STUDIES ON ROOT COLONIZATION OF APPLE PLANT BY POTENTIAL Pseudomonas SPECIES ...

182 Pages·2013·5.09 MB·English

STUDIES ON ROOT COLONIZATION OF APPLE
PLANT BY POTENTIAL Pseudomonas
SPECIES IN REPLANT SITES

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by

KUMARI MANORMA

Submitted in partial fulfilment of the requirements
for the degree of

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1985

COLLEGE OF FORESTRY
Dr. Yashwant Singh Parmar University
of Horticulture and Forestry, Nauni,
Solan - 173230 (H.P.), INDIA
2013
Dr Mohinder Kaur Department of Basic Sciences
Professor College of Forestry
Dr Y S Parmar University of Horticulture
and Forestry, Nauni-173230, Solan (HP)

CERTIFICATE-I

This is to certify that the thesis titled, “Studies on root colonization of
apple plant by potential Pseudomonas species in replant sites”
submitted in partial fulfilment of the requirements for the award of degree of
MASTER OF SCIENCE MICROBIOLOGY to Dr Yashwant Singh Parmar
University of Horticulture and Forestry, Nauni, Solan (HP) is a record of
bonafide research work carried out by Kumari Manorma (F-2010-25-M)
under my guidance and supervision. No part of this thesis has been submitted
for any other degree or diploma.

The assistance and help received during the course of investigations
has been fully acknowledged.

Place: Nauni-Solan Dr Mohinder Kaur
Dated: Major Advisor
CERTIFICATE-II

This is to certify that the thesis titled, “Studies on root colonization of
apple plant by potential Pseudomonas species in replant sites”
submitted by Kumari Manorma (F-2010-25-M) College of Forestry, Dr
Yashwant Singh Parmar University of Horticulture and Forestry, Nauni-Solan
in partial fulfilment of the requirements for the award of degree of MASTER
OF SCIENCE MICROBIOLOGY has been approved by the Advisory
Committee after the thesis viva-voce examination in collaboration with the
internal examiner.

Dr Mohinder Kaur Dr Rajesh Kaushal
Major Advisor Internal Examiner

Members of Advisory Committee

Dr Girish Sharma Dr Nivedita Sharma

Dr Neerja Rana

Dr G.S. Shamet
Dean’s Nominee

Professor and Head
Dept of Basic Sciences, Nauni-Solan

Dean
College of Forestry, Nauni-Solan
CERTIFICATE-III

This is to certify that all the mistakes and errors pointed out by the
external examiner have been incorporated in the thesis titled, “Studies on
root colonization of apple plant by potential Pseudomonas species in
replant sites” submitted to Dr Y S Parmar University of Horticulture and
Forestry, Nauni, Solan (HP) by Kumari Manorma (F-2010-25-M) in partial
fulfilment of the requirements for the award of degree of MASTER OF
SCIENCE MICROBIOLOGY.

Major Advisor

Dr Mohinder Kaur
Professor

Dr C.K. Shirkot
Professor and Head
Department of Basic Sciences
Dr Y S Parmar, UHF, Nauni-173230, Solan (HP)
ACKNOWLEDGEMENT

Pride, praise and perfection belong to Almighty alone, so first of all I would like to offer my heartiest
salutation at the lotus feet of the Supreme Being for the physical and mental strength, bestowed upon me and
in whose faith I was able to complete this task.

I feel privileged to express my deep sense of gratitude, appreciation and heartfelt thanks to my dignified
chairmain Dr(Mrs.) Mohinder Kaur major advisor of my advisory committee for her expert guidance,
constructive criticism, present help, immaculate suggestions and live encouragement during the entire course of
this investigation which led me on and on to achieve my destination successfully.

I emphatically extend my heartiest thanks to the worthy members of my advisory committee Dr. Girish
Sharma, Dr. Nivedita Sharma and Dr. Neerja Rana for their full cooperation, encouragement and valuable
suggestions during the investigation and manuscript preparation.

Thanks are due to Anjali mam and Anju Sharma mam for their help during molecular characterization.

I owe profound debt to Dr C.K. Shirkot Professor and Head Department of Basic Sciences for his
thoughtful assistance and encouragement during the course of investigation.

A big part of my thanks also goes to the Sharma family of Maggota (Rohru) for cooperation during my
research trial for providing plants and site in their orchards.

To my Parents, I owe more than I can express. Their endless sacrifices, the numerous times when they
remained awake so that I could sleep their unrelenting support, encouragement and understanding have helped
me to reach where I stand today.

I would like to thanks DST for providing necessary chemicals and instruments related to my research work.

I am highly thankful for the cooperation and affection which I received from all my respected seniors
especially Sapna, Shweta, Ranjana, Kalpana, Himani, Sheetal and Pankaj. I also extend my thanks to my
junior Vijay and propitious company of all my classmates for their co-operation and help. I feel immense
pleasure of acknowledging propitious company of Bhawna all through the session.

I would like to express my appreciation to laboratory and office staff of Dept of Basic Sciences for their
kind help.

Finally a word of appreciation goes to DPT computers for metamorphosizing this manuscript with deep
interest and well in time.

For any errors or inadequacies that may remain in this work, of course, the responsibility is entirely my
own.

