ISOLATION AND

CHARACTERIZATION OF
Pseudomonas fluorescens FROM RICE
FIELDS FOR THE BIOLOGICAL
CONTROL OF Rhizoctonia solani
CAUSING SHEATH BLIGHT IN RICE

BIYYANI SUMAN
B.Sc. (Ag.)

MASTER OF SCIENCE IN AGRICULTURE

(AGRICULTURAL MICROBIOLOGY)

2015

ISOLATION AND CHARACTERIZATION OF

Pseudomonas fluorescens FROM RICE FIELDS
FOR THE BIOLOGICAL CONTROL OF
Rhizoctonia solani CAUSING SHEATH
BLIGHT IN RICE

BY
BIYYANI SUMAN
B.Sc. (Ag.)

THESIS SUBMITTED TO THE PROFESSOR JAYASHANKAR

TELANGANA STATE AGRICULTURAL UNIVERSITY IN
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
AWARD OF THE DEGREE OF

MASTER OF SCIENCE IN AGRICULTURE

(AGRICULTURAL MICROBIOLOGY)

CHAIRMAN: Dr. A. VIJAYA GOPAL

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY

COLLEGE OF AGRICULTURE
RAJENDRANAGAR HYDERABAD-500 030
PROFESSOR JAYASHANKAR TELANGANA STATE
AGRICULTURAL UNIVERSITY
2015

DECLARATION
I, BIYYANI SUMAN, hereby declare that the thesis entitled, ISOLATION
AND CHARACTERIZATION OF Pseudomonas fluorescens FROM RICE
FIELDS FOR THE BIOLOGICAL CONTROL OF Rhizoctonia solani CAUSING
SHEATH BLIGHT IN RICE submitted to Professor Jayashankar Telangana State
Agricultural University for the degree of MASTER OF SCIENCE IN
AGRICULTURE in the major field of Agricutural Microbiology, is the result of
original research work done by me. I also declare that no material contained in the thesis
or any part there of has not been published earlier in any manner.

Date:

(BIYYANI SUMAN)

Place: Hyderabad

I.D. No. RAM/2013-76

CERTIFICATE

Mr. BIYYANI SUMAN has satisfactorily prosecuted the course of research and the
thesis entitled ISOLATION AND CHARACTERIZATION OF Pseudomonas

fluorescens FROM RICE FIELDS FOR THE BIOLOGICAL CONTROL OF

Rhizoctonia solani CAUSING SHEATH BLIGHT IN RICE submitted is the result of
original research work and is of sufficiently high standard to warrant its presentation to the
examination. I also certify that neither the thesis nor its part there of has been previously
submitted by him for a degree of any University.

Date:

(Dr. A. VIJAYA GOPAL)

Chairman

LIST OF CONTENTS

Chapter No.

Title

INTRODUCTION

II

REVIEW OF LITERATURE

III

MATERIAL AND METHODS

IV

RESULTS AND DISCUSSION

SUMMARY AND CONCLUSIONS

LITERATURE CITED
APPENDICES

Page No.

LIST OF TABLES

Table
No.

Page

Title

No.

4.1

Location details of the isolates from Rice crop along with GPS No. ,
Latitudes, Longitudes, Elevation and Soil type

4.2

Pseudomonas population in the rice rhizosphere at different places

4.3

Cultural and morphological characteristics

fluorescens isolates on Kings B medium

4.4

Naming of the Pseudomonas fluorescens isolates according to the

place of collection

4.5

Biochemical characterization of Pseudomonas fluorescens isolates

from rice rhizosphere of Rangareddy district
Screening of Pseudomonas fluorescens isolates for Phosphate
solubilisation in vitro

4.6

of

Pseudomonas

4.7

Screening of Pseudomonas fluorescens isolates for Plant Growth

Promoting Activities in vitro

4.8

Antagonistic activity of Pseudomonas fluorescens against Rhizoctonia

Solani by dual culture method

4.9

Percent germination and Vigour index of Pseudomonas fluorescens

strains treated seeds

4.10

Influence of Pseudomonas fluorescens isolates on plant height of rice

4.11

Influence of Pseudomonas fluorescens isolates on number of tiller in

rice

4.12

Influence of Pseudomonas fluorescens isolates on Panicle number and

Leaf Area Index (LAI) of rice

4.13

Influence of Pseudomonas fluorescens isolates on Chlorophyll content

of rice

4.14

Score (0-9 scale) of sheath blight and grain yield per plant (gms)

4.15

Percent Disease Index (PDI) of rice Inoculated with Rhizoctonia

Solani

4.16

Reduction in disease severity of rice infected with Rhizoctonia Solani

at different intervals

LIST OF ILLUSTRATIONS

Illustration

Page

Title

No.

No.

4.1

Microbial population count of Pseudomonas in the rice

rhizospheric soils of Rangareddy district

4.2

Screening of Pseudomonas fluorescens isolates for Phosphate

solubilization

4.3

Screening of
production

4.4

Antagonistic activity of Pseudomonas fluorescens isolates against

Rhizoctonia solani

4.5

Plant height (cm) of rice at 30, 60 and 90 DAT as influenced by

Pseudomonas fluorescens

4.6

Tiller number of rice at 30, 60 and 90 DAT as influenced by

Pseudomonas fluorescens

4.7

Panicle number of rice at 90 DAT as influenced by Pseudomonas

fluorescens

4.8

Leaf area index of rice at 90 DAT as influenced by Pseudomonas

fluorescens

4.9

Chlorophyll content of rice at 90 DAT as influenced by

Pseudomonas fluorescens

4.10

Grain yield per plant (gms)

4.11

Percent Disease Index of rice at 60,70 and 80 DAT

4.12

Reduction in Disease Severity of rice at 60, 70 and 80 DAT

Pseudomonas fluorescens

isolates for

IAA

ACKNOWLEDGEMENT

He that trusteth in the Lord, mercy shall compass him about.

.I will redeem thee out of the hand of the terrible

Psalms 32:10
Jeremiah 15:21

First and foremost, I offer my obeisance to the Lord God Almighty for his boundless
blessing, which accompanied me in all the endeavors.
I am pleased to place my profound etiquette to Dr. A. Vijaya Gopal, Assistant Professor and
Head, Department of Agricultural Microbiology and Bioenergy, College of Agriculture,
Rajendranagar, Hyderabad and esteemed chairman of my Advisory Committee for his learned
counsel, unstinted attention, arduous and meticulous guidance on the work in all stages. His keen
interest, patient hearing and constructive criticism have installed in me the spirit of confidence to
successfully complete the task.
I deem it my privilege in expressing my fidelity to Dr. R. Subhash Reddy, Associate Dean
and University Head, Department of Agricultural Microbiology and Bioenergy, College of
Agriculture, Rajendranagar and the member of my advisory committee for the continuous support of
my M. Sc study and research, for his patience, motivation, enthusiasm and immense knowledge.
I humbly extend my profound gratitude to the member of my advisory committee Dr. M.
Shiva Shankar, Professor, Department of Agronomy, College of Agriculture, Rajendranagar,
Hyderabad for his constant support and valuable suggestions offered during the course of research
work.
I avail this opportunity with humbleness to sincerely thank to Dr. S. Triveni, Assistant
Professor, Department of Agricultural Microbiology and Bioenergy, College of Agriculture,
Rajendranagar for her affection, well versed counsel and pragmatic suggestions during the course of
research.
I cordially offer my indebted remarks to Mrs. Sridevi (Agricultural Officer), Mrs. Neeraja,
Ms. Sharanya ,Ms. Aparna, Mr. Jaffer, Mr. Venkataiah and workers Salamma, Santhosha,
Jayamma and Mohammed Department of Agricultural Microbiology and Bioenergy, College of
Agriculture, Rajendranagar, Hyderabad for their constant encouragement and valuable suggestions,
timely help and co-operation during the course of the study.

Words are not enough to express my whole-hearted and affectionate gratitude to my beloved
parents Mr. B.N. Kantaiah and Mrs. B.N. Laxmikantha, and my loving sister Usha ,brother-in-law
Sunil and son-in-law Shreyanck for their unbounding love, unparallel affection and unstinted
encouragement throughout my educational career and without whose invaluable moral support, the
thesis would not have seen the light of the day.
I equally owe my heartfelt thanks to ever loving Brother D.Venkateshwar Rao and sister-inlaw D.Latha and D.Amrutha who also have a hand in framing this beautiful future of mine.
I am greatly beholden and owe a deep sense of honor to my brother Ramarao and uncles
families especially Basavaiah, Peraiah, Narayana and Ramaiah for their companionship in my
personal and professional life co-operation, unparalleled affection, moral support and persistant
encouragement.
I am indebted to my seniors Damodara chari, Nissi Paul, Kavya reddy , Sushma devi and
Himabindu my collegue Arun, Bagwan, Gouri, Manasa, Aneesh and Gireesh my juniors Prasanna,
Vinod, Nagaraju, Thirumal and Bhavya for their support and cooperation during the course of
study.
I cherish the loving moments, great help and sweet sacrifices of my friends, Laxman, Sai,
Shiva, Sathish, Sai santhosh, Sunjeev, Thirupam, Rakesh, Siddu, Bhaskar, Shiva, B.Vinay,
G.Vinay, Srinivas, Rama Krishna ,Mahesh, Govardhan, Jeevula naik, Hari, Jawahar lal, Sai,
Kalyan, Chouhan, Rathod, Harish and my seniors Thirupathi, Ravi, Santhosh, Venu, Rambabu,
Ramprasad for providing a stimulating and fun environment in which to learn and grow.
I am thankful to PJTSAU for providing financial assistance in the form of fellowship
during my course of study.
My greatest regards to the Almighty for bestowing upon me the courage to face the
complexities of life and complete this project successfully.
Finally, I wish my humble thanks to one and all who have directly or indirectly
contributed to the conduct of the study.
Place:
Date:

( Suman Biyyani)

LIST OF SYMBOLS AND ABBREVIATIONS

Cm

Centimetre

&

And

Metre

Gram

Kg

Kilogram

Tonn

Ha

Hectare

kg ha -1

Kilogram per hectare

t ha -1

Tonn per hectare

Kmph

Kilometre per hour

dS m-1

Decisiemen per metre

At the rate of

Sem

Standard error of mean

CD(P=0.05)

Critical difference at 5 percent level

DAT

Days after transplanting

et al.

And others

Hours

Per cent

NS

Non significant

Degree Celsius

Nitrogen

P2O5

Phosphorus

K2O

Potassium

OC

Organic carbon

EC

Electrical conductivity

viz.,

Namely

Degrees

Minutes

Seconds

SSP

Single super phosphate

MOP

Muriate of potash

Max

Maximum

Min

Minimum

day -1

Per day

Mm

Millimetre

RDF

Recommended dose of fertilizers

FYM

Farmyard manure

Fig.

Figure

hour-1

Per hour

Ppm

Parts per million

ml lit -1

Millilitre per litre

cm 2

Square centimetre

Nitrate

RDF

Recommended dose of fertilizer

No.

Number

vis--vis

In relation to

PGPM

Plant growth promoting microbes

CFU

Colony forming unit

Spp.

Species

PSB

Phosphate solubilizing bacteria

PSBF

Phosphate solubilizing biofertilizers

PSM

Phosphate solubilizing microbes

PGPR

Plant growth promoting rhizobacteria

PDA

Potato dextrose agar

KB

KingB medium

CRD

Completely Randomized Design

R1

Replication 1

R2

Replication 2

R3

Replication 3

NA

Nutrient agar

SPAD

Soil plant analytical development

Mg

Milligram

IAA

Indole acetic acid

NO3

Author
Title of the thesis

:
:

BIYYANI SUMAN
ISOLATION

AND

CHARACTERIZATION

OF

Pseudomonas fluorescens FROM RICE FIELDS FOR THE

BIOLOGICAL

CONTROL

OF

Rhizoctonia

solani

CAUSING SHEATH BLIGHT IN RICE

Degree

MASTER OF SCIENCE IN AGRICULTURE

Faculty

AGRICULTURE

Discipline

AGRICULTURAL MICROBIOLOGY

Major Advisor

Dr.A.VIJAYA GOPAL

University

PROFESSOR JAYASHANKAR TELANGANA STATE

AGRICULTURAL UNIVERSITY

Year of submission

2015

ABSTRACT
Sheath blight is a devastating disease caused by Rhizoctonia solani Kuhn in rice
crop. Biocontrol agents have great demand now-a-days as they are replacing chemical
pesticides to a large extent as they are cost effective, ecofriendly and easily available.
Pseudomonas fluorescens is one among them which not only enhances the plant growth but
also controls the fungal pathogens by production of anti fungal metabolites. Present
investigation is focussed towards isolation of fluorescent Pseudomonas from rice
rhizosphere and in vitro, in vivo evaluation of antagonistic activity of these isolated
fluorescent Pseudomonas against Rhizoctonia solani causing sheath blight in rice crop.
Thirty Pseudomonas fluorescens isolates were isolated from rice rhizospheric soils
of twenty two villages of Parigi and Doma mandals of Rangareddy district, Telangana as
the district has severe sheath blight incidence. Latitude, longitude, elevation, GPS numbers
of the place of collection of soil samples are recorded and point map has been generated by
using Arc GIS software and rectification of the toposheets was done by Erdas image 9.3
software. The isolates are purified by observing under UV light and they were culturally,
morphologically and biochemically identified as Pseudomonas fluorescens according to
Bergeys manual of Determinative Bacteriology. Biochemical characterization revealed
that all the isolates were positive for catalase, oxidase, citrate utilization, gelatine
liquefaction, denitrification and negative for indole tests. These isolates were screened in
vitro for Plant growth promoting attributes like phosphate solubilizatin, siderophore, IAA,
ammonia and HCN production. Results revealed that 76.67% isolates solubilized
phosphorous and produced siderophores. 93.33% isolates produced IAA and all the thirty
isolates i.e., 100% produced ammonia and HCN.
All the isolates were further screened in vitro for antagonistic activity against the
fungal pathogen Rhizoctonia solani causing sheath blight and found that all isolates

inhibited the fungal pathogen and highest inhibition was found with the organism DMP1
(53.43%).
Pot culture experiment was conducted for in vivo evaluation of Pseudomonas
fluorescens against the challenge inoculated rice crop by using various methods of
inoculation. Results revealed that seed treatment method of application of Pseudomonas
fluorescens with the isolate DMP1 effectively controlled and reduced the disease severity
of 42.56% at 60 DAT, 41.12% at 70 DAT and 40.81% at 80 DAT caused by the
Rhizoctonia solani compared with the other methods of inoculation. Seed treated plants
especially with the organism DMP1 not only controlled the disease but improved the
growth parameters like plant height (56 cm, 74 cm and 82.6 cm at 30,60 and 90 DAT
respectively), Leaf area index (4.71 at 60 DAT), chlorophyll content (39.60 and 48.17 at 30
and 60 DAT ) and yield parameters like number of tillers (13.7, 17.3 and 17.3 at 30,60 and
90 DAT), panicles (13.7 at 90 DAT) and grain yield per plant (32.69 gms) followed by root
dipping and foliar method of application of biocontrol agents. When the methods of
inoculation are compared seed treatment method improved the growth and yield parameters
in the challenge inoculated rice crop. When the challenge inoculated plants are scored for
the disease, it was observed that the seed treated method of inoculation to the rice showed
resistance towards the disease with a score of 1.2.

Chapter I

INTRODUCTION
Rice (Oryza sativa) is India's pre-eminent crop, it is the most widely consumed staple
food for a large part of the world's human population, especially in Asia. India is one of the
world's largest producers of rice, accounting for 20% of all world rice production.
Moreover, this country has the biggest area under rice cultivation, as it is one of the
principal food crops. India is one of the leading producers of this crop, with a production,
productivity of 160.90 million tons and 2424 kg per hectare respectively (Indiastat, 201314). Rice is the staple food of the people of the eastern and southern parts of the country.
Rice is the most important grain with regard to human nutrition and caloric intake,
providing more than one fifth of the calories consumed world wide by humans.
Sheath blight of rice caused by Rhizoctonia solani Kuhn is a serious threat in
rice growing areas. A modest estimation of losses due to sheath blight disease alone in
India has been up to 54.3% (Rajan, 1987; Roy, 1993). Both local and high yielding
varieties are susceptible to this disease (Naidu, 1989). The pathogen attacks the rice plant at
maximum tillering stage by sclerotium, the primary source of inoculum that over winter in
soil and plant debris (Ou, 1985).
Sheath blight caused by a soilborne fungus, is considered to be the most economically
significant fungal rice disease in the world (Groth et al. 1988, Cu et al. 1996). Yield loss
can occur at any stage but is higher when infection occurs at panicle initiation, booting and
flowering (Sharma et al. 1990, Cu et al. 1996). Sheath blight infection from panicle
initiation to flowering resulted in yield loss by reducing the mean grain weight and the
number of filled grains (Cu et al. 1996). Sheath blight also interferes with grain filling
(Marchetti, 1983) and can reduce rice yield by 39%, but that loss can increase to 50% in
terms of kg/ha of milled whole grain rice because grains can be weakened and subsequently
break during milling. A possible 46% yield loss in milled rice has been estimated if sheath
blight lesions reach 90% of the plant height (Ahn and Mew, 1986).
High levels of nitrogenous fertilizers, double cropping, high plant densities and
growing of early maturing, short-stature, high tillering and susceptible cultivars have

intensified the severity of this disease in most rice-growing regions in the world (Lee and
Rush, 1983). The disease is alarming due to its intensive cultivation of modern high
yielding varieties with high doses of nitrogenous fertilizers. Crop with a high plant density
and close canopy associated favors disease build up from panicle initiation onwards. Poor
weed management practices and increase in frequency of irrigation have aggravated,
incidence of the disease due to modified micro climatic conditions (Srinivas et al. 2013).
Sustainable agriculture is vital in todays world as it offers the potential to meet our
agricultural needs, something that conventional agriculture fails to do. Microbial
populations are responsible for instrumental to fundamental processes that drive stability
and productivity of agro-ecosystems. The contributions of Plant Growth Promoting
Rhizobacteria (PGPR) were emphasized clearly for safe and sustainable agriculture
development (Singh et al. 2011).
The use of chemical fertilizers and pesticides caused an incredible harm to the
environment. These agents are both hazardous and may persist and accumulate in natural
ecosystems an answer to this problem is replacing chemicals with biological approaches,
which are considered more environment friendly in the long term. One of the emerging
research area for the control of different phytopathogenic agents is the use of biocontrol
plant growth promoting rhizobacteria (PGPR), which are capable of suppressing or
preventing the phytopathogen damage (Nihorembere et al. 2011).
Beneficial rhizobacteria that stimulate plant growth are usually referred to as plant
growth promoting rhizobacteria or PGPR. PGPR are a heterogeneous group of bacteria that
can be found in the rhizosphere, at root surfaces and in association with roots. They can
improve the extent or quality of plant growth by direct and/or indirect methods. In last few
decades, a large array of bacteria including species of Pseudomonas, Azospirillum,
Azotobacter, Klebsiella, Enterobacter, Alcaligens, Arthobacter, Burkholderia, Bacillus and
Serratia have been isolated and reported to enhance plant growth. (Glick, 1995; Okon and
Labanderagonzalez, 1994). These beneficial bacteria produces secondary metabolites which
includes antibiotics, extracellular enzymes, hydrogen cyanide (HCN), siderophores, and
phytohormones ( Hayat et al. 2010).

