
|
7.8
|
11.3
|
27.3
|
5.9
|
14.5
|
25.3
|
8.3
|
15.5
|
30.1
|
|
T2 Fertilizer Culture
|
8.7
|
17.3
|
37.5
|
8.2
|
14.5
|
29.7
|
6.2
|
14.7
|
26.2
|
9.1
|
17.2
|
35.8
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table IV Effect of Pseudomonas fluorescens
on Root Length of Oryza sativa, Capsicum annum, Hibiscus esculentus, Spinacia oleracea
|
Treatments
|
Plant Height (cm)
|
|
R1
|
R2
|
R3
|
R4
|
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
|
Control
|
1.2
|
2.3
|
4.3
|
1.03
|
2.5
|
5.9
|
1.01
|
2.9
|
3.7
|
1.3
|
2.1
|
7.1
|
|
T1 Fertilizer
|
1.5
|
3.2
|
6.2
|
1.7
|
3.9
|
6.7
|
1.3
|
3.3
|
4.2
|
1.9
|
2.3
|
7.3
|
|
T2 Fertilizer Culture
|
1.7
|
9.4
|
6.7
|
2.1
|
4.5
|
7.5
|
1.7
|
4.2
|
4.9
|
2.2
|
2.9
|
7.7
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table V Effect of Pseudomonas fluorescens on Shoot Biomass
of Oryza sativa, Capsicum annum, Hibiscus esculentus, Spinacia oleracea
|
Treatments
|
Plant Height (cm)
|
|
R1
|
R2
|
R3
|
R4
|
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
|
Control
|
0.002
|
0.42
|
1.89
|
0.10
|
0.30
|
1.63
|
0.005
|
0.011
|
1.3
|
0.076
|
0.13
|
1.5
|
|
T1 Fertilizer
|
0.027
|
0.49
|
1.60
|
0.11
|
0.37
|
1.67
|
0.059
|
0.93
|
1.7
|
0.109
|
0.19
|
1.7
|
|
T2 Fertilizer Culture
|
0.16
|
0.9
|
2.1
|
0.22
|
0.52
|
1.99
|
0.14
|
1.24
|
2.3
|
0.117
|
1.5
|
1.99
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table VI Effect of Pseudomonas fluorescens on Shoot Biomass
of Oryza sativa, Capsicum annum, Hibiscus esculentus, Spinacia oleracea
|
Treatments
|
Plant Height (cm)
|
|
R1
|
R2
|
R3
|
R4
|
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
10
|
20
|
30
|
|
Control
|
0.004
|
0.081
|
1.32
|
0. 001
|
0. 011
|
1.11
|
0.002
|
0.9
|
1.42
|
0.017
|
1.39
|
2.35
|
|
T1 Fertilizer
|
0.007
|
0.019
|
1.71
|
0.001
|
0.012
|
1.30
|
0.004
|
0.13
|
1.03
|
0.019
|
1.45
|
2.72
|
|
T2 Fertilizer Culture
|
0.030
|
0.9
|
1.98
|
0.002
|
0.015
|
1.92
|
0.14
|
1.8
|
2.9
|
0.12
|
1.75
|
3.22
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
R1-Oryza sativa; R2-Capsicum annum; R3-Spinacia oleracea; R4-Hibiscus
esculentus
Identification of bacterial isolate:
Morphological Analysis:
Nutrient
agar was used for the bacterial isolation and distinct colony type was
evaluated further (Table 1).
Dual Plate Technique:
Antifungal
activity of P. fluorescens
was tested against A. terreus, Verticillium, Circinella.
Maximum zone of inhibition was obtained in A. terreus sp. (14.8) followed by Circinella sp.
(11.4) and there was no zone of inhibition observed on Verticillium sp. (Table II).
Pot Culture Technique:
Biometric observation:
The
effect of P. fluorescens
inoculum on plants of Oryza sativa, Capsicum annum, Hibiscus esculentus,
Spinacia oleracea was
recorded on 10, 20 and 30 days intervals of plant height, root biomass, root
length, shoot biomass datas were presented in Tables.
