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CHARACTERIZATION OF
Pseudomonas fluorescens FROM RICE
FIELDS FOR THE BIOLOGICAL
CONTROL OF Rhizoctonia solani
CAUSING SHEATH BLIGHT IN RICE
BIYYANI SUMAN
B.Sc. (Ag.)
2015
BY
BIYYANI SUMAN
B.Sc. (Ag.)
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
CERTIFICATE
Mr. BIYYANI SUMAN has satisfactorily prosecuted the course of research and the
thesis entitled ISOLATION AND CHARACTERIZATION OF Pseudomonas
Date:
LIST OF CONTENTS
Chapter No.
Title
INTRODUCTION
II
REVIEW OF LITERATURE
III
IV
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
4.3
4.4
4.5
4.6
of
Pseudomonas
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
Score (0-9 scale) of sheath blight and grain yield per plant (gms)
4.15
4.16
LIST OF ILLUSTRATIONS
Illustration
Page
Title
No.
No.
4.1
4.2
4.3
Screening of
production
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
Pseudomonas fluorescens
isolates for
IAA
ACKNOWLEDGEMENT
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)
Cm
Centimetre
&
And
Metre
Gram
Kg
Kilogram
Tonn
Ha
Hectare
kg ha -1
t ha -1
Kmph
dS m-1
At the rate of
Sem
CD(P=0.05)
DAT
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
MOP
Muriate of potash
Max
Maximum
Min
Minimum
day -1
Per day
Mm
Millimetre
RDF
FYM
Farmyard manure
Fig.
Figure
hour-1
Per hour
Ppm
ml lit -1
cm 2
Square centimetre
Nitrate
RDF
No.
Number
vis--vis
In relation to
PGPM
CFU
Spp.
Species
PSB
PSBF
PSM
PGPR
PDA
KB
KingB medium
CRD
R1
Replication 1
R2
Replication 2
R3
Replication 3
NA
Nutrient agar
SPAD
Mg
Milligram
IAA
NO3
Author
Title of the thesis
:
:
BIYYANI SUMAN
ISOLATION
AND
CHARACTERIZATION
OF
CONTROL
OF
Rhizoctonia
solani
Faculty
AGRICULTURE
Discipline
AGRICULTURAL MICROBIOLOGY
Major Advisor
Dr.A.VIJAYA GOPAL
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).
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
2.2
2.3
2.4
2.5
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
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
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
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.
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
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.
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.
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
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)
: Rice
Variety
: Tellahamsa
Season
: Rabi, 2014-15
No. of treatments
: 10
No. of replications : 3
Pot size
: 30 cm30 cm
RDF
Description
No infection
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
Raster
Geometric correction
Polynomial window
Resample window
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
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.
Chapter IV
4.2
4.3
4.4
4.5
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).
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.
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.
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)
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
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).
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).
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
Isolates
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
70
60
50
40
30
20
10
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
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
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
10
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
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
(c)MR test
light
(d)VP test
(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
Isolate PVP2
Isolate DMP1
Isolate DBP
Plate 4.5 Antagonistic activity of Pseudomonas fluorescens against Rhizoctonia
solani
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
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
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
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
Ammonia
production
HCN
production
++
+
+
++
+
+++
+
+
++
+
++
+
+
++
+
+
+
+++
+
+
++
+
+
++
++
+
+
+
+
++
+++
++
+
++
+
++
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
++
+
+
+
+++
+++
+
+++
+
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
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 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
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
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
Treatments
Number of
panicles
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
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
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
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
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
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
Chapter V
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
5.00 g
10.00 g
15.00 g
1000 ml
7.0 0.2
1.00 g
10.00 g
0.50 g
0.20 g
0.10 g
0.016 g
1000 ml
11.0
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
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
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
10.00 g
1.00 g
18.00 g
1000 ml
7.2 0.2
0.10 g
9.10 g
9.50 g
20.00 g
0.01 g
1000 ml
6.8
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
1.00 g
10.00 g
0.50 g
0.20 g
0.10 g
1.00 g
1000 ml
6.8 7.0
10 g
10 g
1.50 g
1.50 g
15.0 g
7.0
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
1.0 g
2.0 g
25 ml
100 ml
c) Counter stain
2.5% safranin in ethanol
Distilled water
10 ml
100 ml
95 ml
5 ml
50 ml
1 ml