Pseudomonas fluorescens bacteria, a major constituent of Rhizobacteria, encourage

the plant growth through their diverse mechanisms (Noori and Saud, 2012). Pseudomonas
fluorescens encompasses a group of common, nonpathogenic saprophytes that colonize
soil, water and plant surface environments. It is a common gram negative, rod-shaped
bacterium. As its name implies, it secretes a soluble greenish fluorescent pigment called
fluorescein, particularly under conditions of low iron availability. It is an obligate aerobe,
except for some strains that can utilize NO3 as an electron acceptor in place of O2. It is
motile by means of multiple polar flagella. Pseudomonas fluorescens has simple nutritional
requirements and grows well in mineral salts media supplemented with any of a large
number of carbon sources (Palleroni, 1984).
Several strains of Pseudomonas fluorescens have been successfully used for the
biological control of rice sheath blight (Mew and Rosales, 1986; Rabindran and
Vidhyasekaran, 1996; Vidhyasekaran and Muthamilan, 1999). Mechanisms of biological
control of plant pathogens by fluorescent pseudomonads generally involve competition for
nutrients, production of bacterial metabolites such as iron chelating siderophores, hydrogen
cyanide (HCN), antibiotics, extracellular lytic enzymes and induced systemic resistance
(OSullivan and OGara, 1992; Van Loon et al. 1998).
Fluorescent pseudomonads representing group of PGPR can promote growth and
suppress plant pathogens by multiple mechanisms. Their applicability as biocontrol agents
has drawn wide attention because of production of secondary metabolites such as
siderophores, antibiotics, volatile compounds, HCN, enzymes and phytohormones (Gupta
et al. 2001). They can be utilized in low input sustainable agricultural applications, such as
biocontrol, on account of their ability to synthesize secondary metabolites with antibiotic
properties and many of such antibiotics produced have a broad spectrum activity but strain
to strain variations do exist (Raaijimakers et al. 2002). These secondary metabolites include
2, 4-diacetylphloroglucinol (DAPG), phenazine (Phz), pyrrolnitrin, oomycin A,
viscosinamide, pyoluteorin and hydrogen cyanide (HCN).
In the present study keeping in view the importance of Pseudomonas fluorescens as
biocontrol agents they have been successfully isolated and used as seed treatment, foliar
spray and root dipping for the biological control of Sheath blight of rice caused by
Rhizoctonia solani Kuhn. Present work preceded with the following objectives

Objectives of investigation:
1. To isolate and enumerate Pseudomonas fluorescens from different rice fields
2. To characterize Pseudomonas fluorescens isolates for their morphological, cultural and
biochemical properties
3. To assess antagonism of Pseudomonas fluorescens for the biocontrol of Rhizoctonia
solani
4. Green house/pot culture study for the control of sheath blight

Chapter II

REVIEW OF LITERATURE
The present investigation was carried out to characterize 30 Plant growth
promoting rhizobacterial isolates from Parigi and Doma mandals of Rangareddy district
of Telangana along with the GPS readings and studied morphological, biochemical
characters and their biocontrol activity against sheath blight in rice crop. The literature
pertaining to these aspects is reviewed and presented below under the appropriate heads,
which will provide an overview of the current status of the research work on these
aspects.
2.1

Isolation of Pseudomonas fluorescence from rice rhizosphere

2.2

Biochemical characterization of Pseudomonas fluorescence

2.3

Screening of pure isolates for PGPR properties

2.4

To find efficient PGPR isolates for bio control activity

2.5

Green house/Pot culture study

2.1 ISOLATION OF Pseudomonas fluorescence FROM RICE

RHIZOSPHERE
Microorganisms are generally found in nature as mixed populations. To study
the specific role played by a specific microorganism in the environment, it should be
isolated as pure culture. Pure culture not only involves isolation but also maintenance in
the artificial medium.
Aarab et al. (2015) isolated three hundred and five bacteria from the rhizosphere
of rice fields in Northwestern Morocco, of which one hundred and thirty six
rhizobacteria were tricalcium phosphate solubilizers. Analysis of 16S rDNA partial
sequencing demonstrated that these nine bacteria belong to three genera: Aeromonas,
Pseudomonas and Enterobacter.
Sarathambal et al. (2015) isolated diazotrophs from rhizosphere of semi-arid
tropical grasses with their latitude and longitude readings and evaluated their
inoculation effects on the growth of rice plants under in vitro and in vivo conditions.
RameshKumar et al. (2014) isolated and analyzed the genetic diversity of
fluorescent pseudomonads associated with the rice rhizosphere. Based on 16S rDNA
sequence similarity the isolates were designated as Pseudomonas plecoglossicida (11),
P.monteilii (3), P.mosselii (3), P.libaniensis (4), and P.aeruginosa (4).
Ahmed et al. (2013) isolated eighteen bacterial strains from roots and

rhizosphere of rice. Three bacterial strains (Azospirillum brasilense strain R1,

Azospirillum lipoferum strain RSWT1 and Pseudomonas strain Ky1) were used to
inoculate rice variety JP 5 at two experimental sites in Swat (Agriculture Research
Institute (N) Mingora and Udigram).
Shivalingaiah and Umesha (2013) isolated ten strains of flurosecent
Pseudomonads from the rhizosphere soils of rice growing areas in Karnataka, India. All
the ten tested isolates of Pseudomonads were positive for the production of
siderophores, hydrogen cyanide, indole acetic acid, chitinase, -1,3-glucanase, cellulase,
salicylic acid production, and phosphate solubilization.
Manjunatha et al. (2012) isolated 92 fluorescent pseudomonads from the
rhizosphere soil of paddy using specific medium. All the isolates showed Gramnegative reaction and were rod shaped, 66 isolates produced pigment in PAF medium
and showed fluorescence under UV light.
Lawongsa et al. (2012) isolated Pseudomonas fluorescens R21 from rhizosphere
of rice in Thailand, Pseudomonas fluorescens F113 from rhizosphere of sugar beet in
Ireland and studied the in vitro antagonistic activity against phytopathogens.
Sharma et al. (2012) isolated, screened and characterized the PGPR from the
rhizosphere soil of rice fields from different areas of Kashipur region in Uttarakhand,
India. Ten isolates of bacteria, designated as PGB1, PGB2, PGB3, PGB4, PGB5, PGB6,
PGB7, PGB8, PGB9 and PGB10 were successfully isolated and characterized.
Twenty eight bacterial cultures were isolated by Preeti et al. (2011) from Korea
and they were characterized by various microscopic and cultural examinations. Out of
which four isolates were identified as Pseudomonas spp. and others were Bacillus
subtilis.
Mishra et al. (2010) isolated two PGPR isolates from the rhizosphere soil of
Pyrethrum (Chrysanthemum cineraefolium) designated as MA-2 and MA-4 and
identified as Bacillus subtilis and Pseudomonas fluorescens and reported that they
increased the productivity of Pelargonium graveolens.
Saravanakumar et al. (2009) isolated PGPR from different agroecosystems of
Tamil Nadu, India, and were tested for their efficacy against the sheath rot pathogen
Sarocladium oryzae under in vitro, glasshouse and field conditions. The results revealed
the significant performance by strains Pf1, TDK1 and PY15 compared to other strains.
Further, the combination of Pseudomonas strains Pf1, TDK1 and PY15 was more
effective in reducing sheath rot disease in rice plants compared to individual strains.

2.2 BIOCHEMICAL CHARACTERIZATION OF Pseudomonas

fluorescence
Anitha and Kumudini (2014) isolated Pseudomonas from fifteen rhizospheric
samples from the field's of rice, chilly, ragi, beans and garden soils from different
regions of India. They characterized morphologically and biochemically and concluded
as genus Pseudomonas.
Hongsheng et al. (2014) isolated a novel phenol-degrading bacterium SKDP-1
from crude oil contaminated soil, Guano oil field in the northeast Shandong Dongying,
East China. The biochemical tests indicated that strain SKDP-1 was Gram-negative and
utilized glucose, citrate and starch not gelatine. Both Voges-Proskauer and H2O2 tests
were positive and the SKDP-1 strain identified as Pseudomonas.
Kumar et al. (2014) isolated and characterized PGPR from the rhizospheric soil
of wheat from different areas of Uttar Pradesh, India. Twenty isolates were isolated,
biochemically characterized and screened for plant growth promoting traits like IAA
production, ammonia production, siderophore production and phosphate solubilization.
Out of twenty isolates twenty, six and ten produced ammonia, siderophore and
solubilized phosphate on Pikovskayas agar medium.
Ahad et al. (2014) investigated nine different strains for biochemical
characterization and were tested for their PGPR properties. 16 S rDNA amplification
was done and restriction profiling was done using two endonuclease i.e., msp1 and taq1.
Depending upon banding pattern of all the nine strains dendrogram was created using
NTsys software. A clear cut difference was seen in genetic diversity among the strains
Pseudomonas was found to be the most effective strain among all.
Fifty Pseudomonas fluorescens and 28 Rhizobium strains were isolated from
rhizospheric soil and root nodules of pigeonpea, biochemically characterized and tested
for their tolerance to fungicides, insecticides and heavy metals under in vitro conditions.
They characterized agrochemical and heavy metal compatible P. fluorescens and
Rhizobium strains which can be combined with biocontrol agents as a seed treatment or
soil application for the management of soil or seed borne diseases. (Basha et al. 2014).
One hundred and forty strains of Pseudomonas were isolated from potato
rhizosphere by Deshwal et al. (2013). Strains were isolated on Kings B medium and
Cetrimide agar medium and various biochemical tests were done. Out of one hundred
and forty isolated Pseudomonas strains, thirty seven strains were identified as P.
fluorescens.
Kumari et al. (2013) isolated forty two rhizobacterial isolates and the isolates

were characterized biochemically and found belonging to genera Pseudomonas (19),

Bacillus (22) and Serratia (1).
Paramageetham and Babu (2013) isolated fluorescent pseudomonads from forest
litter of Seshachalam hills the first ever biodiversity reserve. Among the 34 isolates four
isolates were tentatively identified as Pseudomonas fluorescens based on morphological
and biochemical characters.
Biochemical characteristics of fluorescent Pseudomonas showed that all ten
isolates were positive to catalase, amylase, gelatinase and siderophore production,
while three isolates (Pf5, Pf6 and Pf9) were oxidase positive, nine isolates (Pf1, Pf2,
Pf3, Pf4, Pf6, Pf7, Pf8, Pf9, and Pf10) were tolerant to 6.5% NaCl. (Saravanan et al.
2013).
Thirty five (Pf1 to Pf35) isolates of Pseudomonas fluorescens were isolated
from the rizosphere of rice fields by Meera and Balabaskar (2012). Among these, seven
(Pf4, Pf6, Pf8, Pf11, Pf13, Pf15 and Pf20) isolates showed bright fluorescence under
UV light which were further tested. All the cultural and biochemical studies confirmed
them to be P. fluorescens. The isolates also showed positive response for siderophore
production and plant growth promoting activity on rice cultivar ADT 36.
Showkat et al. (2012) collected rhizosphere soil from different wheat growing
regions of Kashmir valley which were evaluated for presence of Pseudomonas
fluorescens using Kings B medium. Based on colony morphology, siderophore
production and biochemical tests, out of 136 rhizosphere soil samples, only 52 isolates
were identified as Pseudomonas fluorescens.
Supraja et al. (2011) isolated fifteen bacterial isolates from rhizospheric soils of
redgram and maize crops in the Rangareddy district. These test isolates were
biochemically characterized and identified as fluorescent Pseudomonads.
Negi et al. (2011) collected six hundred native cold-tolerant rhizospheric
bacterial isolates from Uttarakhand Himalayas, of which three hundred and thirty six
were confirmed as fluorescent Pseudomonas spp. On the basis of specific biochemical
tests, these were characterized into three major groups: P. fluorescens (308), P.
aeruginosa (20), and P. putida (8).
Nathan et al. (2011) isolated Pseudomonas spp., a potent PGPR in the
rhizosphere. Through appropriate microbiological and biochemical methods, the study
demonstrated the presence of fluorescent and non-fluorescent Pseudomonads in the
rhizosphere of chemical intensive rice growing environments. Augmentation of such
PGPR including, Pseudomonads in the rice ecosystems will ensure a healthy micro

climate for rice.

Wasi et al.(2010) isolated strain SM1 from soil contaminated with domestic and
industrial wastes of Aligarh city was characterized on the basis of morphological,
cultural and biochemical properties and presumptively identified as Pseudomonas
fluorescens.
A proteobacterium was isolated from rhizosphere soil by Rekha et al. (2010) and
it was identified using morphological, cultural and biochemical characteristics as
Pseudomonas fluoresecens.
Megha et al. (2007) isolated 52 fluorescent pseudomonads from soils of
different forests in Western Ghats of Uttara Kannada District, Karnataka. 37 isolates
were identified upto species level based on biochemical and physiological characters as
Pseudomonas fluorescens.

2.3 INVITRO SCREENING OF PURE ISOLATES FOR PGPR

PROPERTIES
Plant growth promoting rhizobacteria (PGPR) are commonly used as inoculants
for improving the growth and yield of agricultural crops, however screening for the
selection of effective PGPR strains is very critical. This study focusses on the screening
of effective PGPR strains on the basis of their potential for in vitro Phosphate
solubilisation, IAA production and plant growth promoting activity under gnotobiotic
conditions.
2.3.1 Mineral Phosphate Solubilization
Phosphate solubilizing bacteria (PSB) were isolated, identified and characterized
by Baliah and Begum (2015). Further, biology of the selected strains was also studied.
The study revealed that the population level of PSB was higher in the rhizosphere soils
of cluster bean. The selected strains were identified as Bacillus and Pseudomonas spp.
Thirty one Pseudomonas sp. were isolated from rhizospheric soil of rice. Out of
thirty one, nine isolates were able to solubilize Ca3(PO4)2 on Pikovskyas agar medium.
Isolate MGR38 was identified as P. fluorescens which showed 83.33% efficiency in
phosphate solubilization and the other isolates MGR31, MGR37, MGR39,MGR40,
MGR41, MGR42, MGR43 and MGR44 showed 33.33, 57.14, 50, 50, 16.16, 40, 60 and
80 % efficiency of phosphate solubilization respectively. (Roy et al. 2013)
Alemu (2013) isolated twelve Pseudomonas fluorescence species from
rhizospheric soil of faba bean and tested for phosphate solubilization. All tested isolates
of Pseudomonas fluorescence species had a potential of phosphate solubilization on

Pikovskayas media. He summarized that all Pseudomonas fluorescens spp. can be used
as bio-fertilizers for soil fertility improvement.
Twenty six Pseudomonas spp. were isolated and identified by Prasad et al.
(2013) and were in vitro screened for PGPR properties like Phosphate solubilization,
siderophore, IAA, HCN productions, antagonistic activity against Rhizoctonia solani,
Sclerotium rolfsii. The results revealed that all Pseudomonas isolates were positive for
IAA production, 76.9% for phosphate solubilization, 92.3% for ammonia, 88.46% for
siderophores, 80.76% for HCN productions. Eight isolates showed inhibition potential
against Rhizoctonia solani and Sclerotium rolfsii.
Belkar and Gade (2012) isolated fifteen isolates of Pseudomonas fluorescens
from the acidic and alkaline rhizospheric soils of different field crops of Vidarbha and
Konkan region. All the cultural and biochemical studies confirmed them to be P.
fluorescens. The isolates showed positive response for siderophore production and
phosphate solubilization, while negative for IAA and HCN production.
Pseudomonas fluorescens K - 34 solubilized tricalcium phosphate and produced
substantial amount of soluble phosphorus (968.5 mg / l) in Pikovskaya s (PVK) broth
as compared to others and exhibited the production of indole acetic acid (IAA),
siderophore, cell wall degrading enzyme activities and growth inhibition against fungal
and bacterial pathogens. (Parani and Saha, 2012).
Panhwar et al. (2012) isolated phosphate-solubilizing bacteria (PSB) from
aerobic rice grown in Penang Malaysia and determined the biochemical properties of
the isolates such as, organic acids, enzymes, indoleacetic acid (IAA), siderophore
production and its antagonistic effect against pathogen Rhizoctonia solani. The highest
P solubilizing activity (69.58%) was found in PSB9 strain grown in NBRIP plate.
Singh et al .(2012) isolated a total of 93 isolates .Out of 93 isolates 30 isolates
were selected for their evaluation of phosphate solubilizing potential on pikovskayas
media. Out of 30 isolates five isolates showed positive result on pikovskayas medium
producing clear zones.
Ramezanpour et al. (2011) isolated bacterial strains from the rhizosphere of
paddy fields in three Northern Provinces (Mazandaran, Gollestan and Guillan) of Iran.
The plant growth promoting properties (indoleacetic acid production, phosphate
solubilization and siderophore production) and genetic diversity of isolated
Pseudomonas strains were examined. Isolated strains showed high ability of IAA
production, phosphate solubilization and siderophore production. Nevertheless, the

strains were distributed into 11 genotypes, including five groups of fluorescent

Pseudomonads.
Vyas and Gulati (2009) isolated nineteen phosphate-solubilizing fluorescent
Pseudomonas strains of P. fluorescens, P. poae, P. trivialis and Pseudomonas spp.
produced gluconic acid, oxalic acid, 2-ketogluconic acid, lactic acid, succinic acid,
formic acid, citric acid and malic acid in the culture filtrates during the solubilization of
Tricalcium phosphate, Mussoorie rock phosphate, Udaipur rock phosphate and North
Carolina rock phosphate. The strains differed quantitatively and qualitatively in the
production of organic acids during solubilization of phosphate substrates.

2.3.2 Production of Plant Growth Promoting Substances (PGPS)

Bumunang and Babalola (2014) screened species of Pseudomonas, Aeromonas,
Sphingomonas, Burkholderia, Stenotrophomonas, Achromobacter, Ewingella and
Bacillus in vitro for plant growth promoting traits such as ammonia production, catalase
activity, indole acetic acid production, Phosphate solubilisation and antifungal activity.
All the thirty two rhizobacterial strains tested in this study were positive for catalase
activity, ammonia production and IAA production; 90.6% were positive for phosphate
solubilization, 34.3% indicated antifungal activity.
Seventy eight strains of P. fluorescens isolated from the rhizosphere of Banana
by Ratnakar (2014). Eighteen strains were selected for their plant growth promoting
activity. They were tested for in vitro plant growth promotion activities such as
phosphate solubilisation, siderophore production, IAA production and fungal cell wall
degrading enzyme production. All the strains were positive for siderophore and IAA
production. Ten strains were positive for phosphate solubilisation. Six strains possess
the ability to produce chitinase, cellulase and protease.
Nehra et al. (2014) isolated PGPR strains from the rhizospheric soil of the
cotton plant from various places of Haryana, India. Isolate exhibiting maximum PGP
traits was identified as Pseudomonas fluorescens based on the biochemical
characterization, phenotypic microarray analysis and 16S rDNA sequencing. The isolate
was screened in vitro for their plant growth promoting traits like production of
indoleacetic acid (IAA), ammonia production, hydrogen cyanide (HCN) production,
siderophore production, phosphate solubilization. The isolate exhibited positive results
for ammonia and siderophore production and phosphate solubilization.
Singh et al. (2013) isolated Pseudomonas spp. from nine different rhizospheric
soils of wheat & pigeon pea. Among the 21 Pseudomonas spp. four different

Pseudomonas spp. isolates (YSY-13, YSY-15, YSY-17 and YSY-19) exhibited

maximum plant growth promoting and heavy metal tolerant activities.
Souza et al. (2013) isolated three hundred and thirty six bacterial strains
belonging to the genera Agrobacterium, Burkholderia, Enterobacter, and Pseudomonas
spp. Siderophore and indolic compound producers were widely found among isolates,
but one hundred and one isolates were able to solubilize phosphate.
Shruti et al. (2013) isolated Fluorescent Pseudomonas strains were from the soil
samples collected from Bhojia Institute of Life Sciences, Budh, Baddi. The
Pseudomonas sp. strain showed positive effect by increasing both roots and shoots
length of Rice plant as compared with control. Both dry shoots and dry root weight of
plants inoculated by the Pseudomonas spp. strain exhibited positive result as compared
with control. Similarly, both wet shoots and dry shoot weight of plants inoculated by
Pseudomonas spp. strain also showed the positive results as compared with control.
Pseudomonas fluorescens and Bacillus subtilis were isolated and designated as
PF -1 to PF 10 and BS - 1 to BS 10 by Sivasakthi et al., (2013). The efficiency of P.
fluorescens and B. subtilis for IAA production, Gibberellic acid production, Siderophore
production and Phosphate solubilization was estimated. The maximum Indole acetic
acid (IAA) and Gibberellic acid (GA) production by P. fluorescens was recorded by the
isolate PF - 8 and the minimum production was observed in PF - 4 isolate. The IAA
production, Gibberellic acid production and siderophore production by B. subtilis was
low when compared to P. fluorescens. The maximum phosphate solubilization was
recorded by the isolate BS 8.
Selected strains PW-99, PW-136, PW-2, PW-56, PW-18, PW-43, PW-5 and
PW-104 effectively showed four major PGPR activities such as Indole Acetic Acid
(IAA), Hydrogen cyanide (HCN), Siderophore and Phosphorous solubilisation. In
present study, nine treatments were prepared. Each seed coated with 108 cfu of
Pseudomonas culture. Results suggested that all Pseudomonas strains enhanced plant
growth in rice plant. P. fluorescens PW-5 produced maximum shoot, root and dry
weight by 157.72, 408.06 and 233.84% respectively as compared to control. (Deshwal
and Kumar, 2013).
Fifty one Pseudomonas spp. were isolated from the rhizospheric soil samples of
different crops. They were screened initially on the basis of their antagonistic activity,
and fifteen Pseudomonas fluorescens designated FP1-FP15 were selected. These
isolates were then tested in vitro for specific PGPR traits. Of the 15 isolates, FP6 was
found to be promising for all PGPR attributes. (Bakthavatchalu et al. 2012).

Fourteen isolates identified as a non-pathogenic Pseudomonas sp that produced

IAA and promoted enhancement of root length, shoot length, or number of lateral root
by Wahyudi et al. (2011). Among those 14 isolates, 8 isolates showed phosphate
solubilizing activity, 12 isolates capable of producing siderophore and six isolates were
observed to have chitinolytic activity.
Ten strains of fluorescent Pseudomonads were screened for their plant growth
promoting activity based on their ability to produce hydrogen cyanide (HCN),
siderophores, proteases, indole acetic acid (IAA), broad spectrum antifungal activity
against pathogenic fungi and phosphate solubilization. The results indicated that most of
the isolates tested possess plant growth promoting traits. These isolates can be used as
potential biofertilizers and also as biocontrol agents. (Suresh et al. 2010)
Ashrafuzzaman et al. (2009) isolated ten isolates of bacteria and were
successfully characterized. Isolates PGB4, PGT1, PGT2, PGT3, PGG1 and PGG2
induced the production of indole acetic acid (IAA), whereas only PGT3 isolate
was able to solubilize phosphorus. Among the ten isolates, PGB4 and PGG2
were found almost equally better in all aspects such as dry matter production,
plant height and root length of rice, and IAA production. Isolate PGT3 was also
found to be promising in IAA production having an additional property of
phosphate solubilization.

2.4. TO FIND EFFICIENT PGPR ISOLATES FOR BIO CONTROL

ACTIVITY
2.4.1 Production of siderophores
Siderophore is an iron binding ligand and an uptake protein, needed to transport
iron into the cell. It has been suggested that the ability to produce specific siderophore
and to utilize a broad spectrum of siderophore, may contribute to the root colonizing
ability of different rhizobacteria.
Marathe et al. (2015) isolated two siderophore producing bacterial strains from
waste water sample and labeled them as RM1 and RM2. Among these two isolates,
RM1 strain was found to produce more siderophore. 16s rRNA analysis and
biochemical characterization, identified the RM1 strain as Pseudomonas aeruginosa 6A
(bc4). Siderophore production was determined by more sensitive and reliable Chrome
azurol-S (CAS) method.

Chemical and spectrophotometric assays showed that P.

aeruginosa 6A strain produced 99% siderophore units.