Determination of plant height:
All
the three periods of observation, P. fluorescens treated plants showed better growth than
untreated. The bio fertilizer treatment
exhibited the moderate effect. The
increase in plant height was more between 20 to 30 days, this indicating the
stimulatory effect P. fluorescens
during this period of crop growth. But we
have noted on the 30th day observation in table III.
Determination of root length:
All
the three periods of observation, P. fluorescens treated plants showed better growth than
untreated. The bio fertilizer treatment exhibited the moderate effect. The increase in root length was more between 20
to 30 days, this indicating the stimulatory effect P. fluorescens during this period of crop
growth. But we have noted on the 30th day
observation in table IV.
Determination of shoot biomass:
All
the three periods of observation, P. fluorescens treated plants showed better growth than
untreated. The bio fertilizer treatment exhibited the moderate effect. The
increase in shoot bio mass was more between 20 to 30 days, this indicating the
stimulatory effect P. fluorescens
during this period of crop growth. But we
have noted on the 30th day observation in table V.
Determination of root biomass:
All
the three periods of observation, P. fluorescens treated plants showed better growth than
untreated. The bio fertilizer treatment exhibited the moderate effect. The
increase in root bio mass was more between 20 to 30 days, this indicating the
stimulatory effect P. fluorescens
during this period of crop growth. But we
have noted on the 30th day observation in table VI.
In
this study Rhizosphere bacteria such as P. fluorescens
was isolated from rhizosphere soil collected from
agricultural land. Plant growth
promoting activity of this strain was tested on four edible plants such as Oryza sativa, Capsium annuum, Hibisus esclentus, Stinacia
oleracea.
The
possible role of PGPR in restricting mineral nutrients and thus alleviating
soil salinity stress during plant growth has been studied salt stressed plants
had significantly decreased plant growth compared to those from non saline
soil. The inoculated plants compared to
the inoculated plants under soil salinity conditions had an increased
antioxidant activity. The results
suggested that inoculation of salt stressed plants with PGPR strains could
alleviate salinity stress (Han and Lee, 2005).
The
prospect of manipulating crop rhizosphere microbial
populations by inoculation of beneficial bacteria to increase plant growth and
controlling different pathogens have shown considerable promise and found to be
safe on human health compared with chemical fertilizers. (Han et al, 2005).
Biocontrol
property of P. fluorescens was tested
against three fungal pathogens such as A.
terreus (ircinella and verticillium and P. fluorescens showed maximum zone of inhibition against A. terreus (14.8mm)
followed by Circinella (11.4mm) and there was no activity
found against Verticillium species.
REFERENCE:
1.
Han, H.S., and Lee, K.D., 2005. Physiological response of soybean-
inoculation of brady rhizobium japonicum with PGPR in
saline soil conditions. Journal of
Agriculture and Biological science.3:216-221
2.
Han, J.L., Sun, X., Dong, L., Cai, X., Sun,
H., Yang, Y., Wang and Song, w., 2005.Characterization of a novel plant growth
–promoting bacteria strain defftia sturuhatensis HR4 both as a diazotroph
and a potential biocontrol agent against various
plant pathogens. Systs. Appl. Mibio.
28:66-76
3.
Javed, M., Akhtar, hafiz Naeem Ashar,Shahzad,K.,and
Arshad,M.,2009.Role of plant growth promoting rhizobacteria
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and yield of wheat Pak. J.Bio.4(1):381-390.
4.
Holt, J.G., Krieg, N.R., Sneuth, A., Staley. J.T.,
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Wilkins, Baltimore. USA
5.
Joseph,B., Ranjan,
R., Patra, R., Lawrence., 2007. Characterization of
plant growth promoting rhizobacteria associated with chick pea (Cicer arietinum L.).
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production: ISSN.1735-6814
6.
Kirankumar, R., Jagadeesh, K.S., and
Krishnaraj,P.U.,2008. Screening and selection of PGPR
isolates against TMV on tomato. J. Soil Biol .
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7.
Nelson, L.M., 2004.Plant growth promoting rhizobacteria
(PGPR); Prospects of new inoculants online. Crop management, DIO:10:1094\ (m-2004-0301-05RM