Naureen et al. (2015) screened bacterial strains isolated from the rhizosphere of
rice plants, and observed antagonistic activity towards the fungal pathogen, Rhizoctonia
solani. Correlation analysis with different metabolites produced by these bacteria
revealed that antagonism was strongly correlated with the quantity of siderophores
produced by individual strains, and was increased under iron-limiting conditions.
Selected high-siderophore-producing strains were found to promote the growth of rice
plants, possibly via the solubilization of soil phosphates, nitrogen fixation and the
production of phytohormones. These same PGPR also conferred resistance against
sheath blight disease, which resulted in significant yield increase in infected plants.
Fifty nine Pseudomonas fluorescens were isolated by Mandal and Kotasthane
(2014) from the rhizosphere and non rhizospheric soil of cave, forest, fallow land and
agriculture field in Chhattisgarh region. The amounts of siderophore produce by P.
fluorescens isolates were screened in iron deficient succinate media and most of them
were found positive for the production of much siderophores. One of the isolate from
Pakhanjore area P3 produced highest siderophore.
Saranraj et al. (2013) isolated and characterized Pseudomonas fluorescens by
Gram staining, motility test, plating on Kings B medium and bio-chemical tests. The
ten Pseudomonas fluorescens isolates obtained from the rhizosphere of paddy were
tested for their efficiency of IAA production. The maximum IAA production was
recorded by the isolate PF-8. The minimum production of IAA was found in PF-4
isolates. The isolate Pseudomonas fluorescens (PS-8) showed maximum Siderophore
production and the least Siderophore production was showed by the Pseudomonas
fluorescens isolate PS-4.
Diazotrophic bacteria were isolated from the rhizosphere of rice plants in two
districts (Parsa and Bara) of Nepal by Shrivastava (2013). Their plant growth promoting
characters were also analysed. It was observed that 64.3% of them showed IAA
production, 32.1% phosphate solubilization, 53.6% siderophore production whereas
10.7% isolates showed all the plant growth promoting characters.
Gull and Hafeez (2012) examined 28 Pseudomonas bacterial strains, among the
28 strains tested, 14 were found to be siderophore producers. These strains were
evaluated for their biocontrol potential against Rhizoctonia solani using various dual
culture assays. The role of siderophores in the inhibition of R. solani was confirmed by
iron chloride (FeCl3) experiment. Data demonstrated that bacterial strain Mst 8.2
produces more than one antifungal agents but the siderophore production is the key
mechanism involved in the antagonism. Bacterial strains MS-3y, Mst 8.2 and Mst 7.4

were the most effective with more than 70% disease reduction in plant growth of wheat.
The complete 16S rRNA gene sequence analysis demonstrated that Mst 8.2 is a
Pseudomonas fluorescens strain.
Seven isolates were found to produce more than 85% siderophore units.
Amongst them S-11 was found be the most efficient siderophore producer (96% SU). S11 was further characterized and identified as Pseudomonas fluorescens. Physicochemical parameters were evaluated for optimum for production of siderophores
by Pseudomonas fluorescens strain. It was found to produce maximum siderophore at
pH 7 and 290C. (Tailor and Joshi, 2012)
Out of 144 bacteria from cucumber rhizosphere, eight isolates were identified as
Pseudomonas fluorescens. Maleki et al. (2010) reported that these isolates were selected
for root colonization and PGP properties. Among these CV-6 strain was produced
considerable amounts of siderophore, indole acetic acid and also shown positive
reactions for HCN, catalase, protease and phosphatase.
2.4.2

HCN Production
Hydrogen cyanide (HCN) is a secondary metabolite produced by many

antagonistic Pseudomonas flourescens species from glycine, essentially under

microaerophilic conditions.
Ten different strains of Pseudomonas fluorescens were isolated from Coleus
rhizosphere except the pf1 strain and identified by biochemical tests. The mechanism of
Pseudomonas strains namely the iron-chelating agent (siderophore), volatiles (HCN)
and antibiotic (Fluorescein and pyocyanin) production tests were studied and reacted for
siderophore, antibiotic and HCN production. (Vanitha and Ramjegathesh, 2014)
Fifty five (JS-1.JS-55) isolates of fluorescent Pseudomonas were isolated
from the rhizosphere soil of ground nut fields in Rayalaseema region of Andhra
Pradesh. Based on colony morphology and biochemical tests, out of 120 samples, only
55 isolates were identified as fluorescent Pseudomonas. These were tested for their
ability to produce secondary metabolites such as Hydrogen cyanide (HCN), Ammonia,
Salicylic acid (SA), Indole acetic acid etc. Maximum production of secondary
metabolites (HCN 0.094 Abs 625; SA 4.68 mg/ml; IAA-22.5 g/ml) was found with
fluorescent Pseudomonas. (Jyothi and Reddy, 2014)
Jayamma et al. (2013) isolated 57 bacterial isolates belonging to Bacillus,
Pseudomonas, Azotobacter and Rhizobium from different rhizospheric soils of southern
telangana zone. These isolates were biochemically characterized and screened in vitro

for their plant growth promoting traits like phosphate solubilization, production of
indoleacetic acid (IAA), hydrogen cyanide (HCN) and siderophore. Among the
Pseudomonas 65% produced HCN, 45% solubilized phosphate with highest
solubilization zone of 17 mm and only 25% produced IAA and siderophores.
Ghodsalavi et al. (2013) isolated 40 colonies of bacteria from the rhizosphere of
valerian and the ability of bacteria to produce siderophore, indoleacetic acid (IAA),
hydrogen cyanide (HCN), lipase and protease were tested in vitro. Additionally, the
effects of seven isolated bacteria (belong to Pseudomonas genus) that showed a high
potential of siderophore, IAA, HCN, lipase and protease production on the quantity of
root extracts were investigated under greenhouse condition. Results showed that the
population of Pseudomonas was the highest in comparison to other genera in the
rhizosphere of plant. Isolated bacteria could mostly produce siderophore, lipase, HCN
and protease.
Twenty Pseudomonad strains were isolated by Noori and Saud (2012) from the
rhizosphere soils of paddy areas in Malaysia and were screened for their Plant growth
promoting activity. All the 20 tested isolates of Pseudomonads were positive for the
production of siderophores and HCN, while of the 20 antagonist bacteria strains, 15
strains (75%) showed positive for the production of plant growth-promoting hormone,
IAA. Among the 20 isolates, 18 isolates (90%) produced phosphate solubilization on
NBRIP medium. Following biochemical identification kit, of the 20 isolates, 15 strains
were identified as Pseudomonas fluorescens.
Supraja et al., (2011) fifteen fluorescent Pseudomonas isolated, identified and
characterized from rhizospheric soils of redgram and maize crops in the Rangareddy
district. These test isolates were biochemically characterized and screened in vitro for
their plant growth promoting traits like phosphate solubilization, production of indole
acetic acid (IAA), Hydrogen cyanide (HCN) and siderophore. Due to production of
HCN and siderophores, fluorescent Pseudomonas isolates inhibited the growth of
Fusarium moniliforme. All the 15 isolates inhibited the growth of fungal pathogen
except MPF-1.
Anand and Kulothungan (2010) isolated Pseudomonas fluorescens from
rhizosphere of healthy groundnut plants and they were tested for their ability to produce
secondary metabolites such as Hydrogen cyanide (HCN), Salicylic acid (SA) and iron
chelating Siderophores. Maximum production of secondary metabolites (HCN 0.0.8
Abs 625; SA - 6.14 mg/ml; Siderophores 4.92 mol benzoic acid/ml) were found
with Pseudomonas fluorescens 04.

2.4.3

IAA Production
Twenty one rhizobacteria were isolated from rhizospheric soils of Uttarakhand

Tarai region. The isolated soil bacteria were screened for the production of indole acetic
acid, phosphate solubilization and siderophore production. Out of 21 isolates, 3 isolates
(P3, P9, and P19) were screened for indole-3-acetic acid (IAA) production that are
Gram negative, catalase positive and starch hydrolysis positive. Isolate P19 was the best
IAA producer strain (50.25g/ml) while isolate P3 was lowest IAA producer
(15.25g/ml) comparatively. All three isolates showed siderophore production while
phosphate solubilization was shown by only P19 isolate. (Rai and Sharma, 2015)
The Pseudomonas species were selectively obtained followed by screening for
IAA production. Most efficient species were selected for further studies like
optimization of growth conditions for Pseudomonas, comparative studies for IAA
production and effect of IAA on seed germination. The study shows Pseudomonas
is potential bacterium for IAA production. Further it was observed that within short
period of time considerable enhancement in the germination was observed in case of
seeds treated with IAA. (Kamble and Galerao, 2015)
The soil samples were collected from rhizosphere of onion plants from Botanical
garden, Department of Botany, Annamalai University. Effect of IAA producing
Pseudomonas fluorescens and Bacillus subtilis bacteria on plant growth was studied by
pot culture experiments by using sterilized air dried soil and viable onion seeds . Both
bacteria demonstrated increase in root length, shoot length, root and shoot fresh and dry
weight, on bacterial inoculated onion seeds over control. (Reetha et al. 2014)
Jeyanthi and Ganesh (2013) isolated Pseudomonas fluorescence and identified
as Pseudomonas fluorescence by 16S rRNA gene sequencing after following the
conventional biochemical tests as per Bergeys manual of Systematic Bacteriology.
Cultural and nutritional conditions were optimized for indole acetic acid production.
The effect of L-tryptophan was studied and the highest yield of 58g/ml on 72 hr was
obtained using 0.5mg/ml concentration.
Ramezanpour et al. (2011) isolated 111 strains of fluorescent Pseudomonads
from rhizosphere of rice and characterized by morphological and biochemical methods.
Those isolates were tested for the production of IAA, presence of tryptophan (50mgL-1)
was recorded within the range of 17.7-95.9 g mL-1
Shahi et al. (2011) isolated 114 diazotrophic bacteria from the rice rhizosphere
of five districts of Eastern Uttar Pradesh (India) and screened for plant growth
promoting (PGP) activities. All these isolates showed nitrogenase activity in the range

of 0.23 to 1.72 mol C2H4 mg-1 protein h-1. Further analysis showed that 84 (73.68%)
isolates were Indole-3-acetic acid (IAA) producers; the value of IAA production ranged
from 10.1 to 353.0 g IAA mg-1 protein. IAA production occured solely in the medium
supplemented with tryptophan. P solubilization activity was observed in 28 (24.56%)
isolates, the activity being in the range of 38.50 to 321.0 P released g mg-1protein. 45
(39.46%) isolates were capable of producing siderophore, the range of production being
4.50 to 223.26 g mg-1 protein.
Twelve bacterial strains were isolated and ten strains were identified as
Pseudomonas and two as Azotobacter. All isolates showed IAA production in growth
medium containing tryptophan as a precursor. Maximum IAA production (4.49mg/L)
was detected in isolate A17 where as IAA production in strains A4 and A11 was also
significant. (Ashraf et al., 2011)
Abbas et al. (2010) studied the Plant growth promoting activities of forty
different strains of Pseudomonas fluorescens and Pseudomonas putida, previously
isolated from the rhizosphere of wheat (Triticum aestivum L.) and canola (Brassica
napus L.) and reported that most of the bacterial isolates were able to produce IAA,
HCN, siderophores and also solubilized phosphorus.
Jayasudha et al. (2010) evaluated forty fluorescent Pseudomonads quantitatively
for indole acetic acid (IAA) producing ability in the presence or absence of tryptophan
and growth promotion in groundnut in response to seed treatment with high IAA
producers

was

analyzed

reported

that

IAA

producers

of

the

fluorescent Pseudomonas group showed significant plant growth when compared with
control.
Thirty fluorescent Pseudomonas isolates (15 P.fluorescens and 15 P.
aeruginosa) were characterized on the basis of biochemical tests and plant growthpromoting activities. Pseudomonas fluorescens AK1 and Pseudomonas aeruginosa
AK2 showed the best plant growth-promoting activity. These isolates were tested for
their ability to produce indole acetic acid in pure culture in the absence and presence of
L-tryptophan at 50, 100, 200 and 500g/ml. For both strains, indole production
increased with increases in tryptophan concentration (0.5, 1.2, 4.3 and 9.3 g/ml; and
0.2, 0.7, 3.8, and 8.3 g/ml, respectively). P. aeruginosa AK2 was less effective in
production of indole acetic acid than P. fluorescens AK1. (Karnal, 2009)

2.4.4

Antagonistic Activity
Aly et al. (2015) revealed that among 116 bacterial isolates, nineteen bacterial

isolates showed the highest inhibition percentage against Fusarium oxysporum,

Rhizoctonia solani and Sclerotium rolfsii. Regarding the antifungal activities, results
indicated that isolates B38 and B103 recorded the higher values of chitinase activity,
catecholate type siderophores and NH3. Also data showed that isolate B103 was the
highest producer for HCN followed by the isolates number B13 and B38. Concerning
the production of volatile compounds, data indicated that isolates B103 and B38 showed
considerable inhibition against pathogenic fungal growth. The most potent isolates were
identified as Pseudomonas fluorescens (B103) and Bacillus subtilis (B38).
Totally eight micro flora resembling Pseudomonas fluorescens were isolated and
three isolates were confirmed as P. fluorescens (strain P.f.01, strain P.f.05 and strain
P.f.07). Pseudomonas fluorescens strains P.f 07 were found most effective with the
highest antagonistic activity against three fungal pathogens and show maximum
inhibition of mycelial growth of Fusarium moniliforme (65.45%), Rhizoctonia solani
(68.23%), and Alternaria alternate (48.13%). (Kumar et al. 2014)
Four bacterial strains of Bacillus subtilis and five strains of Pseudomonas
fluorescens were isolated from tomato field soil by Mezeal (2014). The antagonistic
microorganisms against the pathogens were observed by dual culture technique. P.
fluorescens5 isolate was found to show 81.3% and 77.4% of growth inhibition against
R. solani and F. oxysporum respectively while B. subtilis1 77.4% and 73.2% of growth
inhibition against test pathogens respectively.
Akter et al. (2014) isolated 325 bacteria and 14 isolates were found to be
antagonistic against the pathogen, and in the dual culture test selected bacterial isolates
KMB25, TMB33, PMB38, UMB20 and BMB42 showed 68.44%, 60.89%, 60.22%,
50.00% and 48.22% fungal growth inhibition, respectively. These bacterial isolates
were identified as fluorescent pseudomonads by morphological and biochemical
characterization.
Thirty isolates were screened for PGP attributes and isolates showing PGP
properties were further screened for in vitro antagonism against soil borne
phytopathogens viz., Sclerotium rolfsii, Rhizoctonia solani and Fusarium solani.
Results revealed that 50% (15 out of 30 isolates) reacted positively for one or more PGP
properties. A high prevalence of antagonists was found against the three fungal
pathogens. Majority of the bacterial isolates (33%) displayed antagonism through the
production of siderophores or HCN. (Sarvani and Reddy, 2013)

Arumugam et al. (2013) isolated Trichoderma viride and Pseudomonas

fluorescens from the rizhosphere of rice fields by the serial dilution method and tested
against rice pathogens Rhizoctonia solani, Helminthosporium oryzae and Sarcocalsdium
oryzae in dual culture method. The test result revealed that Pseudomonas fluorescens
actively inhibited, Heminthosporium oryzae 75.6%, Rhizoctonia solani 47.8%,
Sarcocalsdium oryzae 68.9%.
Ravindran and Shaike (2013) isolated Rhizoctonia solani from naturally infected
vanilla plants and attempted to minimize the damage caused by the pathogen using
biocontrol agents Trichoderma harzianum and Pseudomonas fluorescens isolated from
soil. The combined inoculation of Trichoderma harzianum with Pseudomonas
fluorescens treatment showed maximum disease suppression followed by the single
inoculation of Pseudomonas fluorescens, Trichoderma harzianum, Pseudomona putida
and Trichoderma virens respectively in decreasing order.
Ten isolates of bacteria, designated as PGB1, PGB2, PGB3, PGB4, PGB5,
PGT1, PGT2, PGT3, PGG1 and PGG2, were successfully isolated and characterized by
Manivannan et al. (2012). Subsequently, to investigate the PGPR isolates for their
antagonistic activity against phytopathogenic fungi such as Fusarium oxysporum,
Rhizoctonia solani and Sclerotium rolfsii. Furthermore, most of the PGPR isolates
shown antifungal activity against Fusarium oxysporum, and Rhizoctonia solani, and
only one against Sclerotium rolfsii.
Anitha and Das (2011) isolated Pseudomonas fluorescens and Trichoderma sp.
from rhizosphere soil and the antagonistic activity of isolates was observed against
Rhizoctonia solani. Rice plants infected with R. solani were treated with Biocontrol
agents along with arbuscular mycorrhizal (AM) fungi and/or sprayed with hormonal
inducers (Salicylic acid). Plants treated with biocontrol agents alone showed moderate
growth. Biocontrol agents Trichoderma and P. fluorescens when applied along with
salicylic acid showed appraisable increase in the biometric parameters in rice against R.
solani and decreased the percentage of rate of infection compared with control and other
treatments.
Malhotra et al. (2011) evaluated thirteen biocontrol fungi and 4 bacterial strains
against Rhizoctonia solani using dual culture technique. The results showed that among
the fungal species Gliocladium virens and T. harzianum (T8) were the most effective
isolates and inhibited R. solani mycelial growth by 74.82% and 73.33% respectively.
Among the bacterial strains maximum growth inhibition was caused by P. fluorescens
P.f.1 (73.33%) followed by P. fluorescens P.f.2 (62.22%).

Afsharmanesh et al. (2010) reported that Fluorescent pseudomonads able to

produce secondary antifungal metabolites can inhibit soil-borne plant pathogens. For
this reason the antagonistic activity of Pseudomonas fluorescens UTPF5 against R.
solani AG-4 was assessed in bean under in vivo and in vitro conditions. Production of
some secondary metabolites nonvolatile metabolites on growth of the fungus were
observed in UTPF5 and their impact on mycelial growth of R. solani was also studied.
The results showed that UTPF5 could inhibit the growth of R. solani both in vitro and in
vivo, and suppress the disease by 33.34% and 14.29% by soil drenching and seed
treatment, respectively. Production of HCN, siderophore and protease and involvement
of siderophore, volatile and nonvolatile metabolites on growth of the fungus were
observed in UTPF5.
Fluorescent Pseudomonads isolated from rice seedlings were used to screen for
their antagonistic ability against the pathogens Magnaporthe grisea and Rhizoctonia
solani. Among 10 isolates, strain P.f 003 gave significantly higher inhibition of mycelial
growth of M. grisea and R. solani. (Reddy et al. 2010)
Dev and Dawande (2010) evaluated the antagonistic property of Trichoderma
spp. and Pseudomonas fluorescens against Rhizoctonia solani and found that the
mycolytic enzymes produced by the antagonists suppressed the growth of Rhizoctonia
solani.
Two hundred isolates from cotton rhizosphere isolated by Fallahzadeh and
Ahmadzadeh (2010), out of which 39% pertained to fluorescent Pseudomonads. Dual
culture assays were conducted against Rhizoctonia solani AG4 for these isolates to
evaluate the ability of antibiotic production. In greenhouse studies, all of the isolates
significantly suppressed the disease on plant.
Among 20 strains of P. fluorescens isolated from rhizosphere of rice seedlings,
one particular strain P.f 05 was highly effective in inhibiting mycelial growth of rice
pathogens Magnaporthe grisea and Rhizoctonia solani. Four secondary metabolites
were identified on Kings B medium through thin layer chromatography with Rf values
of 0.22, 0.35, 0.42 and 0.51. Of these four, one particular metabolite was found to
inhibit the mycelial growth of two rice pathogens significantly higher compared to other
three metabolites. Of interest, this metabolite was further characterized by HPLC, NMR
and Mass Spectroscopy and identified as 2, 4-diacetyl-phloroglucinol (DAPG). (Reddy
et al., 2009)
Fifteen rhizobacterial fluorescent Pseudomonas isolates obtained from rice in
the region of Andhra Pradesh, India. In all 10 strains of Pseudomonas fluorescens were

selected based on preliminary screening of all these isolates for antifungal activity
against rice fungal pathogens (Pyricularia oryzae and Rhizoctonia solani)., inhibited the
growth of rice fungal pathogens in Fe deficient Kings B medium that varied from (3 to
58% inhibition). Among these Pf 003 strain completely inhibited the mycelial growth of
two rice pathogens both in presence and absence of FeCl3 which indicated the
siderophore mediation along with antifungal metabolites. (Reddy and Reddy, 2009)
Rini and Sulochana (2007) isolated twenty-six isolates of Trichoderma spp. and
56 isolates of fluorescent pseudomonads from Kerala were evaluated for their
antagonistic activity against R. solani under in vitro conditions. Different isolates
showed varying degrees of antagonism. The two most antagonistic isolates against R.
solani were T. pseudokoningii TR17 and T. harzianum TR20. Of the fluorescent
pseudomonads, Pseudomonas fluorescens isolates P28 and P51 showed the greatest
inhibition against R. solani.
Ahmadzadeh et al. (2006) evaluated biological control activity of 47 fluorescent
Pseudomonas spp., against certain soil-borne phytopathogenic fungi such as,
Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var.
parasitica, Pythium sp. and Fusarium sp. The results indicated that 66%, 40.42%,
63.82%, 48.94% and 27.65% of strains revealed antagonistic activity against R. solani,
M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively.

2.5. POT CULTURE STUDY FOR THE CONTROL OF SHEATH

BLIGHT
Saranya and Sowndaram (2014) studied the plant growth promoting (PGP)
activity and antagonistic activity of two rhizobacteria Pseudomonas fluorescens and
Rhizobium sp., isolated from rhizosphere area of rice, against two major rice
pathogens and found both of them showing best PGP activities. The cross streak
studies revealed that the complete inhibition of mycelia growth of Rhizoctonia solani
(85%) and partial inhibition of Sarocladium oryzae (45%) against two rhizobacteria.
Usharani et al. (2014) studied the application of systemic resistance inducing
chemicals with Pseudomonas fluorescens at 15th and 30th day to improve the sheath
blight disease management in rice. The combined activity of Pseudomonas fluorescens
with salicylic acid applied on 30th day showed maximum growth and yield.
Three bacterial strains (Azospirillum brasilense R1, Azospirillum lipoferum
RSWT1 and Pseudomonas Ky1) were inoculated in rice variety JP-5 at control lab
environment and field. Plant growth promotion was observed in all inoculated

treatments over non-inoculated, which was evident from increase in root area, root
length, number of tillers, straw and grain yields and total weight of plant. Inoculation
with Azospirillum lipoferum RSWT1 and Pseudomonas Ky1 increased grain weight by
18.5% and 13.8% respectively. (Midrarullah et al. 2014)
Biswas and Datta (2013) isolated four strains of Trichoderma viride (TV-01,
TV-02, TV-03 and TV-04), one each of T. harzianum (TH-01) and Pseudomonas
fluorescens (PF-01) were evaluated against sheath blight disease of rice grown under
upland condition in Tripura. Only Pseudomonas fluorescens, when applied both in soil
as a supplement along with FYM and on foliage as spray, were effective in minimizing
the disease. In poisoned food technique using cell-free culture filtrates, P. fluorescens
showed maximum inhibition (56.3%) of radial growth of mycelium of Rhizoctonia
solani.
Singh et al. (2013) studied combined activity of Trichoderma harzianum and
Pseudomonas fluorescens-27 applied as seedling root dip and foliar spray against the
sheath blight of rice caused by Rhizoctonia solani under glass house conditions. The
results showed that combined activity of seedling root dip with Trichoderma harzianum
and Pseudomonas fluorescens-27 and foliar spray with Trichoderma harzianum was the
most effective in reducing the disease severity.
Sivakamasundari and Usharani (2012) isolated Pseudomonas fluorescens in the
phyllosphere and rhizosphere of rice grown at Cuddalore district. Studies on the
combined effect of Pseudomonas fluorescens and salicylic acid application during the
inoculation of Rhizoctonia solani revealed the application of Pseudomonas fluorescens
at seed and soil level together with application at salicylic acid on 30th day augmented
the plant defence enzyme system to a maximum level.
The antagonistic strain of P. fluorescens was applied as suspension in pot
experiment by different methods viz. seed, root, soil, and their integration methods
seed+root, root+soil, seed+soil and seed+root+soil. The control treatments were
inoculated control (only pathogen inoculated) and uninoculated control (neither
pathogen nor antagonist inoculated). The population dynamics of the pathogen and
antagonist in brinjal rhizosphere soil showed that the crop receiving seed+root+soil
treatment had the lowest population recovery of the pathogen 26106 cfu/g (7.33) and
correspondingly highest population recovery of the antagonist 179.67106 cfu/g (8.25).
The yield, yield attributes and physiological and biochemical parameters were also
found to be best performing in the seed+root+soil treatment of the antagonist
suspension indicating its potential as PGPR. (Chakravarty and Kalita, 2012)

The IR50 rice seeds were treated with the different doses of talc based
Pseudomonas fluorescens (@ 2.5, 5, 7.5, 10 g/kg) under pot conditions by Shyamala
and Siva kumaar (2012). The least incidence (20%) was observed when the talc based
inoculums applied at 10 g/kg seed. The talc based formulation of P. fluorescens (@1,
1.5, 2, 2.5 kg/ha) was sprayed on 15 day old IR 50 rice plants in pot conditions. The
results revealed the rice blast control was achieved by spraying P. fluorescens @ 1.0
kg/ha. The increasing dose of talc based inoculums when applied on foliage increased
the phyllosphere population of P. fluorescens and controlled rice blast. The maximum
disease control was achieved when inocula was applied at 2.5 kg/ha.
In pot culture experiment on rice variety Tapaswini the bioefficacy of
Trichoderma viride (Bangalore isolate) was observed to be significantly higher
followed by Gliocladium virens (Bhubaneswar isolate). Among the bioagents,
Bangalore isolate of T. viride was found very effective in restricting the growth and
sclerotia production of R.solani by 67.94% and 68.62%, respectively as compared
to that of the control. (Lenka et al. 2012)
In pot trial, Pseudomonas fluorescens tested in combination with salicylic acid
was highly efficient in management of rice blast diseases. Application of P.fluorescens
along with salicylic acid significantly increased the disease resistance. The results
indicate that the combined biological and chemical inoculation showed a better response
to fight against rice blast pathogen P.oryzae than the treatment alone. (Shyamala and
Sivakumaar, 2012)
Surendran et al. (2011) isolated Pseudomonas fluorescens from different
locations in Kuttanad and screened against rice sheath blight disease. Three effective
strains, viz., PF43, PF46 and PF47 were tested individually and also in combination
against sheath blight under field conditions. The results indicated the combined
application of the three strains was found to be effective for sheath blight disease
management during rabi 2009-10, kharif 2010 and rabi 2010-11.
Anitha and Das (2011) isolated Pseudomonas fluorescens and Trichoderma sp.
and the antagonistic activity of isolates was observed against Rhizoctonia solani. Rice
plants infected with R. solani were treated with biocontrol agents along with arbuscular
mycorrhizal (AM) fungi and/or sprayed with hormonal inducers (Salicylic acid). Plants
were harvested on 14 and 28 days after pathogen infection growth rate of the plant was
measured. Biocontrol agents Trichoderma and P. fluorescens when applied along with
salicylic acid showed appraisable increase in the biometric parameters in rice against R.
solani and decrease the percentage of rate of infection compared with control and other

treatments.
Tiwari and Thrimurty (2009) isolated seven Pseudomonas fluorescens isolates
collected from Chhattisgarh region and screened by dual culture method for their
antagonistic ability against Magnaporthe grisea and Rhizoctonia solani. Results showed
that maximum inhibition (76.3%) was recorded with the isolate pfr2 against M. grisea
whereas in case of R. solani inhibition (75.2%) was recorded with isolate pfr1.When the
strains grown under mist chamber and field conditions, P. fluorescens isolate pfr1
promoted the shoot length and number of tillers in rice and also effectively reduced the
blast and sheath blight severity when applied as seedling treatment with one or two
foliar sprays.
Singh and Sinha (2009) isolated five strains of Pseudomonas fluorescens (Pfr 1,
Pfr5, FLP 19, FLP 82 and FLP 90) and screened against Rhizoctonia solani. In dual
culture test, Pfr 1 showed more inhibition against Rhizoctonia solani. Among all strains
maximum reduction in disease severity (67.8%) along with increase in grain yield
(31.6%) was recorded when R. solani was applied 7 days after application of Pfr1.
Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of
rice were tested for their antagonistic effect towards the rice sheath blight fungus
Rhizoctonia solani. Among them, PfMDU2 was the most effective in inhibiting
mycelial growth of R. solani in vitro because of the highest -1,3-glucanase activity,
siderophore production, salicylic acid (SA) production and HCN production. The
significant relationship between the antagonistic potential of P. fluorescens against R.
solani and its level of -1,3 -glucanase, SA and HCN was observed (Kumar et al., 2004)

Chapter III

MATERIAL AND METHODS

The present study was carried out at the Department of Agricultural
Microbiology & Bioenergy, College of Agriculture, Rajendranagar, Hyderabad on
Isolation and characterization of Pseudomonas fluorescence from rice fields for the
biological control of Rhizoctonia solani causing sheath blight in rice. Soil samples
were collected from Parigi and Doma mandals of Rangareddy district, Telangana. The
materials used and methods employed in the investigation are outlined below.
The general laboratory techniques followed in the present study were those
described by Cappuccino and Sherman (1992), Nene and Thapliyal (1993) and Aneja
(2001) for preparation of media, sterilization, isolation and maintenance of bacterial
cultures, with slight modifications wherever necessary.
3.1. GLASSWARE
Petri plates, test tubes, glass slides, conical flasks of different capacities i.e.,
1000 ml, 750 ml, 500 ml, 250 ml, pipettes of 1 ml, 2.2 ml, 5 ml, 10 ml beakers and
measuring cylinders of 10 ml, 50 ml, 100 ml, 500 ml and 1000 ml were used. All the
glassware used was of Borosil made.
3.1.1 Cleaning of Glassware
Glassware was first washed with a detergent, then cleaned with tap water and
finally placed in the Chromic acid solution prepared with following composition:
Potassium dichromate

60 g

Conc. H2SO4

60 ml

Distilled water

1000 ml

The glassware were kept in the cleaning solution for 24 h and then thoroughly
washed with running tap water before its final cleaning with distilled water and dried.
3.1.2 STERILIZATION OF GLASSWARE
Glassware was sterilized in hot air oven at 1600C for 2 h before use. Media,
distilled water, etc., were sterilized in an autoclave at 15 lb psi (1210C) for 20 min.

3.2 EQUIPMENT AND APPARATUS USED

Hot air oven and autoclaves were used for sterilization of glassware and media
respectively. BOD incubators were used for incubating cultures at different
temperatures. Cultures were stored and maintained in a refrigerator. The pH was
measured by using digital pH meter. Cyclomixer was used for homogenization during
serial dilution. Plate mixer was used for spread plate technique. Centrifuge was used for
making cell-free cultures. Hi-media zonal scale was used to measure the zone of
inhibition around the colonies during phosphate solubilization, biocontrol activity.
Samples were weighed using a single pan electric balance. Compound electron
microscope was used to observe the morphology of bacterial cultures. Double
distillation unit was used for sterilization of water. Laminar air flow was used for
maintenance of aseptic techniques.
3.3 CHEMICALS USED
The chemicals used in the present investigation were of analytical and laboratory
grade. The pH of the media was adjusted to the required level using 0.1N NaOH, 0.1N
Hcl and 0.1N KOH. Formaldehyde 10% solution was used to fumigate the laminar air
flow chamber and incubators for disinfection.
3.4 ISOLATION OF Pseudomonas fluorescence
3.4.1 Soil Sample Collection
Samples were collected from different rhizospheric soils of rice plants of Parigi
and Doma mandals of Rangareddy district, Telangana state.
Twenty two (22) rhizospheric soil samples of rice crop plants up to a depth of 10
to 15 cm were collected from Parigi and Doma mandals of Rangareddy district,
Telangana. The soil intimately adhering to the roots was collected and mixed to provide
a composite soil sample.
The GPS data (Latitude 0N and Longitude 0E) were collected at each sampling
site distributed over the entire two mandals of Rangareddy district of Telangana state.
3.4.2 Isolation of Pseudomonas fluorescence
For isolation of Pseudomonas fluorescence, the method proposed by Vlassak et
al. (1992) was followed. In this procedure 10 g of soil from each soil sample was taken
in a conical flask to which 90 ml of normal saline was added. The sample was agitated

for 15 minutes on a vortex and serial dilutions of soil suspensions were prepared. Serial
dilutions prepared for the rhizobacteria are given below.
For Pseudomonas spp. 10-2 to 10-5 dilutions were taken and 0.1 ml of
respective dilutions were spread on sterilized petri plates containing specific media i.e.,
Kings B (Pseudomonas spp.) and the petri plates were incubated at room temperatures
(280C 20C) for 24-72 h. Media composition is presented in appendix I.
Two replicates were maintained for each dilution. The plates were examined
daily up to 3 days for bacterial colonies. Pure cultures of isolated colonies were
obtained by the streak plate method.
3.5 IDENTIFICATION OF BACTERIAL ISOLATES
3.5.1. Morphological Characterization
All the 30 isolates were checked for their purity and then studied for the colony
morphology and pigmentation. The cell shape and Gram reaction were also recorded as
per the standard procedures given by Barthalomew and Mittewar (1950).
3.5.2. Grams Staining
A drop of sterile distilled water was placed in the center of glass slide. A loopful
of inoculum from young culture was taken, mixed with water, and placed in the center
of the slide. The suspension was spread out on slide using the tip of inoculation needle
to make a thin suspension. The smear was dried in air and fixed through mild heating by
passing the slide 3 to 4 times over the flame. The smear was then flooded with Crystal
violet solution for 1 min and washed gently with flow of tap water. Then the slide was
flooded with Iodine solution. After incubation at room temperature for 1 min, Iodine
solution was drained out followed by washing with 95% decolorizer. After that, it was
washed with water within 15 to 30 sec and blot carefully. The smear was incubated with
Saffranin solution for 1 min. The slide was washed gently in flow of tap water and dried
in air. The slide was examined under microscope at 100X power with oil immersion and
data was recorded.
3.5.3 Colony Morphology
Morphological characteristics of the colony of each isolate were examined on
Kings B medium and incubated for according to isolate. Cultural characterization of
isolates observed by different characteristics of colonies such as shape, size, elevation,
surface, margin, color, odor, pigmentation etc., were recorded.

3.5.4 Biochemical and Physiological Characterization

Different biochemical tests were performed as per the procedure described by
Aneja (2001). The protocols followed are briefly outlined below.
3.5.4.1 Indole Production
Sterilized Hydrogen Sulfide-Indole-Motility agar (SIM agar) slants or
Tryptophan broth tubes were inoculated with the overnight cultures of the isolates and
incubated for 48 h at 28 20C. Following incubation, 10 drops of Kovacs indole
reagent were added to each tube. The isolates showing production of red colour were
recorded as positive for indole production.
3.5.4.2 Catalase Test
This test was performed to study the presence of catalase enzyme in bacterial
colonies. Pure isolates (24 h old) were taken on glass slides and one drop of H2O2 (30
%) was added. Appearance of gas bubble indicated the presence of catalase enzyme.
3.5.4.3 Oxidase Test
The overnight cultures of the test isolates were spotted on plates poured with
sterile Trypicase Soy Agar (TSA) and the plates were incubated for 24 h at 28 20C.
After incubation, 2-3 drops of N, N, N, N- tetramethyl-p-phenylenediamine
dihydrochloride (Wursters reagent) were added on to the surface of growth of each test
organism. The isolates showing change of colour to maroon were noted as oxidase
positive.
3.5.4.4 Gelatin liquefaction
The overnight cultures of the test isolates were inoculated to sterilized nutrient
gelatin deep tubes and incubated for 24 h at 28 20C. Then the tubes were kept in the
refrigerator for 30 minutes at 40C. The isolates showing liquefied gelatin were taken as
positive and those which resulted in solidification of gelatin on refrigeration were
recorded as negative for the test.
3.5.4.5 Methyl Red Test
Sterilized glucose-phosphate broth tubes were inoculated with the test culture
and incubated at 28 20C for 48 h. After incubation five drops of Methyl red indicator
was added to each tube and gently shaken. Red colour production was taken as positive
and yellow colour production was taken as negative for the test.
3.5.4.6 Voges Prauskers Test
To the presterilized glucose-phosphate broth tubes, test cultures were inoculated
and incubated at 370C for 48 h. After incubation ten drops of Baritts reagent A was

added and gently shaken followed by addition of ten drops of Baritts reagent B.
Development of pink colour in the broth was taken as positive for the test.
3.5.4.7 Citrate Utilization
Isolates were streaked on Simmons citrate agar slants and incubated at 28 20C
for 24 h. Change in colour from green to blue indicates the positive reaction for citrate
utilization.
3.5.4.8 Starch Hydrolysis
Sterile starch agar plates were spotted with 10 l overnight broth cultures of the
isolates and incubated at 28 20C for 24-48 h. After incubation, the plates were flooded
with iodine solution. The formation of a transparent zone around the colony was taken
as positive reaction for the test.
3.5.4.9 Hydrogen Sulfide Test
Sterilized Hydrogen Sulfide- Indole-Motility agar (SIM agar) stabs were
inoculated along the wall of the tubes with overnight cultures of the isolates and
incubated for 48 h at 28 20C. Visualization of black colour along the line of
inoculation indicated a positive reaction for the test.
3.5.4.10 Denitrification
Sterilized nitrate broth tubes inserted with Durhams tube in inverted position
were inoculated with overnight grown cultures of the test organisms and incubated at
250C for 10-15 days. After incubation, the isolates which showed accumulation of gas
in the Durhams tubes were scored as positive for denitrification.
3.5.4.11 Carbohydrate Utilization test
All the pure bacterial isolates were screened for the carbohydrate fermentation
abilities using four different carbohydrates (lactose, sucrose, dextrose and mannitol) in
peptone broth medium. Bacterial isolates were inoculated in broth containing specific
carbohydrate. The change in colour of peptone broth was observed for utilization of
particular carbohydrate present in broth.
3.6 SCREENING OF Pseudomonas fluorescens ISOLATES FOR PGPR
ACTIVITIES.
Pure isolates were isolated by streaking isolates on respective media plates and
screened for following plant growth promotion properties.
3.6.1 Phosphate Solubilization
Sterilized Pikovskayas agar was poured as a thin layer on to the sterilized petri
plates and incubated for 24 h, after solidification. After incubation the pikovskayas

plates were spot inoculated with 30 isolates of Pseudomonas spp. incubated at 28 20C
for 4-5 days. Formation of a clear zone around the colonies was considered as positive
result for phosphate solubilisation. It was calculated by following formula
PSE (Phosphate Solubilization Efficiency) =

Z C x 100

Z- Clearance zone including bacterial growth

C- Colony diameter
3.6.2 Ammonia production
The isolates were tested for ammonia production by inoculating the isolates in to
10 ml of pre-sterilized peptone water in the test tubes. The tubes were incubated for 4872 h at 36 20C. Nesslers reagent (0.5 ml) was added in each tube. Change in colour of
the medium from brown to yellow colour was taken as positive test for ammonia
production.
3.6.3 Indole Acetic Acid Production
The selected antagonistic bacterial strains were grown in 5 ml of nutrient broth
medium in test tube or 24 hrs. After incubation period, bacterial culture were harvested
and centrifuged at 10000xg for 15 min at 40c. Two drops of orthophosphoric acid were
added to 2 ml of cell free supernatant and the development of colour was observed the
presence of a pink colour indicate positive reaction for indole acetic acid and yellow
colour indicate negative reaction (Reetha et al. 2014).
3.7 SCREENING OF EFFICIENT PGPR ISOLATES FOR BIOCONTROL
ACTIVITY
3.7.1

Siderophore Production
Siderophore production was estimated qualitatively. 0.5% of cell free culture

supernatant was added to 0.5 ml of 0.2% aqueous Ferric chloride solution. Appearance
of orange or reddish brown color indicated the presence of siderophore (Yeole and
Dube 2000).
3.7.2 Hydrogen Cyanide Production
The HCN production was tested by the method of Castric and Castric (1983).
Medium plates i.e., Kings B (Pseudomonas spp.) were prepared separately and
incubated for 24 h. One ml of culture of each test isolate was inoculated on respective
media plates separately. A disc of whatman filter paper No.1 of the diameter equal to
the petri plate size, impregnated with alkaline Picric acid solution (0.5% Picric acid
(w/v) in 1% Sodium carbonate) was placed in the upper lid of the inoculated petri plates

under aseptic condition. The control plate did not receive the inoculum. The plates were
incubated-upside up at 28 20C for 48-72 h. Change in color from yellow to light
brown, moderate or strong reddish brown was taken as indication of HCN production.
3.7.3 Antagonistic Activity
Pure fungal isolates of Sheath blight disease causing soil phytopathogen viz.,
Rhizoctonia solani, were obtained from the Department of Plant Pathology, College of
Agriculture, Rajendranagar.
Antagonistic activity was verified by following dual culture technique
(Skidmore and Dickinson, 1976). First, the bacterial isolates were streaked on
respective media plates and incubated at respective temperature and time. Loopful of
each bacterial isolate was streaked on the Potato dextrose agar plate at one end, which
was pre-inoculated with 5 days old, 5 mm mycelial disc of test pathogen at the other
end. Control plate was maintained by placing only pathogen mycelial disc on the plate
without bacteria.
The assay plates were incubated at 28 10C for 5 days and observations were
made on inhibition of mycelial growth of the test pathogens. For each bacterial isolate
three replications were maintained with suitable controls.
The percent growth inhibition over control was calculated by using the formula:
Percent Inhibition =
Growth of pathogen in control (mm)-growth of pathogen in treatment (mm) x100
Growth of pathogen in control (mm)
Note: The percent inhibition in control is taken as zero percent.
3.8 POT CULTURE STUDY FOR THE CONTROL OF SHEATH BLIGHT
3.8.1. Percent germination and Vigour index
Seedling vigour and seed germination of the Pseudomonas fluorescens treated
seeds was determined by the standard roll towel method (International Seed Testing
Association, 1996). The seeds were immersed in test solution for 6 h at room temperature.
The seeds were then placed on coarse blotter paper sheets and covered with a moistened
blotter and rolled. The roll was kept on a butter paper sheet and rolled as a bundle and
incubated in a growth chamber at 25 10C with 80% relative humidity. After 10 days of
incubation, percent germination was recorded and root length and shoot length were
measured. Vigor index was calculated using the formula
Vigour index = % germination x seedling length (shoot length + root length)

3.8.2. METHODS OF APPLICATION OF Pseudomonas fluorescens

3.8.2.1. Seed treatment
Rice seeds were surface sterilized with 2% Sodium hypochlorite solution and
soaked in a double volume of bacterial suspension (9x108 cfu ml-1). After 24 h, the
bacterial suspension was drained and the seeds were dried under shade for 30 min. The
seeds were allowed to sprout for another 24 h before sowing (Vidhyasekaran et al. 1997).
After 25 days, the seedlings were pulled out from the pots and transplanted at the rate of
four seedlings per hill and two hills per pot (30 cm diameter containing 10 kg of clay soil)
3.8.2.2. Root dipping
Rice seedling in bundles (approximately 200 seedlings) were dipped in 250 ml of
bacterial suspension (9x108 cfu ml-1) for 2 h, ensuring that roots alone were immersed in
the inoculum and planted in pots.
3.8.2.3. Foliar application
25 ml of bacterial suspension (9x108 cfu ml-1) per pot was sprayed 30 days after
transplanting (Vidhyasekaran et al. 1997).
3.8.3. Preperation of inoculum of Rhizoctonia solani
Three hundred grams of barley seed sterilized were put in Erlenmeyer flask, then in
each flask 5 mm mycelial disc from a 4 days old culture of Rhizoctonia solani on PDA
was placed in the flasks and they were incubated at room temperature (26 20C) for 3
weeks (Kazempour, 2004).
3.8.4. DETAILS OF THE TREATMENTS USED IN THE FIELD EXPERIMENT
Crop

: Rice

Variety

: Tellahamsa

Season

: Rabi, 2014-15

No. of treatments

: 10

No. of replications : 3
Pot size

: 30 cm30 cm

Experimental design : CRD

RDF

: 120: 60: 40 NPK kg ha-1

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

3.8.5. GROWTH CHARACTERS

3.8.5.1. Plant height
The plant height was recorded by measuring the total height from the base of the
plant to the tip of the top most panicle at the time of harvest and is expressed in
centimeters.
3.8.5.2. Total number of tillers hill-1
In each pot the number of tillers were counted on each plant at the time of
maturity and expressed as total number of tillers hill-1
3.8.5.3. Number of productive tillers hill-1
The ear bearing tillers, which produced healthy panicles were counted and
expressed as number of productive tillers hill-1
3.8.5.4. Grain yield per plant (g)
The harvested plants from pots were threshed manually and each pot yield was
separately sun dried, cleaned by winnowing and weighed. Grain yield was computed at
14 percent moisture content and expressed in kg ha-1.

3.8.5.5. Measurement of chlorophyll content (SCMR Values)

The SPAD-502 (Soil plant analytical development) meter was used for
measuring the relative chlorophyll content of leaves. The chlorophyll content was
measured for recent fully expanded leaves. Mean of the five values from each hill was
obtained. This meter enables obtaining instant readings without destroying the plant
tissue.
3.8.6. Leaf area index
Leaf area was estimated at 60 and 90 DAT. The area of total leaves was
measured with digital leaf area meter (LI-3100) and expressed in cm2. Leaf area index
was calculated by using the formula as proposed by Watson (1952).
Leaf area
LAI =
Unit ground area
3.8.7. Scoring of sheath blight
Sheath blight incidence was recorded on 15th day after last spraying, using a 0-9 grade
SES scale (IRRI., 2002).
Scores

Description

No infection

Vertical spread of lesions up to 20% of plant height

Vertical spread of lesions up to 21-30% of plant height

Vertical spread of lesions up to 31-45% of plant height

Vertical spread of lesions up to 46-65% of plant height

Vertical spread of lesions up to 66-100% of plant height

3.8.8. Reduction in disease severity

Development of symptoms was observed and recorded 7 days after inoculation as
grades 09 (IRRI., 2002). Based on the grades, the disease index was calculated using the
following formula

Disease index = Total grade / No. of sheath observed x 100 / maximum grade
3.9. MAPPING OF RHIZOSPHERIC SOIL SAMPLES OF PARIGI AND DOMA
MANDALS OF RANGAREDDY DISTRICT
3.9.1. Computer hardware and software
Scanner was used for scanning of the toposheet (56G/16, 1:50,000 scale) plate
pertaining to the study area and saved as JPEG format and imported into Erdas software
as .img format for further use. For digital image processing and analysis of Remote
sensing data HP desktop having 4GB RAM work station with ERDAS imagine version
9.3 software was used. Geographic Information System (GIS) software, Arc GIS
version 9.3 was used for mapping the processed image. The facilities available in
Remote sensing and GIS cell of Water Technology Centre (WTC), PJTSAU,
Rajendranagar, Hyderabad was used for this purpose.
3.9.2. Topographical maps of the study area
The Survey of India (SOI) topographical map of 56G/16 of 1:50,000 scale
covering Parigi and Doma mandals of Rangareddy district, Telangana was used as
reference maps for demarcating study area.
3.9.3. Equipments and software used
3.9.3.1. Global Position System (GPS) (Model- Garmn72H)
Global Position System (GPS) which is a constellation of 24 satellites (Plate 3.1)
orbiting the Earth at a very high altitude of 20,200 km, allows anyone with a GPS
receiver to determine the precise 3-D location. It offers advantages of accuracy, speed,
versatility and economy while in use as an aid for position based data collection. GPS
owes its popularity to the dependable high accuracy with which position and time can
be determined. The GPS was conceived as a ranging system from known positions of
satellites in space to unknown positions on land, sea and space. GPS uses pseudo ranges
derived from the broadcast satellites. The pseudo ranges were derived either from
measuring the travel time of the (coded) signal and multiplying it by its velocity or by
measuring the phase of the signal. The antenna detects the electromagnetic waves
arriving from the satellites, converts the wave energy into an electric current, amplifies
the signal strength and sends the signals to the receiver electronics.

The GARMIN, GPS72H GPS receiver (plate) in stand-alone mode was used to
collect the information regarding the geographical location (latitudes and longitudes) of
the soil sampling sites during the present information. Data entered in MS Excel sheet
i.e., data in degrees, minutes and seconds are converted to degree decimals format.
Ex: Latitude 1700739.1 (in degrees, minutes and seconds), 10 = 60, 1 = 60. Here
17007 39.1 = 17+7/60+39.1/3600 = 17.1280 (Latitude in degree decimals)
3.9.4. Delineation of the study area
Study area was delineated with the help of administrative boundaries of mandal
map of Rangareddy district. The study area latitudes ranged from 1700N to 17015N
and longitudes ranged from 7704539.1E to 7800E. The procedure followed for geo
referencing of 56G/16 toposheet presented in Fig 3.1 by using Erdas 9.3 software
3.9.5. Preparation of point map
The location of soil samples were collected in study area by using a hand held
GPS instrument GARMIN GPS72H receiver. The GPS technology proved to be very
useful for enhancing the spatial accuracy of the data integrated in the GIS.
GPS corrections which are misplaced could be rectified by correction factor.
Dams, four road junctions, important land marks etc. were selected in the selected area
and the GPS readings are noted and corrected on the map which we prepared as per the
procedure indicated in the flow chart. (Fig 3.2)
The Arc GIS version 9.3 software was used in the study. Based on the location
data obtained, prepared point feature showing the position of samples in MS Excel
format and linked with the spatial data by join option in the Arc Map. The steps for
generation of point map by using Arc GIS 9.3 indicated in flow diagram in Fig. 3.2 and
the resultant map generated is presented in Plate 4.1

Plate 3.1 Satellites orbiting the Earth

Open the Erdas image software

Open toposheet to be rectified

Raster

Geometric correction

Polynomial window

Projection: Geographic (Lat/Lon), Spheroid name: WGS 1984

Datum type: WGS 1984

Give GCP points by zooming in corner points

Add X Ref, Y Ref values by

keyboard

Resample window

Output file name

Generates rectified toposheet

Fig: 3.1 Flow diagram of rectification of the toposheets in Erdas image 9.3 software

Open the Arc GIS software

Add excel file containing the information on latitude, longitude details in degree
decimals and select the work sheet in table of contents containing the above
information

Also add boundary layer and merged toposheets of the study area

Tools

Add XY data

X-field-Longitudes
Y-field-Latitudes

Co-ordinate system- import same from other file like

rectified toposheeets
Projection: Geographic (Lat/Lon), Spheroid name:
WGS 1984
Datum type: WGS 1984
Point map gets displayed on window as event file

Export and save it as shape file by giving suitable file name

Point map along with boundary layer can be exported and saved as .Pdf or
.jpeg format

Fig: 3.2 Flow diagram for preparation of point map in Arc GIS software.

3.10. STATISTICAL ANALYSIS

The data obtained in different experiment was statistically analyzed using
Completely Randomized Design (CRD) as per the procedures given by Snedecor and
Cochran (1967) and Panse and Sukhatme (1985).
Data on different characters viz., growth and yield components and yield, were
subjected to analysis of variance procedures as outlined for CRD. Statistical
significance was tested by Fvalue at 0.05 level of probability and critical difference
was worked out where ever the effects were significant.

Chapter IV

RESULTS AND DISCUSSION

Plant growth promoting rhizobacterial strains collected from different rhizospheric soils
of Parigi and Doma mandals of Telangana state, were confirmed for purity and maintained in the
department of Agricultural Microbiology & Bioenergy, College of Agriculture, Rajendranagar.
The isolates were characterized, identified based on morphological and biochemical
characteristics and studied for their functional plant growth promoting activities and their
biological control of sheath blight disease on rice crop. The results obtained on these aspects are
presented under the headings.
4.1

Isolation of Pseudomonas fluorescens from rice rhizosphere

4.2

Biochemical characterization of Pseudomonas fluorescens

4.3

Screening of pure isolates for PGPR properties

4.4

To find efficient PGPR isolates for bio control activity

4.5

Pot culture study for the control of sheath blight of rice

4.1 Isolation of Pseudomonas fluorescens from the rice rhizospheric soils

of Parigi and Doma mandals of Rangareddy district
4.1.1 Collection of soil samples by using GPS
Soil samples were collected from the twenty two sites in Parigi and Doma mandals
of Rangareddy district with their GPS numbers, latitude, longitude and elevation (Table
4.1) and the resultant point map generated, showing the place of collection i.e., villages
(Plate 4.1).
4.1.2 Isolation of Pseudomonas fluorescens
Twenty two rhizospheric soils of rice obtained from different villages of Parigi and
Doma mandals of Rangareddy district, Telangana state were used for the isolation of
Pseudomonas fluorescens (Table 4.1). The microbial population in the rhizospheric soils of
rice of different places were counted (Table 4.2). Maximum Pseudomonas population was
found in the black rice rhizospheric soils of Rangapur, Doma mandal (6.7) and next to it is
5.8 found in Kishtapur of Doma mandal. The Pseudomonas population in the soils
collected from different places ranged between 1.2-6.7 cfu x 107g-1 soil (Fig 4.1). Thirty
bacterial isolates were isolated from the rhizosphere of rice from twenty two villages of
Parigi and Doma mandals of Rangareddy district, Telangana state.

Microbial community in the plant rhizospheric soil is highly dynamic and relative
abundance of the microbial population in the plant rhizosphere depend upon the plant
species, soil types and environmental conditions. Several studies have reported that plant
roots release variety of organic compounds such as sugars, amino acids, organic acids,
fatty acids, enzymes, auxins and hydrogen cyanide (Neumann and Romheld, 2001).
Recently, it has been shown that difference in root exudation at different stages of plant
development influence the microbial community in the rhizosphere. (Graystone et al.
1998).
As the Pseudomonas bacteria isolated depend on the soil type, environmental
conditions and mainly on the root exudates. The population number varied from 1.2-6.7
cfu x 107g-1 soil because as they have been collected from different stages of the rice crop
and different soil types. In the rhizospheric and non- rhizospheric soils of crop plants, the
bacterial counts varied. Rhizospheric soil composed of more number of Pseudomonas
bacterial population compared with the non-rhizospheric soil.
Similar results obtained with Panpatte et al. (2014) isolated 10 fluorescent
Pseudomonas from rhizospheric and non rhizospheric soils of different crops of Vadodara
locality. Rameshkumar et al. (2014) isolated fluorescent pseudomonads associated with the
rice rhizosphere.
Ten strains of flurosecent Pseudomonads were isolated from the rhizospheric soils
of rice growing areas in Karnataka, India (Shivalingaiah and Umesha, 2013). Manjunatha
et al. (2012) isolated 92 fluorescent pseudomonads from the rhizosphere soil of paddy
using specific medium.
4.1.3 Cultural and morphological characterization
A total of thirty Pseudomonas fluorescens isolates were isolated, culturally and
morphologically characterized on Kings B agar medium.
The Pseudomonas isolates took about 24 h incubation to establish their full growth
on Kings B agar medium (Vlassak et al. 1992) (Plate 4.2a). All the isolates developed
small to medium, smooth, glistening colonies. Out of the total 30 isolates, 8 isolates
(PRP1, PVP1, DBP, DMoP, DBoP, PKP, PSP2 and DGP2) showed yellowish green
pigmentation, 12 isolates (PLP, DOP, DTP1, DDP, DGP1, DPP, DRP, DMuP, PSmP,
PGuP1, PGP2 and PVP2) showed light green pigmentation, 5 isolates (DKP, PGP1,
DMP1, DMP2 and PGuP2) showed bluish green and 5 isolates (PGoP, PSP1, DPiP, PRP2

and DTP2) showed dark green pigmentation under UV light. These isolates were Gram
negative, rods without sporulation when observed under microscope (Table 4.3).
These isolates were identified as Pseudomonas fluorescens (30 isolates) based on
their colony morphology on Kings B agar medium, cell morphology and Gram reaction.
The bacterial isolates were named as PRP1, PVP1, DBP, DMoP, DBoP, PKP, PSP2,
DGP2, PLP, DOP, DTP1, DDP, DGP1, DPP, DRP, DMuP, PSmP, PGuP1, PGP2, PVP2,
DKP, PGP1, DMP1, DMP2, PGuP2, PGoP, PSP1, DPiP, PRP2 and DTP2 according to the
soil type, crop, village and mandal (Table 4.4).
Microscopic studies of bacteria showed that all isolates were gram negative, rod
shaped and non-spore forming cells which were morphologically corresponding to wellknown Pseudomonas species. The isolated bacteria observed under UV light showed
yellowish green, dark green, bluish green and the majority of isolates had light green
fluorescence (Plate 4.2b). Exhibition of fluorescence was the peculiar character of
fluorescent Pseudomonads, hence the isolates were confirmed.
Similar results were observed with Wasi et al. (2010) isolated strain SM1 and
characterized on the basis of morphological, cultural and biochemical properties and
presumptively identified as Pseudomonas fluorescens.
A proteobacterium was isolated from rhizosphere soil and it was identified using
morphological, cultural and biochemical characteristics as Pseudomonas fluorescens.
(Rekha et al. 2010).

4.2 Biochemical characterization of Pseudomonas fluorescens

After the study of cultural and cell morphology, the isolates of the Pseudomonas
fluorescens (30 isolates) were characterized with different biochemical tests viz., IMVIC
test, oxidase test, catalase test, carbohydrate utilization test, denitrification, H2S
production, starch hydrolysis, gelatin liquefaction and ammonia production.
Results presented in Table 4.5 reveal that all the 30 isolates of Pseudomonas
fluorescens were negative for indole production and for methyl red test 21 isolates i.e.,
PRP1, DBP, DMoP, DBoP, PSP2, DGP2, PLP, DOP, DDP, DPP, DRP, DMuP, PGuP1,
PVP2, DMP1, DMP2, PGuP2, PGoP, PSP1, DPiP and DTP2 showed positive results
(Plate 4.2c). For Voges Prauskers test 22 isolates i.e., PRP1, PVP1, DBP, DMoP, PKP,
PSP2, DGP2, DDP, DGP1, DPP, DRP, DMuP, PGuP1, PGP2, PVP2, DKP, DMP2,
PGuP2, PGoP, DPiP, PRP1 and DTP2 are positive (Plate 4.2d). All the thirty
Pseudomonas fluorescens isolates were positive for gelatin liquefaction (Plate 4.2e)

Starch was hydrolysed by only 12 isolates i.e., PVP1, PLP, DOP, DTP1, DMoP,
DMuP, DMP1, DBoP, PRP2, PGP2, PSP2 and DTP2 (Plate 4.2f).
All the isolates showed positive for citrate utilization (Plate 4.3a) and oxidase test
(Plate 4.3b). For TSI test 19 isolates i.e., PVP1, DBP, DMoP, PKP, PSP2, DGP2, DOP,
DTP1, DGP1, DPP, DMuP, PSmP, PGuP1, PGP2, DKP, DMP2, PGoP, PSP1 and DPiP
showed positive results (Plate 4.3c).
For H2S test 26 Pseudomonas fluorescens isolates PRP1, PVP1, DMoP, DBoP,
PKP, PSP2, DGP2, PLP, DOP, DTP1, DDP, DGP1, DPP, DRP, DMuP, PSmP, PGuP1,
PGP2, PVP2, PGP1, DMP1, DMP2, PGoP, DPiP, PRP1 and DTP2 were positive (Plate
4.3d) and all the isolates were positive for catalase test (Plate 4.3e), denitrification (Table
4.5).
In carbohydrate utilization test glucose, galactose and lactose sugars were used and
results are recorded (Table 4.5). Glucose was utilized by the 19 isolates DKP, PGP1,
PGoP, PLP, PSP1, DOP, DTP1, DBP, DPP, DMoP, DPiP, DMP1, DBoP, PGuP1, PRP2,
PGP2, PVP2, DGP2 and DMP2. Galactose was utilized by the 22 isolates PRP1, DKP,
PGP1, PVP1, PLP, PSP1, DDP, DBP, DPP, DMoP, DPiP, DRP, DMuP, DMP1, PSmP,
PGuP1, PKP, PRP2, PVP2, PSP2, DGP2 and PGuP2. Lactose was utilized by the 18
isolates PRP1, PGP1, PVP1, PGoP, PLP, PSP1, DTP1, DDP, DGP1, DPP, DMoP, DRP,
PGuP1, PKP, PGP2, PVP2, DTP2 and DGP2.
All the isolates showed negative reaction for indole production test and positive for
citrate utilization and gelatine liquefaction i.e., utilized citrate and liquified gelatine. The
isolates of P. fluorescens produced gelatinase enzyme in nutrient broth agar media
supplemented with gelatine substrate as 1% and gelatinase belongs to proteolytic enzyme
resulting in gelatinous hydrolysis. The P. fluorescens organisms produce the enzymes
catalase, oxidase and hence showed positive for the test. These tests confirm biochemically
the isolates as Pseudomonas fluorescens.
Akter et al. (2014) isolated 325 bacteria and 14 of them were identified as
fluorescent pseudomonads by morphological and biochemical characterization. Fifty
Pseudomonas fluorescens and 28 Rhizobium strains were isolated from rhizospheric soil
and root nodules of pigeonpea, biochemically characterized and identified as Pseudomonas
fluorescens and Rhizobium (Basha et al. 2014).
Anitha and Kumudini (2014) isolated Pseudomonas from fifteen rhizospheric
samples from different regions of India. They characterized morphologically and
biochemically and concluded as genus Pseudomonas. Paramageetham and Prasada Babu

(2013) isolated fluorescent pseudomonads from forest litter of Seshchalam hills. Among
the 34 isolates four isolates were identified as Pseudomonas fluorescens based on
morphological and biochemical characters.
Thirty five isolates of Pseudomonas fluorescens were isolated from the rizosphere
of rice fields by Meera and Balabaskar (2012). Among these, seven isolates were showed
bright fluorescence under UV light were further tested and all the cultural and biochemical
studies confirmed them to be P. fluorescens. Kumar et al. (2010) isolated root nodulating
Sinorhizobium fredii KCC5 and Pseudomonas fluorescens LPK2 and studied their
physiological and biochemical characterization which confirms the purity of these isolates.

4.3 Screening of Pseudomonas fluorescens isolates for PGPR activities

The isolated Pseudomonas fluorescens isolates were tested for their purity and
preserved in the department of Agricultural Microbiology & Bioenergy, College of
Agriculture, Rajendranagar, Hyderabad. Isolates were further screened for Plant growth
promoting activities in vitro.
4.3.1 Screening of pure isolates for PGPR properties
Plant growth promoting rhizobacteria (PGPR) are naturally occurring soil bacteria
that aggressively colonize plant roots & benefit plants by providing growth promotion. They
are reported to influence the growth, yield & nutrient uptake both by direct and indirect
mechanisms. Bacteria are most abundant among the PGPR. The use of PGPR offers an
attractive way to replace chemical fertilizers, pesticides. PGPR also modify root functioning,
improve plant nutrition and influence the physiology of the whole plant.
For identification of efficient PGPR strains with biocontrol and multiple activities,
rhizospheric isolates of Pseudomonas fluorescens were isolated and further studied to
understand their Plant Growth Promoting Properties (PGPR) under in vitro conditions. Pure
isolates were screened for mineral phosphate solubilization, ammonia and IAA production.
4.3.1.1 Phosphate solubilization ability of Pseudomonas fluorescens isolates
Among 30 Pseudomonas fluorescens isolates 23 isolates were able to solubilize
phosphate on Pikovskayas media (Plate 4.3a) containing Tri calcium phosphate (TCP) in
the range of 6 mm to 17.8 mm and depicted in Table 4.6. Among 23 isolates PVP1
recorded the highest solubilization zone (17.8 mm) followed by PRP2 (15.1 mm), DGP1
and DMuP (15.0 mm), DKP (14.8 mm), PSmP (14.7 mm), least was shown by DTP2 (6

mm) and the isolates which doesnt show solubilization are PGP1, PGoP, DPP, DPiP,
DBoP, PGP2 and DGP2 (Fig 4.2).
Isolates of Pseudomonas fluorescens species differ in the ability to produce
phosphatase enzyme and production of organic acids and hence showed different
solubilization efficiency.
Most of phosphorus in soil is present in the form of insoluble phosphates and
cannot be utilized by the plants (Pradhan and Sukla, 2006) and Phosphate solubilizing
bacteria solubilize the insoluble phosphates present in the soil by the phosphatase enzyme
activity and organic acid production. PGPR have been shown to solubilize precipitated
phosphates and enhance phosphate availability to rice that represent a possible mechanism
of plant growth promotion under field conditions (Verma et al. 2001). Pseudomonas spp. is
having P-solubilization activity of mineral phosphate solubilization by different organic
acid production such as succinic acid, mallic acid, oxalic acids etc.
Phosphate solubilizing bacteria (PSB) were isolated, identified and characterized by
Baliah and Begum (2015) and the selected strains were identified as Bacillus and
Pseudomonas spp. Alemu (2013) isolated twelve Pseudomonas fluorescens species and
tested for phosphate solubilization. All tested isolates of Pseudomonas fluorescens species
have a potential of phosphate solubilization on Pikovskayas media.
Twenty six Pseudomonas spp. were isolated and identified by Prasad et al. (2013)
and were in vitro screened for PGPR properties like Phosphate solubilization. The results
revealed that all Pseudomonas isolates were 76.9% positive for phosphate solubilization.
Upadhyay and Srivastava (2010) reported that Pseudomonas fluorescens strain solubilized
phosphorus and synthesized IAA.
Belkar and Gade (2012) isolated fifteen isolates of Pseudomonas fluorescens from
Vidarbha and Konkan region. The isolates showed positive response for siderophore
production and phosphate solubilization.
4.3.2 Production of plant growth promoting substances
Plant growth promoting substances such as auxins, gibberellins, ethylene substances
are produced by plant growth promoting rhizospheric bacteria. These are directly involved in
plant growth promotion. In the present study thirty Pseudomonas fluorescens isolates were
screened for indole acetic acid production for selection of efficient growth promoting
bacterial strain.

4.3.2.1 Indole acetic acid production

Among thirty bacterial isolates IAA production varied with supplementation of Ltryptophan. Out of thirty isolates only twenty eight showed positive for IAA production
(Plate 4.3e) of which highest was shown by PGP2 (62.1 g ml-1), followed by DTP1 (50.8 g
ml-1), DMoP (49.8 g ml-1) and the least was shown by the isolate PGuP1. The isolates which
doesnt showed IAA production are PKP and DGP2 (Table 4.7 and Fig 4.3).
IAA may function as important signal molecule in the regulation of plant
development. Isolates from the rhizosphere are more efficient auxin producers than isolates
from the bulk soil (Sarwar and Kremer, 1992) and IAA production by PGPR can vary among
different species and strains, and it is also influenced by culture condition, growth stage and
substrate availability (Mirza et al. 2001) and hence the Pseudomonas fluorescens isolates
showed different range of IAA production from 21.5 to 62.1 g ml-1
Similarly out of 21 isolates, 3 isolates (P3, P9, and P19) were screened for indole3-acetic acid (IAA). Isolate P19 was the best IAA producer strain (50.25 g ml-1) while
isolate P3 was lowest IAA producer (15.25 g ml-1) comparatively (Rai and Sharma,
2015). Pseudomonas fluorescens isolated by Jeyanthi and Ganesh (2013) and the effect of
L-tryptophan was studied and the highest yield of 58 g ml-1 on 72 hr was obtained using
0.5 mg ml-1 concentration.
Effect of IAA producing Pseudomonas fluorescens and Bacillus subtilis bacteria on
plant growth was studied. Both bacteria demonstrated increase in root length, shoot length,
root and shoot fresh and dry weight, on bacterial inoculated onion seeds over control
(Reetha et al. 2014).
Hussain and Srinivas (2013) isolated 35 isolates Pseudomonas and Azotobacter
each from rhizosphere of Acacia nilotica and Albizia lebbeck and reported 70% of the
isolates of Azotobacter and Pseudomonas produced IAA.
Verma et al. (2010) evaluated Rhizobium spp., Pseudomonas fluorescens, Bacillus
megaterium and Azotobacter chroococcum for plant growth promoting properties. All the
bacterial strains were found to be positive for IAA and phosphate solubilization.
4.3.2.2 Ammonia production
The isolates were tested for ammonia production by inoculating the isolates into 10
ml of sterilized peptone water in the test tubes.
Ammonia production was shown by all the isolates of which strong (+++) ammonia
production was shown by two isolates (DMP1, PLP), moderate (++) production was shown

by nine isolates i.e., PRP1, PVP1, DTP1, DBP, DMoP, PGuP1, PGP2, PVP2 and PGuP2
and the remaining nineteen isolates i.e., DBoP, PKP, PSP2, DGP2, DOP, DDP, DGP1,
DPP, DRP, DMuP, PSmP, DKP, PGP1, DMP2, PGoP, PSP1, DPiP, PRP1 and DTP2
showed weak (+) production of ammonia (Table 4.7 and Plate 4.3c)

4.4. To find efficient PGPR isolates for bio control activity

4.4.1 Production of Siderophores
Siderophore is one of the biocontrol mechanisms belonging to PGPR groups under
iron limiting condition. PGPR produces a range of siderophore which have a very high
affinity for iron. Therefore, the low availability of iron in the environment would suppress
the growth of pathogenic organisms including plant pathogenic fungi (Whipps, 2001).
Out of thirty isolates twenty three isolates produced siderophores (Table 4.7).
Strong production (+++) was shown by the nine isolates i.e., DKP, PVP1, DDP, DGP,
DPP, DMoP, DPiP, PGup1 and PGP2. Moderate production (++) was shown by eight
isolates i.e., PGP1, DOP, DTP1, DRP, DMP1, PSmP1, PVP2 and DGP2, Weak production
(+) was shown by six isolates i.e., PLP, DBoP, PSP2, DTP2, DMP2 and PGuP2 where as
seven isolates doesnt produce siderophores i.e., no production () (Plate 4.3b)
The production of siderophore that sequester iron in the root environment, making
it less available to competing deleterious microflora vary among the P. fluorescens
isolates because of the type and quantity of siderophore produced and this is the reason
siderophore production varied among the isolates.
Similar results obtained with by Alemu and Alemu (2015) twelve P. fluorescens
isolates were assessed in vitro for their plant growth promoting activity based on their
ability to produce hydrogen cyanide (HCN), siderophores, indole acetic acid (IAA),
ammonia and phosphate solubilization. The results indicated that most of the isolates tested
possess plant growth promoting traits.
Mandal and Kotasthane (2014) isolated fifty nine Pseudomonas fluorescens and the
amount of siderophore produced by P. fluorescens isolates were screened in iron deficient
succinate media and most of them were found positive for the production of siderophores.
Saranraj et al. (2013) isolated and characterized Pseudomonas fluorescens and
were tested for their efficiency of siderophore production. The isolate Pseudomonas
fluorescens (PS-8) showed maximum siderophore production and the least siderophore
production was showed by the Pseudomonas fluorescens isolate PS-4.

Anand and Kulothungan (2010) also showed that Pseudomonas fluorescens

isolated from Groundnut (Arachis hypogeae) produced maximum amount of secondary
metabolites (HCN and siderophores).
4.4.2 HCN Production
Hydrogen cyanide (HCN) is a secondary metabolite produced by many antagonistic
Pseudomonas flourescens species from glycine. All the thirty isolates of Pseudomonas
flourescens produced HCN under microaerophilic conditions (Plate 4.3d).
Out of thirty isolates, four isolates i.e., PRP1, PSP2, DTP2 and DMP2 proved to
produce strong (+++) for HCN production, moderate production (++) of HCN was
produced by the isolates DKP, PVP1, PLP, DMuP and PKP and remaining twenty one
isolates produced weak HCN (+) and the results are mentioned in the Table 4.7.
Jyothi and Reddy (2014) fifty five isolates of fluorescent Pseudomonas were tested
for their ability to produce secondary metabolites such as Hydrogen cyanide (HCN),
ammonia, salicylic acid (SA), indole acetic acid etc. Maximum production of secondary
metabolites (HCN 0.094 Abs 625; SA 4.68 mg ml-1; IAA-22.5 g ml-1) was found with
fluorescent Pseudomonas isolates.
Twenty Pseudomonad strains were isolated by Noori and Saud (2012) and screened
for their plant growth promoting activity. All the 20 tested isolates of Pseudomonads were
positive for the production of siderophores and HCN.
Supraja et al. (2011) isolated fifteen fluorescent Pseudomonas and the isolates
screened in vitro for their plant growth promoting traits like phosphate solubilization,
production of indole acetic acid (IAA), Hydrogen cyanide (HCN) and siderophore. Due to
production of HCN and siderophores, fluorescent Pseudomonas isolates inhibited the
growth of Fusarium moniliforme.
Ahmadzadeh and Sharirifi-tehrani (2009) identified the production of HCN by six
isolates out of the 41 selected Pseudomonads having good antagonistic activity against
Rhizoctonia solani.
4.4.3 Antagonistic Activity
Antagonistic activities of thirty isolates were checked against Rhizoctonia solani
under in vitro conditions using dual culture plate technique on PDA medium. All the thirty
Pseudomonas flourescens isolates inhibited Rhizoctonia solani maximum percent
inhibition 53.43% was shown by DMP1 on par results were found with DBP 51.53%

followed by PVP2 48.26%. 40-50% range inhibition was shown by the isolates DBoP
(45.41%), DTP1 (44.32%), PLP (42.76%), DMuP (42.75%) and the remaining twenty
three isolates showed inhibition within the range of 24 to 30% (Table 4.8 and Fig 4.4). The
least percent inhibition 24.89% was shown by the isolate PGP2 (Plate 4.5)
In our study isolated P. fluorescens also produced HCN, siderophores which was
responsible for the antagonistic activity of P. fluorescens against R. solani. The isolates
also inhibited the R. solani in dual culture method due to the production of secondary
metabolites.
The inhibitory effect of antagonistic bacteria such as B. subtilis and P. fluorescens
against growth reduction of phytopathogenic fungi may be due to the production of
hydrolytic enzymes that can degrade cell walls, iron-chelating siderophores, and several
cyclic lipodepsipeptides (Kim et al. 2008).
Kumar et al. (2014) isolated three Pseudomonas fluorescens and studied the
antagonistic activity against Rhizoctonia solani. Pseudomonas fluorescens strains P.f 07
were found most effective with the highest antagonistic activity against the fungal
pathogen and show maximum inhibition of mycelial growth of Rhizoctonia solani
(68.23%).
Dev and Dawande (2010) evaluated the antagonistic property of Trichoderma
spp. and Pseudomonas fluorescens against Rhizoctonia solani, which revealed that
the

mycolytic

enzymes produced by these antagonists suppressed the growth of

Rhizoctonia solani.

4.4 Pot culture study for the control of sheath blight of rice
The results for the biological control of sheath blight of rice experiment was
conducted during Rabi, 2014-15 with Completely Randomized Design, in the green house,
College of Agriculture, Rajendranagar, Hyderabad are presented and discussed here.
Experimental data were statistically analyzed apportioned under various headings and
subheadings, furnished in tables and illustrated through figures wherever necessary. The
experimental data on various growth parameters, yield attributes, scoring, percent disease
index and reduction in disease severity are given in the tables.
4.4.1 Screening and selection of Pseudomonas fluorescens isolates
In the preliminary screening of 30 Pseudomonas fluorescens isolates for

antagonistic activity against Rhizoctonia solani under in vitro conditions, three strains
DMP1 (53.43%), DBP (51.53%) and PVP2 (48.26%) were found to inhibit mycelia growth
significantly (Table 4.7). These three isolates were used for the pot culture study.
4.4.2 Influence of Pseudomonas fluorescens on growth parameters of rice
4.4.2.1 Percent germination
The effect of DBP, DMP1 and PVP2 on germination of rice was conducted on
water agar plates. The results showed that highest percent germination was found with the
seeds inoculated with PVP2 i.e., 100% followed by DBP (98.35%) on par results found
with DMP1 (96.65%) when compared with the control (90%) (Table 4.9).
The isolates obtained in our study when seed treated with the rice grains enhanced
germination percentage. Pseudomonas fluorescens is one among the PGPR having the
ability to produce plant growth regulators such as gibberlins, cytokinins and indole acetic
acid etc. induced higher percentage of germination in the seeds when compared with the
untreated control.
Inoculated rice in the nursery by Pseudomonas flourescens (PGPR Pf) bacterial
inoculation caused an increase in seed germination, seedling vigour characters, yield and
most of its attributes (Elekhtyar, 2015).
4.4.2.2 Vigour index
The seedling vigour significantly increased on seed treatment with the
Pseudomonas fluorescens isolates DBP, DMP1 and PVP2. The seed vigour index in
untreated control was 2368.59 which was increased to 2696.50 (isolate DBP), 3089.31
(PVP2) and 3115.98 (DMP1) when the seeds were treated with P. fluorescens isolates. The
efficiency of the strains varied in terms of vigour index was given in the Table 4.9.
P. fluorescens treated seedlings placed on the roll towel observed to have an
increased vigour index of 3115.98 with the isolate DMP1 when compared with untreated
control (2368.59) due to the production of higher amount of plant growth promoting
attributes by DMP1. The vigour index with isolates PVP2 and DMP1 were on par.
Shivalingaiah and Umesha (2013) studied the effect of P. fluorescens on seed
germination and seedling vigour, along with the in vivo disease protection under
greenhouse conditions showed significant improvement over the controls. Nandakumar et.
al. (2001) found two P. fluorescens strains PF1 and FP7 inhibited mycelia growth of

sheath blight fungus Rhizoctonia solani and increased the seedling vigour of rice plants in
vitro.
4.4.2.3 Plant height
Plant heights at 30, 60 and 90 DAT are measured and the values are mentioned in
Table 4.10 and Fig 4.5. Plant height at 30 DAT was found significantly highest in T5 (seed
treatment with DMP1 + Rhizoctonia solani inoculation) 56 cm followed by 52 cm with T6
(root dipping with DMP1 + Rhizoctonia solani inoculation) and on par results are found
with T8 (seed treatment with DBP + Rhizoctonia solani inoculation) 51.67 cm and T2 (seed
treatment with PVP2 + Rhizoctonia solani inoculation) 51.33 cm, and least was found with
control (T1: inoculation of Rhizoctonia solani alone) i.e., 42 cm.
Plant height at 60 DAT was found highest in T5 (seed treatment with DMP1 +
Rhizoctonia solani inoculation) 74 cm on par with 72 cm T8 (seed treatment with DBP +
Rhizoctonia solani inoculation) and T2 (seed treatment with PVP2 + Rhizoctonia solani
inoculation) followed by T6 (root dipping with DMP1 + Rhizoctonia solani inoculation)
70.70 cm on par with T3 (root dipping with PVP2 + Rhizoctonia solani inoculation) and
least was found with control (T1: inoculation of Rhizoctonia solani alone) i.e., 57.3 cm
(Table 4.10).
Plant height at 90 DAT was found significantly highest in T5 (seed treatment with
DMP1 + Rhizoctonia solani inoculation) 82.6 cm followed by T8 (seed treatment with DBP
+ Rhizoctonia solani inoculation) 82.1 cm. Next highest to it is 78.9 cm with T6 (root
dipping with DMP1 + Rhizoctonia solani inoculation) on par with T2 (seed treatment with
PVP2 + Rhizoctonia solani inoculation) 78.3 cm and T9 (root dipping with DBP +
Rhizoctonia solani inoculation) 78.0 cm and least was found with control (T1: inoculation
of Rhizoctonia solani alone) i.e., 68.3 cm (Table 4.10).
Seed treatment, root dipping and foliar application with plant growth promoting
rhizobacteria bioformulations significantly enhanced the growth of rice plants compared
with the control. Seed treatment has shown effective growth with the organisms DMP1,
DBP and PVP2 when considered individually compared with root dipping and foliar spray
as the inocula to rest of them has been added at later stage. When the treatments are
considered, T5 seed treatment with DMP1 had shown best plant growth promotion as it is
having the ability to produce siderophores, IAA, Ammonia and HCN followed by T8 i.e.,
seed treatment with DBP having IAA, ammonia and HCN producing ability.
Similarly a 20% increase in the plant height of 30 day old rice plants was observed
in response to PGPR application (Ashraffuzzaman et al. 2009).

Treatment with Pseudomonas fluorescens strains promoted plant growth in terms of

increased plant height and number of tillers and ultimately grain yield (Nandakumar et al.
2001). Application of Pseudomonas fluorescens had positive influence on growth
parameters of rice such as plant height, root length, shoot weight, root weight and number
of tillers per hill (Seenivasan, 2011).
4.4.2.4 Number of tillers per plant
Number of tillers at 30, 60 and 90 DAT are counted and mentioned in Table 4.11
and Fig 4.6. Number of tillers at 30 DAT was found significantly highest with the
treatment T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation) 13.7 followed
by 12.3 with T6 (root dipping with DMP1 + Rhizoctonia solani inoculation), next highest
tiller number is 11 with T8 (seed treatment with DBP + Rhizoctonia solani inoculation) and
the least was found with control (T1: inoculation of Rhizoctonia solani alone) 8.3.
Number of tillers at 60 DAT was found significantly highest with the treatment T5
(seed treatment with DMP1 + Rhizoctonia solani inoculation) 17.3 (Table. 4.11) next is
16.3 with T6 (root dipping with DMP1 + Rhizoctonia solani inoculation) followed by 15.3
with T8 (seed treatment with DBP + Rhizoctonia solani inoculation) and least was found
with control (T1: inoculation of Rhizoctonia solani alone) 11.3.
Number of tillers at 90 DAT was found significantly highest 17.3 with the
treatment T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation) followed by
14.7 with T8 (seed treatment with DBP + Rhizoctonia solani inoculation) on par with T7
(foliar spray with DMP1 + Rhizoctonia solani inoculation), T2 (seed treatment with PVP2
+ Rhizoctonia solani inoculation) 13.7 and least was found with control (T1: inoculation of
Rhizoctonia solani alone) 11 (Table 4.11).
In our study along with the inhibition of Rhizoctonia solani, seed treated plants
have shown good plant growth promoting traits compared with the root dipped and foliar
sprayed plants.
Besides plant height, increase in the number of tillers was reported in the rice plants
treated with the plant growth promoting rhizobacteria (Salamone et al. 2012).
Tiwari and Thimurthy (2009) reported that isolate Pseudomonas fluorescens Pfr1
promoted the shoot length and number of tillers in rice and also effectively reduced the
sheath blight severity when applied as seedling treatment with one or two foliar sprays.

4.4.2.5 Number of panicles per plant

Number of panicles at 90 DAT was found highest 13.7 with the treatment T5 (seed
treatment with DMP1 + Rhizoctonia solani inoculation) on par with T8 (seed treatment
with DBP + Rhizoctonia solani inoculation) 13.3 followed by 12.7 with T2 (seed treatment
with PVP2 + Rhizoctonia solani inoculation), T6 (root dipping with DMP1 + Rhizoctonia
solani inoculation) and least was found with control (T1: inoculation of Rhizoctonia solani
alone) 8.7 (Table 4.12 and Fig 4.7).
Elekhtyarthe (2015) reported similar results. The application of Pseudomonas
flourescens (PGPR Pf) in the permanent field improved the yield and its attributes. Such as
number of panicles m-2, number of grains panicle-1, percentage of filled grains, 1000 grain
weight, nitrogen uptake in grain (Kg fed-1), nitrogen uptake in straw (Kg fed-1), grain yield
and straw yield.
4.4.2.6 Leaf Area Index (LAI)
LAI values at 60, 90 DAT was given in the Table 4.12 and Fig 4.8. LAI at 60 DAT
was found significantly highest with the treatment T5 (seed treatment with DMP1 +
Rhizoctonia solani inoculation) 4.71 followed by 4.60 with T6 (root dipping with DMP1 +
Rhizoctonia solani inoculation) and least was found with control (T1: inoculation of
Rhizoctonia solani alone) 4.04.
LAI at 90 DAT was found highest significance with the treatment T5 (seed
treatment with DMP1 + Rhizoctonia solani inoculation) 2.3 followed by 2.04 with T8 (seed
treatment with DBP + Rhizoctonia solani inoculation), 1.90 with T6 (root dipping with
DMP1 + Rhizoctonia solani inoculation) and least was found with control (T1: inoculation
of Rhizoctonia solani alone) i.e., 1.44 (Table 4.12).
Leaf area was also found highest with the seed treated plants as the plant height and
leaf area are correlated. LAI at 90 DAT was decreased when compared with the LAI at 60
DAT as the crop reached period of harvest.
The boosting effect of Pf1 formulation on plant growth was evidenced by the fact
that the plant height, leaf area index, root length and dry matter production were the
maximum in P. fluorescens treated plants. (Meena and Marimuthu, 2012). Results of the
experiment demonstrated that dual inoculation of Azospirillum brasilense and P.
fluorescence rhizobacteria in lowland rice cultivation improved the leaf area index (LAI)
under low irrigation and nitrogen conditions. (Niknejhad et al. 2013)

4.4.2.7 Chlorophyll content

Chlorophyll content with SPAD meter was measured at 30, 60 and 90 DAT and
was mentioned in Table 4.13 and Fig 4.9. Chlorophyll content at 30 DAT was found
highest with the treatment T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation)
39.6 on par with T6 (root dipping with DMP1 + Rhizoctonia solani inoculation) 39.4, T8
(seed treatment with DBP + Rhizoctonia solani inoculation) 37.87, T9 (root dipping with
DBP + Rhizoctonia solani inoculation) 37.13 and least was found with control (T1:
inoculation of Rhizoctonia solani alone) i.e., 32.7.
Chlorophyll content at 60 DAT was found highest with the treatment T5 (seed
treatment with DMP1 + Rhizoctonia solani inoculation) 48.17 on par with T6 (root dipping
with DMP1 + Rhizoctonia solani inoculation) 47.87, T7 (foliar spray with DMP1 +
Rhizoctonia solani inoculation) 47.5, T8 (seed treatment with DBP + Rhizoctonia solani
inoculation) 47.43, T9 (root dipping with DBP + Rhizoctonia solani inoculation) 46.6, T2
(seed treatment with PVP2 + Rhizoctonia solani inoculation) 45.7 and least was found with
control (T1: inoculation of Rhizoctonia solani alone) i.e., 40.6 (Table 4.13).
Chlorophyll content at 90 DAT was found significantly highest with the treatment
T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation) 42.13. Next highest was
T6 (root dipping with DMP1 + Rhizoctonia solani inoculation) 37.13 followed by T2 (seed
treatment with PVP2 + Rhizoctonia solani inoculation) 35.27, T9 (root dipping with DBP +
Rhizoctonia solani inoculation) 33.29 and T7 (foliar spray with DMP1 + Rhizoctonia solani
inoculation) i.e., 30.79 (Table 4.13).
Chlorophyll content increased with increasing crop upto 60 DAT and then
decreased as the crop is inoculated with the Rhizoctonia solani and the result was found
best with the method seed treatment and treatment T5 as the organism DMP1 is showing
plant growth promotion as well as disease resistance more compared with the other
treatments.
The growth attributes namely, leaf area, chlorophyll content and consequently the
total biomass were also increased due to PGPR inoculation (Mia, 2010). Lenin and
Jayanthi (2012) reported that root inoculation of PGPR strains significantly increased
germination rate, vigour index and chlorophyll content compared with the control.
4.4.3 Scoring of sheath blight
Scoring of sheath blight was done according to 0-9 grade scale by inoculating

Rhizoctonia solani at 60 DAT (maximum tillering stage) and covered the plants with
polythene sheet (Plate 4.6a). Diseased plants with symptoms are scored (Plate 4.6b) and T5
(seed treatment with DMP1 + Rhizoctonia solani inoculation) showed resistance towards
sheath blight incidence with a score of 1.2 found significant. Followed by T6 (root dipping
with DMP1 + Rhizoctonia solani inoculation) with score of 2.0 and 2.4 with T8 (seed
treatment with DBP + Rhizoctonia solani inoculation) and more susceptibility to sheath
blight was observed with the treatment T1 i.e., control (inoculation with Rhizoctonia solani
alone) having score of 5.5 (Table 4.14).
Seed treatment method has shown resistance towards the disease as the inocula
have been added earlier in the beginning of the crop compared with the root dipping and
foliar spray. When the treatments were compared seed treatment with DMP1 (T5) showed
best resistance towards the disease i.e., with the organism DMP1 followed by root dipping
with DMP1 (T5) and seed treatment with DBP (T8) had shown similar effect as the
organisms were producing HCN and siderophores which showed antagonistic activity
towards Rhizoctonia solani.
Nadarajah et al. (2014) isolated two field isolates of Rhizoctonia solani from
infected paddy plants in Malaysia. Isolate 1802 was more virulent based on the disease
severity index values. This study shows that the disease severity index is a better mode of
scoring for resistance compared to lesion length.
4.4.4 Grain yield per plant
Grain yield was found highest with the treatment T5 (seed treatment with DMP1 +
Rhizoctonia solani inoculation) which was significant with other treatments 32.69 gms
followed by T8 (seed treatment with DBP + Rhizoctonia solani inoculation) 31.00 gms and
the least was found with control (T1: inoculation of Rhizoctonia solani alone) i.e., 20.47
gms (Table 4.14 and Fig 4.10).
In our study grain yield was found highest in the seed treated plants as they showed
more resistance towards the fungal pathogen Rhizoctonia solani.
Bacterization of rice cultivars with P. fluorescens enhanced plant height, number of
tillers, and grain yields from 3 to 160% (Sakthivel and Gnanamanickam, 1987).
Of the three rates viz., 2, 4 and 8 g/litre of the bacterial bio-agent (P. fluorescens)
tested, higher rate (8 g/litre) was found highly effective in reducing disease severity
(60.0%), incidence (35.6%) and increasing grain yield (33.8%) and 1000- grain weight
(12.9%) (Singh and Sinha, 2005).

4.4.5 Percent Disease Index (PDI)

Percent Disease Index (PDI) was calculated at 60, 70 and 80 DAT and was
mentioned in Table 4.15 and Fig 4.11. PDI at 60 DAT was found significantly best with
the treatment T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation) 33.69%
followed by 40.31% with T6 (root dipping with DMP1 + Rhizoctonia solani inoculation),
T8 (seed treatment with DBP + Rhizoctonia solani inoculation) 40.93%, T2 (seed treatment
with PVP2 + Rhizoctonia solani inoculation) 42.76% and T9 (root dipping with DBP +
Rhizoctonia solani inoculation) 42.8%. The disease index was found highest 58.79% with
the control (T1: inoculation of Rhizoctonia solani alone) (Plate 4.6c).
PDI at 70 DAT was found significantly highest with the treatment T5 (seed
treatment with DMP1 + Rhizoctonia solani inoculation) 36.70% followed by 43.34% with
T6 (root dipping with DMP1 + Rhizoctonia solani inoculation), T8 (seed treatment with
DBP + Rhizoctonia solani inoculation) 43.38%, T2 (seed treatment with PVP2 +
Rhizoctonia solani inoculation) 44.36. The disease index was found highest i.e., 62.23%
with the control (T1: inoculation of Rhizoctonia solani alone) (Table 4.15).
PDI at 80 DAT was found significantly highest with the treatment T5 (seed
treatment with DMP1 + Rhizoctonia solani inoculation) 38.65% followed by 45.28% with
T8 (seed treatment with DBP + Rhizoctonia solani inoculation), T6 (root dipping with
DMP1 + Rhizoctonia solani inoculation) 46.18% and T2 (seed treatment with PVP2 +
Rhizoctonia solani inoculation) 47.34%. The disease index was found 66.98% i.e., highest
with the control (T1: inoculation of Rhizoctonia solani alone) (Table 4.15).
In our study, application of the biocontrol agents as seed treatment reduced the PDI
to a maximum extent, as the fungal pathogen Rhizoctonia solani is a soil borne and seed
treatment inhibited the pathogen from the initial stages of plant growth followed by root
dipping and foliar spray (Plate 4.6c)
In seed treatment the bacterial bioagent appeared to move from seed to roots, stems
and leaves. The fluorescent Pseudomonads could be isolated from aerial parts of plants
grown from seeds treated with the bacteria (Colyer and Mount, 1984).
4.4.6 Reduction in disease severity
Reduction in disease severity was calculated at 60, 70 and 80 DAT (Table 4.16 and
Fig 4.12). In 60 DAT maximum reduction in disease severity was found with the treatment
T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation) 42.56% followed by

31.56% in T6 (root dipping with DMP1 + Rhizoctonia solani inoculation), 30.50% in T8

(seed treatment with DBP + Rhizoctonia solani inoculation) DBP + Rhizoctonia solani
inoculation) DBP + Rhizoctonia solani inoculation) when compared with the control (T1:
inoculation of Rhizoctonia solani alone)
In 70 DAT maximum reduction in disease severity was found with the treatment T5
(seed treatment with DMP1 + Rhizoctonia solani inoculation) 41.12% followed by 30.47%
in T6 (root dipping with DMP1 + Rhizoctonia solani inoculation) on par with 25.29% in T9
(foliar spray with DBP + Rhizoctonia solani inoculation) when compared with the control
(T1: inoculation of Rhizoctonia solani alone) (Table 4.16).
In 80 DAT maximum reduction in disease severity was found with the treatment T5
(seed treatment with DMP1 + Rhizoctonia solani inoculation) 43.81% followed by 34.54%
in T8 (seed treatment with DBP + Rhizoctonia solani inoculation), 33.08% in T6 (root
dipping with DMP1 + Rhizoctonia solani inoculation) when compared with the control (T1:
inoculation of Rhizoctonia solani alone).
Seed treated plants had shown resistance towards the disease and the reduction of
disease severity was found highest with the seed treated plants.
Similar results were observed with Singh and Sinha (2009) reported that maximum
reduction in disease severity (67.8%) along with increase in grain yield (31.6%) was
recorded when Rhizoctonia solani was applied 7 days after application of Pfr 1 followed
by Pfr 5 which resulted in 64.5 and 40.7% reduction in disease severity and incidence
respectively.
Nandakumar et al. (2001) reported that three PGPR strains PF1, FP7 and PB2 were
tested alone and in combinations for suppression of rice sheath blight disease and found
that the average mean of disease reduction was 29.2% for single strains and 45.1% for
mixtures.

Pseudomonas population count

Pseudomonas population
count(cfu107g-1 soil)

7.0
6.0

5.0
4.0
3.0
2.0
1.0

0.0
1

10

11

12

13

14

15

16

17

18

19

20

21

22

Different rhizospheric soils of Rice

Figure 4.1 Microbial population count of Pseudomonas in the rice rhizospheric soils of Rangareddy district
1 Rukanpet, 2- Kishtapur, 3- Gadsingapur, 4- Vidyarayapuri, 5- Govindapur, 6- Laknapur, 7- Sulthanpur, 8 Ootpalle, 9 Thimmai
-palle, 10 Doma, 11 Bachpalle, 12 Godgonpally, 13 Palepally, 14 Mothkur, 15 Pirampally, 16 Rangapur, 17 Mujahidp
-ur, 18 Mailaram, 19 Bompalle, 20 - Syed malkapur, 21 Gudur, 22 Khudwanpur

Isolates

Figure 4.3 Screening of Pseudomonas fluorescens isolates for IAA production

PGuP2

DMP2

DGP2

DTP2

PSP2

PVP2

PGP2

PRP2

PKP

PGuP1

PSmP

DBoP

DMP1

DMuP

DRP

DPiP

DMoP

DPP

DGP1

DBP

DDP

DTP1

DOP

PSP1

PLP

PGoP

PVP1

PGP1

DKP

PRP1

IAA production(g ml-1)

IAA production

70

60

50

40

30

20

10

Phosphate solubilization zone

Phosphate solubilization
18
16
14
12
10
8
6
4
2
0

Isolates
Figure 4.2 Screening of Pseudomonas fluorescens isolates for Phosphate solubilization

Antagonistic activity
60.00

Percent inhibition

50.00
40.00
30.00
20.00
10.00
0.00

Isolates
Figure 4.4 Antagonistic activity of Pseudomonas fluorescens isolates against Rhizoctonia solani

90.00

Plant height (cm)

80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
1

10

Treatments
Figure 4.5 Plant height (cm) of rice at 30, 60 and 90 DAT as influenced by
Pseudomonas fluorescens

18.0

Number of tillers

16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
1

10

Treatments
Figure 4.6 Tiller number of rice at 30, 60 and 90 DAT as influenced by Pseudomonas
fluorescens

16.0

Number of panicles

14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
1

10

Treatments
Figure 4.7 Panicle number of rice at 90 DAT as influenced by Pseudomonas
fluorescens

5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
1

LAI at 60 DAT

LAI at 90 DAT

Figure 4.8 Leaf area index of rice at 90 DAT as influenced by Pseudomonas

fluorescens

10

50.00

Chlorophyll content

45.00
40.00
35.00
30.00
25.00

30 DAT

20.00

60 DAT

15.00

90 DAT

10.00
5.00
0.00
1

10

Treatments
Figure 4.9 Chlorophyll content of rice at 90 DAT as influenced by Pseudomonas
fluorescens

35
30
25
20
15
10
5
0
1

Grain yield per plant (gms)

Figure 4.10 Grain yield per plant (gms)

10

Percent disease index (%)

70.00
60.00
50.00
40.00

PDI at 60 DAT

30.00

PDI at 70 DAT

20.00

PDI at 80 DAT

10.00
0.00
1

10

Treatments
Figure 4.11 Percent Disease Index of rice at 60,70 and 80 DAT

Reduction in disease severity (%)

45.00
40.00
35.00
30.00
25.00

60 DAT

20.00

70 DAT

15.00

80 DAT

10.00
5.00
0.00
1

10

Treatments
Figure 4.12 Reduction in Disease Severity of rice at 60, 70 and 80 DAT

Plate 4.1 Point map showing location details of soil sampling

(a)Pure culture of Pseudomonas fluorescens (b) Pseudomonas fluorescens under UV

on Kings B agar medium

(c)MR test

(e) Gelatin liquefaction

light

(d)VP test

(f) Starch hydrolysis

Plate 4.2 Cultural (a,b) and Biochemical characterization (c,d,e and f) of

Pseudomonas fluorescens isolates

(g) Citrate utilization

(h)Oxidase test

(i)TSI test

(j)H2S production

(k)Catalase test
Plate 4.3 Biochemical characterization (g,h,i,j and k) of Pseudomonas fluorescens
isolates

(a)Phosphate solubilisation

(b)Siderophore production

(c) Ammonia production

(d) HCN production

(e) IAA production

Plate 4.4 Plant growth promoting attributes of Pseudomonas fluorescens isolates

Isolate PVP2

Isolate DMP1

Isolate DBP
Plate 4.5 Antagonistic activity of Pseudomonas fluorescens against Rhizoctonia
solani

a) Rice plants covered with covers to maintain humidity

b) Rice plants showing sheath blight symptoms

c) Comparision of T5 treatment with T7 treatment

Plate 4.6 Pot culture study of antagonistic activity of Pseudomonas fluorescens

against Rhizoctonia solani causing sheath blight in rice

Table 4.3. Cultural and morphological characteristics of Pseudomonas fluorescens isolates on Kings B medium
Isolate

Isolate
name

Size

Shape

Color

Elevation

PRP1

Small

Round

Yellowish
green

Convex

DKP

Medium

Round

Dull white

Convex

PGP1

Small

Round

Bluish white

Convex

PVP1

Small

Round

Yellow

Convex

PGoP

Small

Round

PLP

Small

Irregular

PSP1

Small

Irregular

Green

Convex

DOP

Medium

Round

White

Convex

DTP1

Small

Irregular

White

Convex

10

DDP

Small

Round

Bluish white

Convex

11

DBP

Small

Round

Yellow

Convex

12

DGP1

Small

Irregular

Yellowish
green

Convex

13

DPP

Medium

Irregular

Dull white

Convex

14

DMoP

Small

Round

Yellow

Convex

15

DPiP

Small

Round

Green

Convex

Yellowish
green
Yellowish
green

Convex
Convex

Surface
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny

Margin

Pigmentation

Gram
reaction

Shape

Sporulation

Regular

Yellowish green

Negative

Rod

Negative

Regular

Bluish green

Negative

Rod

Negative

Regular

Bluish green

Negative

Rod

Negative

Regular

Yellowish green

Negative

Rod

Negative

Regular

Dark green

Negative

Rod

Negative

Irregular

Light green

Negative

Rod

Negative

Irregular

Dark green

Negative

Rod

Negative

Regular

Light green

Negative

Rod

Negative

Irregular

Light green

Negative

Rod

Negative

Regular

Light green

Negative

Rod

Negative

Regular

Yellowish green

Negative

Rod

Negative

Irregular

Light green

Negative

Rod

Negative

Irregular

Light green

Negative

Rod

Negative

Regular

Yellowish green

Negative

Rod

Negative

Regular

Dark green

Negative

Rod

Negative

Table 4.3.(cont.)

Isolate

Isolate
name

Size

Shape

Color

Elevation

16

DRP

Small

Round

Dull white

Convex

17

DMuP

Small

Irregular

Dull white

Convex

18

DMP1

Small

Round

Dull white

Convex

19

DBoP

Medium

Round

Yellow

Convex

20

PSmP

Small

Irregular

Dull white

Convex

21

PGuP1

Small

Irregular

Green

Convex

22

PKP

Small

Round

23

PRP2

Small

Irregular

24

PGP2

Medium

Round

25

PVP2

Small

Round

Green

Convex

26

PSP2

Small

Irregular

Yellow

Convex

27

DTP2

Small

Irregular

Green

Convex

28

DGP2

Medium

Round

Yellow

Convex

29

DMP2

Small

Irregular

Dull white

Convex

30

PGuP2

Small

Round

White

Convex

Yellowish
white
Whitish
green
Yellowish
green

Convex
Convex
Convex

Surface
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny
Smooth
shiny

Margin

Pigmentation

Gram
reaction

Shape

Sporulation

Regular

Light green

Negative

Rod

Negative

Irregular

Light green

Negative

Rod

Negative

Regular

Bluish green

Negative

Rod

Negative

Regular

Yellowish green

Negative

Rod

Negative

Irregular

Light green

Negative

Rod

Negative

Irregular

Light green

Negative

Rod

Negative

Regular

Yellowish green

Negative

Rod

Negative

Irregular

Dark green

Negative

Rod

Negative

Regular

Light green

Negative

Rod

Negative

Regular

Light green

Negative

Rod

Negative

Irregular

Yellowish green

Negative

Rod

Negative

Irregular

Dark green

Negative

Rod

Negative

Regular

Yellowish green

Negative

Rod

Negative

Irregular

Bluish green

Negative

Rod

Negative

Regular

Bluish green

Negative

Rod

Negative

Table 4.1. Location details of the isolates from Rice crop along with GPS No. , Latitudes, Longitudes, Elevation and Soil type
S.No.

GPS No.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

50
52
57
61
65
69
108
109
110
111
112
114
115
116
118
119
120
121
123
124
128
129

Latitudes

Longitudes

1709 49.4
775040.5
1708 02.4
774747.3
1709 29.5
774736.8
1709 59.0
775146.2
1711 19.4
775033.9
1712 35.2
774951.5
1709 49.3
775200.8
1707 52.3
775123.9
1706 08.2
775026.8
1705 22.7
775039.4
1706 07.8
775201.1
1705 07.4
775156.9
1704 41.3
774957.8
1703 38.6
775040.8
1702 42.8
775226.2
1701 52.2
775223.8
1703 10.5
775226.3
1704 45.8
775201.0
1706 32.5
775211.5
1709 13.2
775346.9
1706 51.9
775412.9
1708 09.1
775602.5
*R - Rhizospheric soil

Elevation
(feet)
1904
1802
1767
1984
1901
1812
2010
2026
1925
1926
1933
1939
1864
1936
2028
2003
2100
1916
1959
2053
2066
2176

Name of the farmer

Village

Narsingulu
Rukanpet
Nizamuddin
Kishtapur
Krishnaiah
Gadsingapur
Narayana
Vidyarayapuri
Manipal reddy
Govindapur
Venkataiah
Laknapur
Ramaiah
Sulthanpur
Masireddy
Ootpalle
Gopal reddy
Thimmaipalle
N.Venkataiah
Doma
Narsimhumulu
Bachpalle
Balu
Godgonpally
Venkaiah naidu
Palepally
Benya naik
Mothkur
Veerabrahmam
Pirampally
Raju
Rangapur
Devaiah
Mujahidpur
Ramesh
Mailaram
Venkat rao
Bompalle
Basavaiah
Syed malkapur
Kumar
Gudur
Subbareddy
Khudwanpur
**NR - Non rhizospheric soil

Mandal

Soil type

*R/**NR soil

Parigi
Doma
Parigi
Parigi
Parigi
Parigi
Parigi
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Parigi
Parigi
Parigi

Black
Black
Black
Black
Red
Black
Black
Black
Black
Black
Black
Red
Black
Black
Black
Black
Black
Red
Black
Red
Black
Black

R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

Table 4.4. Naming of the Pseudomonas fluorescens isolates according to the place
of collection
S.No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

Area
Rukanpet
Kishtapur
Gadsingapur
Vidyarayapuri
Govindapur
Laknapur
Sulthanpur
Ootpalle
Thimmaipalle
Doma
Bachpalle
Godgonpally
Palepally
Mothkur
Pirampally
Rangapur
Mujahidpur
Mailaram
Bompalle
Syed malkapur
Gudur
Khudwanpur

Mandal
Parigi
Doma
Parigi
Parigi
Parigi
Parigi
Parigi
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Parigi
Parigi
Parigi

Soil type
Black
Black
Black
Black
Red
Black
Black
Black
Black
Black
Black
Red
Black
Black
Black
Black
Black
Red
Black
Red
Black
Black

Crop
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice
Rice

Abbrevation
PRP1, PRP2
DKP
PGP1, PGP2
PVP1, PVP2
PGoP
PLP
PSP1,PSP2
DOP
DTP1, DTP2
DDP
DBP
DGP1, DGP2
DPP
DMoP
DPiP
DRP
DMuP
DMP1, DMP2
DBoP
PSmP
PGuP1, PGuP2
PKP

Table 4.2. Pseudomonas population in the rice rhizosphere at different places

S.No.

Area

Mandal

Soil type

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

Rukanpet
Kishtapur
Gadsingapur
Vidyarayapuri
Govindapur
Laknapur
Sulthanpur
Ootpalle
Thimmaipalle
Doma
Bachpalle
Godgonpally
Palepally
Mothkur
Pirampally
Rangapur
Mujahidpur
Mailaram
Bompalle
Syed malkapur
Gudur
Khudwanpur

Parigi
Doma
Parigi
Parigi
Parigi
Parigi
Parigi
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Doma
Parigi
Parigi
Parigi

Black
Black
Black
Black
Red
Black
Black
Black
Black
Black
Black
Red
Black
Black
Black
Black
Black
Red
Black
Red
Black
Black

*R - Rhizospheric soil
**NR- Non rhizospheric soil

Rhizospheric
/ Non
Rhizospheric
soil
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

Pseudomonas
population (cfu
x 107g-1 soil)
4.2
5.8
2.9
1.2
2.2
2.6
1.8
3.0
1.8
2.6
1.5
3.5
1.4
3.8
2.4
6.7
2.1
5.0
1.6
3.4
2.4
4.1

Table 4.7. Screening of Pseudomonas fluorescens isolates for Plant Growth

Promoting Activities in vitro
Isolate
no.

Isolate
name

Siderophore
production

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

PRP1
DKP
PGP1
PVP1
PGoP
PLP
PSP1
DOP
DTP1
DDP
DBP
DGP1
DPP
DMoP
DPiP
DRP
DMuP
DMP1
DBoP
PSmP
PGuP1
PKP
PRP2
PGP2
PVP2
PSP2
DTP2
DGP2
DMP2
PGuP2
CD
SE(d)

+++
++
+++
+
++
++
+++
+++
+++
+++
+++
++
++
+
++
+++
+++
++
+
+
++
+
+

No production
+ Weak production

IAA
production
g/ml
32.4
31.6
37.7
32.7
31.4
40.6
38.3
45.5
50.8
39.1
33.5
31.5
39.8
49.8
23.0
41.5
29.5
25.2
29.9
24.4
21.5
0
32.4
62.1
36.5
32.7
23.2
0
23.4
27.7
1.854
0.925

IAA - Indole Acetic Acid

++ Moderate production

Ammonia
production

HCN
production

++
+
+
++
+
+++
+
+
++
+
++
+
+
++
+
+
+
+++
+
+
++
+
+
++
++
+
+
+
+
++

+++
++
+
++
+
++
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
++
+
+
+
+++
+++
+
+++
+

HCN Hydrogen Cyanide

+++ Strong production

Table 4.6. Screening of Pseudomonas fluorescens isolates for Phosphate

solubilisation in vitro

Isolate
name
PRP1
DKP
PGP1
PVP1
PGoP
PLP
PSP1
DOP
DTP1
DDP
DBP
DGP1
DPP
DMoP
DPiP
DRP
DMuP
DMP1
DBoP
PSmP
PGuP1
PKP
PRP2
PGP2
PVP2
PSP2
DTP2
DGP2
DMP2
PGuP2
CD
SE(d)

Phosphate solubilisation
zone diameter (mm)
Solubilization Efficiency
Solubilization
Culture diameter
(%)
zone
(mm)
139.1
12.8
9.2
164.4
14.8
9
0.0
0
0
254.3
17.8
7
0.0
0
0
190.0
9.5
5
172.6
10.7
6.2
136.5
8.6
6.3
150.8
9.5
6.3
0.0
0
0
229.2
11
4.8
238.1
15
6.3
150.0
13.5
9
120.0
12
10
0.0
0
0
195.1
8
4.1
202.7
15
7.4
165.0
6.6
4
0.0
0
0
204.2
14.7
7.2
206.7
6.2
3
227.5
9.1
4
215.7
15.1
7
0.0
0
0
208.0
10.4
5
310.0
12.4
4
150.0
6
4
0.0
0
0
226.6
14.5
6.4
188.5
11.5
6.1
0.731
0.364

Table 4.8. Antagonistic activity of Pseudomonas fluorescens against Rhizoctonia

Solani by dual culture method
Isolate no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Isolate name
PRP1
DKP
PGP1
PVP1
PGoP
PLP
PSP1
DOP
DTP1
DDP
DBP
DGP1
DPP
DMoP
DPiP
DRP
DMuP
DMP1
DBoP
PSmP
PGuP1
PKP
PRP2
PGP2
PVP2
PSP2
DTP2
DGP2
DMP2
PGuP2
control
CD
SE(m)

Percent inhibition (%)

39.03
30.33
33.78
38.15
34.33
42.76
37.55
25.69
44.32
34.10
51.53
32.29
33.14
38.64
35.38
35.89
42.75
53.43
45.41
36.22
35.23
37.70
30.62
24.89
48.26
38.49
32.65
37.09
38.01
35.74
0
2.709
0.955

Inhibition zone (mm)

3.00
0.00
2.00
2.00
2.00
4.00
3.00
0.00
4.00
2.00
5.00
2.00
2.00
3.00
3.00
3.00
4.00
5.00
4.00
3.00
2.00
2.00
1.00
0.00
5.00
3.00
2.00
3.00
3.00
2.00

Table 4.9. Percent germination and Vigour index of Pseudomonas fluorescens

strains treated seeds
Organism/strain

Percent germination

Vigour index

Control

90

2368.59

DBP

98.35

2696.50

DMP1

96.65

3115.98

PVP2

100

3089.31

CD

0.781

242.5

SE(m)

0.236

73.2

Table 4.10. Influence of Pseudomonas fluorescens isolates on plant height of rice

Plant height(cm)
Treatments
30 DAT

60 DAT

90 DAT

T1

42.00

57.30

68.20

T2

51.33

72.00

78.30

T3

47.67

70.00

75.40

T4

45.67

59.70

73.30

T5

56.00

74.00

82.60

T6

52.00

70.70

78.90

T7

42.33

64.00

75.60

T8

51.67

72.00

82.10

T9

48.00

63.00

78.00

T10

45.67

61.00

69.30

CD

3.525

2.236

1.250

SE(m)

1.187

0.753

0.421

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

Table 4.11. Influence of Pseudomonas fluorescens isolates on number of tillers in

rice crop
Number of tillers plant-1
Treatments
30 DAT

60 DAT

90 DAT

T1

8.3

11.3

11.3

T2

10.3

14.0

13.7

T3

9.7

13.3

13.3

T4

9.3

12.7

12.0

T5

13.7

17.3

17.3

T6

12.3

16.0

14.3

T7

10.3

14.3

13.7

T8

11.0

15.0

14.7

T9

10.7

13.7

13.3

T10

10.3

12.7

12.3

CD

0.939

1.129

1.129

SE(m)

0.316

0.380

0.380

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

Table 4.12. Influence of Pseudomonas fluorescens isolates on Panicle number and

Leaf Area Index (LAI) of rice

Treatments

Number of
panicles

Leaf Area Index

90 DAT

60 DAT

90 DAT

T1

8.7

4.04

1.44

T2

12.7

4.48

1.71

T3

10.3

4.13

1.60

T4

9.7

4.12

1.53

T5

13.7

4.71

2.30

T6

12.7

4.60

1.90

T7

12.0

4.40

1.89

T8

13.3

4.57

2.04

T9

11.3

4.23

1.78

T10

9.7

4.08

1.70

CD

0.939

0.032

0.039

SE(m)

0.316

0.011

0.014

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

Table 4.13. Influence of Pseudomonas fluorescens isolates on Chlorophyll content

of rice
Chlorophyll content
Treatments

30 DAT

60 DAT

90 DAT

T1

32.70

40.60

23.78

T2

36.07

45.70

35.27

T3

35.40

44.50

29.32

T4

34.53

42.57

27.39

T5

39.60

48.17

42.13

T6

39.40

47.87

37.13

T7

36.63

47.50

30.79

T8

37.87

47.43

40.15

T9

37.13

46.60

33.29

T10

33.33

44.23

25.77

CD

2.596

2.998

2.705

SE(m)

0.874

1.009

0.508

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

Table 4.15. Percent Disease Index (PDI) of rice Inoculated with Rhizoctonia Solani
PDI
Treatments

60 DAT

70 DAT

80 DAT

T1

58.79

62.33

68.96

T2

42.76

44.36

47.34

T3

48.99

52.74

57.04

T4

55.67

57.25

60.08

T5

33.69

36.70

38.65

T6

40.31

43.34

46.18

T7

49.52

53.08

55.53

T8

40.93

43.38

45.28

T9

42.80

46.57

49.29

T10

53.92

59.71

63.45

CD

3.493

3.128

2.209

SE(m)

1.176

1.053

0.744

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

Table 4.16. Reduction in disease severity of rice infected with Rhizoctonia Solani at
different intervals
Percent Reduction

Treatments
60 DAT

70 DAT

80 DAT

T1

0.00

0.00

0.00

T2

27.40

28.84

31.37

T3

16.82

15.39

17.36

T4

8.65

8.16

12.80

T5

42.56

41.12

40.81

T6

31.56

30.47

33.08

T7

15.92

14.85

19.61

T8

30.50

30.41

34.54

T9

27.33

25.29

28.52

T10

5.48

4.21

8.03

CD

0.489

0.578

0.629

SE(m)

0.165

0.195

0.212

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

Table 4.5. Biochemical characterization of Pseudomonas fluorescens isolates from rice rhizosphere of Rangareddy district
S.No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Isolate
PRP1
DKP
PGP1
PVP1
PGoP
PLP
PSP1
DOP
DTP1
DDP
DBP
DGP1
DPP
DMoP
DPiP
DRP
DMuP
DMP1
DBoP
PSmP
PGuP1
PKP
PRP2
PGP2
PVP2
PSP2
DTP2
DGP2
DMP2
PGuP2
+ positive

Indole
test
-

MR VP
test test
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
negative

Carbohydrate utilization
Citrate
Starch
Gelatin
TSI
Catalase Oxidase
H2S
utilization
hydrolysis liquefaction
test
Glucose
Galactose Lactose Denitrification
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
MR-Methyl Red test
VP-Voges Prauskers test
H2S- Hydrogen sulphide test TSI-Triple Sugar Iron test

Table 4.14. Score (0-9 scale) of sheath blight and grain yield per plant (gms)
Treatments

Score

Grain yield plant-1(gms)

T1

5.5

20.47

T2

2.9

28.63

T3

3.1

26.95

T4

4.0

26.37

T5

1.2

32.69

T6

2.0

28.88

T7

2.5

27.82

T8

2.4

31.00

T9

2.8

26.58

T10

3.6

24.73

CD

0.141

0.900

SE(m)

0.047

0.303

T1: Inoculation of Rhizoctonia solani alone

T2: Seed treatment with PVP2 + inoculation of Rhizoctonia solani
T3: Root dipping with PVP2 + inoculation of Rhizoctonia solani
T4: Foliar spray with PVP2 + inoculation of Rhizoctonia solani
T5: Seed treatment with DMP1 + inoculation of Rhizoctonia solani
T6: Root dipping with DMP1 + inoculation of Rhizoctonia solani
T7: Foliar spray with DMP1 + inoculation of Rhizoctonia solani
T8: Seed treatment with DBP + inoculation of Rhizoctonia solani
T9: Root dipping with DBP + inoculation of Rhizoctonia solani
T10: Foliar spray with DBP + inoculation of Rhizoctonia solani

Chapter V

SUMMARY AND CONCLUSIONS

Biocontrol of the disease using plant growth promoting rhizobacteria (PGPR) is
a potential substitute to the presently available chemical control methods and it is
described as cheap and non hazardous strategy to reduce crop damage caused by plant
pathogens. Plant growth promoting rhizobacteria (PGPR) are a group of
microorganisms in the rhizosphere that promote plant growth by increasing nutrient
availability and used as inoculants for biofertilization, phytostimulation and biocontrol.
Pseudomonas fluorescens is one among the PGPR known to inhibit plant pathogenic
fungi by the production of secondary metabolites like antibiotics, Fe-chelating
siderophores, and cyanide. The present investigation focussed towards isolation of
Pseudomonas fluorescens from rice rhizosphere and assessment of antagonistic activity
in vitro and in vivo against Rhizoctonia solani causing sheath blight disease
Parigi and Doma mandals of Rangareddy district, Telangana state were selected
for the isolation of Pseudomonas fluorescens as there was high incidence of Sheath
blight disease. The fungal strain Rhizoctonia solani was obtained from Department of
Plant Pathology, College of Agriculture, Rajendranagar, Hyderabad. The growth media
used were Kings B for Pseudomonas fluorescens and Potato Dextrose Agar for
Rhizoctonia solani.
Twenty two soil samples were collected from twenty two villages of
Rangareddy district.

Preliminary investigations involved isolation of Pseudomonas

fluorescens from rice rhizosphere. Dilution plate technique was followed and spread
plate method was used for isolation and enumeration of rhizobacteria. The obtained
isolates were visualised under UV light and thirty isolates showing fluorescence were
isolated and purified.
The isolates were characterized culturally, morphologically and biochemically
(IMVIC tests, oxidase test, catalase test, carbohydrate utilization test, denitrification,
H2S production, starch hydrolysis, gelatin liquefaction and ammonia production) and
further confirmed as Pseudomonas fluorescens. Further, all the thirty isolates were
screened in vitro for plant growth promoting attributes viz., phosphate solubilization,
ammonia production and IAA production. All the isolates produced ammonia and
strong production was seen in the isolates DMP1 and PLP. Phosphate solubilisation was
found highest with an efficiency of 250% in PSP2 with a zone of 15 mm and highest

zone was recorded in DMuP with a zone of 21 mm and solubilisation was absent in the
isolates PGP1, PGoP, DDP, DPiP, DBoP, PGP2 and DGP2. All the isolates produced
IAA except PKP and DGP2 and the highest IAA production was found with the isolate
PGP2 i.e., 62.8 g ml-1.
All the isolates were further screened for assessment of antagonism viz.,
siderophore production, HCN production and dual culture method. Except eight, all the
isolates produced siderophores and strong production was found with the nine isolates
and moderate production was found in eight isolates. HCN production was observed in
all the thirty isolates and strong production was seen in four isolates. In the dual culture
method all Pseudomonas flourescens isolates inhibited Rhizoctonia solani and
maximum inhibition was found with the isolates DMP1 (53.43%), DBP (51.53%) and
PVP2 (48.26%). The isolates inhibited the R. solani in dual culture method due to the
production of secondary metabolites.
To assess the plant growth promotion of the isolates percent germination and
vigour index was calculated with the seed inoculated rice grains on water agar plates
and roll towel respectively and found 100% germination with the isolate PVP2 and
3115.98 vigour index with DMP1 due to the production of higher amount of plant
growth promoting attributes by DMP1.
The biocontrol activity was further evaluated in pot culture of rice crop.
Rhizoctonia solani was challenge inoculated to the rice crop at the maximum tillering
stage and covered with a polythene sheet to increase the humidity and lead to the rapid
spread of the disease and lesions were observed after 2 days of inoculation. The
biocontrol agents were tested against Rhizoctonia solani challenge inoculated rice crop
by different methods of application viz., seed treatment, root dipping and foliar spray.
Seed treated biocontrol strains significantly improved growth and yield
parameters like plant height, number of tillers, number of panicles, Leaf area index,
chlorophyll content and grain yield per plant. Plant height at 30 DAT was found
significantly highest in T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation)
i.e., 56 cm, plant height at 60 and 90 DAT was found highest in T5 i.e., 74 cm and 82.6
cm respectively. Number of tillers at 30 DAT was found significantly highest in T5
(seed treatment with DMP1 + Rhizoctonia solani inoculation) i.e., 13.7 and number of
tillers at 60 and 90 DAT was found significantly highest in T5 i.e., 17.3 and 17.3
respectively.

Leaf area index at 60 and 90 DAT was found significantly highest with T5 (seed
treatment with DMP1 + Rhizoctonia solani inoculation) i.e., 4.71 and 2.3 respectively.
Leaf area was also found highest with the seed treated plants as the plant height and leaf
area are correlated. LAI at 90 DAT was decreased when compared with the LAI at 60
DAT as the crop reached period of harvest.
Chlorophyll content at 30, 60 DAT was found highest with the treatment T5
(seed treatment with DMP1 + Rhizoctonia solani inoculation) i.e., 39.6, 48.17
respectively and at 90 DAT was found significantly highest in T5 (seed treatment with
DMP1 + Rhizoctonia solani inoculation) i.e., 42.13. Chlorophyll content increased with
increasing crop upto 60 DAT and then decreased as the crop is inoculated with the
Rhizoctonia solani.
Seed treatment, root dipping and foliar application with plant growth promoting
rhizobacteria bioformulations significantly enhanced the growth and yield parameters of
rice plants compared with the control. When the individual treatments are considered,
T5 seed treatment with DMP1 had shown best plant growth promotion as it is having the
ability to produce siderophores, IAA, Ammonia and HCN followed by T8 i.e., seed
treatment with DBP having IAA, ammonia and HCN producing ability
Number of panicles at 90 DAT was found highest in T5 (seed treatment with
DMP1 + Rhizoctonia solani inoculation) i.e., 13.7 and found least with the control 8.7.
Grain yield per plant was found significantly highest in T5 (seed treatment with DMP1
+ Rhizoctonia solani inoculation) i.e., 32.69 gms.
Biocontrol agents showed more resistance towards Rhizoctonia solani when it
was seed treated with the Pseudomonas flourescens inoculum than with root dip and
foliar spray method. Percentage disease incidence at 60, 70 and 80 DAT was found low
with the treatment T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation) i.e.,
33.69, 36.70 and 38.65 respectively. Application of the biocontrol agents as seed
treatment reduced the PDI to a maximum extent, as the fungal pathogen Rhizoctonia
solani is a soil borne and seed treatment inhibited the pathogen from the initial stages of
plant growth followed by root dipping and foliar spray.
Reduction of disease severity at 60, 70 and 80 DAT was found best with the
seed treated treatment T5 (seed treatment with DMP1 + Rhizoctonia solani inoculation)
i.e., 42.56, 41.12 and 40.81 respectively compared with the other methods. Seed treated

plants had shown resistance towards the disease and the reduction of disease severity
was found highest with the seed treated plants.
Pseudomonas fluorescens not only served as biocontrol agent but also served as
PGPR and enhanced the plant growth. Hence, this PGPR serves farmers in many ways
like cost effective in minimising chemical application and ecofriendly as it helped in
plant growth promotion and controlled disease to a large extent.

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APPENDIX I
Composition of different growth media/reagents/indicators used
1. Glucose Broth
Peptone
Beef extract
Glucose
Distilled water
Bromo Cresol Purple (BCP) Solution
pH

5.00 g
3.00 g
5.00 g
1000 ml
15 ml
7.0 0.2

2. Glucose Peptone Agar

Glucose
Peptone
Agar
Distilled water
pH

5.00 g
10.00 g
15.00 g
1000 ml
7.0 0.2

3. Hofers Alkaline Medium

Yeast extract
Mannitol
Dipotassium hydrogen phosphate
Magnesium Sulphate
Sodium chloride
Thymol blue
Distilled water
pH

1.00 g
10.00 g
0.50 g
0.20 g
0.10 g
0.016 g
1000 ml
11.0

4. Lactose Agar Medium

Lactose
Yeast extract
Dipotassium hydrogen phosphate
Magnesium Sulphate
Sodium chloride
Calcium carbonate
Agar
Distilled water
pH

10.00 g
1.00 g
0.50 g
0.20 g
0.10 g
3.00 g
20.00 g
1000 ml
7.0

5. MR VP broth
Buffered peptone
Dextrose
Dipotassium phosphate
Distilled water
pH

7.00 g
5.00 g
5.00 g
1000 ml
6.9 0.2

6. Nutrient Agar
Peptone

5.00 g

Beef extract
Sodium chloride
Agar
Distilled water
pH

3.00 g
5.00 g
18.00 g
1000 ml
6.8 7.2

7. Nutrient Gelatin Agar

Peptone
Beef extract
Gelatin
Distilled water
pH

5.00 g
3.00 g
120.00 g
1000 ml
6.8 7.0

8. Pikovskayas Agar
Yeast extract
Dextrose
Calcium phosphate
Ammonium sulphate
Potassium chloride
Manganese sulphate
Magnesium Sulphate
Ferrous sulphate
Agar
Distilled water
pH

0.50 g
10.00 g
5.00 g
0.50 g
0.20 g
0.0001 g
0.10 g
0.0001 g
20.00 g
000 ml
7.0

9. Plate Count Agar

Casein enzymic hydrolysate
Yeast extract
Dextrose
Agar
Distilled water
pH
10. Potato Dextrose Agar
Potato infusion
Dextrose
Agar
Distilled water
pH
11. Simmons Citrate Agar
Magnesium Sulphate
Ammonium dihydrogen phosphate
Dipotassium phosphate
Sodium citrate
Sodium chloride
Bromothymol blue

5.00 g
2.00 g
1.00 g
0.00 g
1000 ml
7.0 0.2

200.00 g
20.00 g
18.00 g
1000 ml
5.6 0.2

0.20 g
1.00 g
1.00 g
2.00 g
5.00 g
0.08 g

Agar
Distilled water
pH

18.00 g
1000 ml
6.8 0.1

12. Starch Casein Agar

Starch
Casein powder
Agar
Distilled water
pH

10.00 g
1.00 g
18.00 g
1000 ml
7.2 0.2

13. Urea Broth

Yeast extract
Potassium dihydrogen phosphate
Disodium hydrogen phosphate
Urea
Phenol red
Distilled water
pH

0.10 g
9.10 g
9.50 g
20.00 g
0.01 g
1000 ml
6.8

14. Yeast Extract Mannitol Congored Agar

Yeast extract
Mannitol
Dipotassium hydrogen phosphate
Magnesium Sulphate
Sodium chloride
Congo red
Agar
Distilled water
pH

1.00 g
10.00 g
0.50 g
0.20 g
0.10 g
0.025 g
20.00 g
1000 ml
6.8 7.0

15. Yeast Extract Mannitol Broth

Yeast extract
Mannitol
Dipotassium hydrogen phosphate
Magnesium Sulphate
Sodium chloride
Calcium carbonate
Distilled water
pH

1.00 g
10.00 g
0.50 g
0.20 g
0.10 g
1.00 g
1000 ml
6.8 7.0

16. Pseudomonas Agar (For Fluorescein)

Pancreatic digest of casein
Peptide digest of animal tissue
Anhydrous dibasic potassium phosphate
Magnesium sulphate
Agar agar
pH

10 g
10 g
1.50 g
1.50 g
15.0 g
7.0

17. Nitrate Broth

Peptone
Beef extract
Potassium nitrate
Water
pH
18. Kings B agar medium
Peptone
K2HPO4
MgSO4
Glycerol
Agar
Distilled water
19. Gram Stain Solutions
a) Crystal violet solution
Crystal violet
Ammonium oxalate
Ethanol
Distilled water

5.0 g
3.0 g
5.0 g
1000 ml
6.8-7.0

16.0 g
1.6 g
1.6 g
10.0 g
18.00 g
1000 ml

10.0 g
4.0 g
100 ml
1000 ml

b) Grams Iodine solution

Iodine
Potassium iodide
Ethanol
Distilled water

1.0 g
2.0 g
25 ml
100 ml

c) Counter stain
2.5% safranin in ethanol
Distilled water

10 ml
100 ml

d) Ethyl alcohol (Decolouriser)

Ethanol
Distilled water
20. Salkowski Reagent
35% perchloric acid
0.5M FeCl3

95 ml
5 ml

50 ml
1 ml