Place: Nauni, Solan
Date: (Manorma)
CCOONNTTEENNTTSS

Chapter Title Page(s)

1. INTRODUCTION 1-4
2. REVIEW OF LITERATURE 5-32
3. MATERIALS AND METHODS 33-56
4. EXPERIMENTAL RESULTS 57-94
5. DISCUSSION 95-126
6. SUMMARY AND CONCLUSIONS 127-130
7. REFERENCES 131-152
ABSTRACT 153
APPENDICES I-II
LIST OF TABLES

Table Title Page(s)

1. Collection of rhizoshere soil samples of apple from normal 57
and replant sites of orchard at Maggota (Shimla District)
2. Population density of total bacterial and total fluorescent 58
Pseudomonas species present in rhizospheric soil of
apple in normal & replant site of apple orchards at
Maggota (Shimla district)
3. Morphological, physiological and biochemical 60-63
characteristics of bacterial isolates
4. Effect of commonly used Mancozeb and Carbendazim on 64
growth of Pseudomonas isolates on king’s B agar at 280C
after 24 hr of incubation
5. Potential of fluorescent Pseudomonas isolates for 66
production of multifarrous PGP activities
6. Potential of fluorescent Pseudomonas isolates for 68
production of plant growth regulators
7. Potential of fluorescent Pseudomonas isolates for 70
production of antifungal activities against different plant
pathogenic fungi
8. RAPD marker bands with primer OPA-05, OPA-06, OPA- 80
07, OPA-08 that are diagnostic for similarity of seven
Pseudomonas isolates from the rhizosphere of apple in
replant and normal site
9. Total number of amplified fragments and number of 81
monomorphic and polymorphic fragments generated by
PCR using 4 random decamer oligonucleotide primers
10. Similarity matrix showing relationship among 81
Pseudomonas sp. isolates from the rhizosphere of apple
at Maggota
Table Title Page(s)

11. Details of replant site and isolates of fluorescent 82
Pseudomonas species used for field trials at Maggota
12. Effect of cyclic application of selected strains of 83
Pseudomonas aeruginosa isolates Pn-13-San & An-16-
Kul on population density of total bacteria and total
fluorescent Pseudomonas sp. in rhizospheric soil of apple
in replant site of orchard Maggota
13. Effect of cyclic application Pseudomonas isolates Pn-13- 85
San & An-16-Kul on population density of total fungal flora
in rhizospheric soil of apple at Maggota
14. Effect of cyclic application of Pseudomonas aeruginosa 86
Pn-13-San & An-16-Kul on percent survival of apple root
stock in replant site of orchard at Maggota
15. Effect of cyclic application of Pseudomonas aeruginosa 87
Pn-13-San & An-16-Kul on growth of apple in replant site
of orchard at Maggota
16. Effect of Pseudomonas aeruginosa Pn-13-San & An-16- 91
Kul on Nitrogen, Phosphorus & Potassium (N, P, K)
content of rhizosphere soil after nine months plantation
17. Effect of cyclic application of Pseudomonas aeruginosa 92
Pn-13-San & An-16-Kul on chlorophyll content of leaves
after nine and twenty months
18. Effect of cyclic application of Pseudomonas aeruginosa 94
Pn-13-San and An-16-Kul on multifarrous PGP potential
of rhizosphere soil extract
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Plates Title Between
pages

1. A. Growth of Pseudomonas isolate An-16-kul 62-63
showing fluorescent pigment production on King’s

agar
B. Denirification test showing gas production in
semisolid medium by Pseudomonas isolates:

Control(1) An-1-Mag(2) An-2-Mag(3) An-3-Mag(4)
An-4-Mag(5) An-5-Mag(6) An-6-Mag(7) and An-7-
Mag(8)

Lecithinase production by Pseudomonas isolate
C.
An-2-Mag showing opaque zone

D. Liquification of gelatin by isolate Control (1) and
Pn-13-San(2)
2. A. Sensitivity of Pseudomonas isolate Pn-13-San to 64-65
different concentrations of fungicide Mancozeb:
0.25%(1), 0.30%(2) 0.40%(3), 0.50%(4) as

inhibition of growth (clear zone) on King’s agar at

280C after 24 h of incubation
B. Sensitivity of Pseudomonas isolate An-4-Mag to
different concentrations of fungicide carbendazim:
0.04%(1), 0.05%(2), 0.06%(3), 0.075%(4) as
inhibition of growth (clear zone) on King’s agar at
280C after 24 h of incubation
3. A. Siderophore production shown by Pseudomonas 66-67
isolate An-3-Mag by 100µl of 72h old supernatant
on CAS agar at 280C after 48h of incubation

B. Proteolytic activity shown by Pseudomonas
isolate An-7-Mag as mm dia of clear zone
produced on skim milk agar plates by 100µl of 72h

old supernatant at 280C after 48h of incubation

C. Phosphate solubilization shown by Pseudomonas
isolates Pn-13-San(1) An-2-Mag(2) and An-16-
Kul(3)as mm dia of yellow zone produced on

Pikovskaya’s agar plates by 100µl of 72h old
supernatant at 280C after 24h of incubation
D. Ammonia production shown by Pseudomonas
isolates: Control(c) Pn-13-San(1) An-16-Kul(2) An-
1-Mag(3) An-2-Mag(4) An-3-Mag(5) An-4-Mag(6)

An-5-Mag(7) An-6-Mag(8) An-7-Mag(9) and Ar-3-
Mag(10) expressed as color change of 24h old
culture broth at 280C after addition of Nessler’s

reagent
E. HCN production shown by Pseudomonas isolate
An-16-kul
Plates Title Between
pages

4. Antifungal activity shown by Pseudomonas isolates 70-71
against
A. Pythium ultimum An-16-kul(1) and Pn-13-san(2)
B. Alterneria solani by An-1-Mag
C. Dematophora necatrix by An-16-kul
5. Agarose gel exhibiting amplified DNA PCR products of 74-75
Pseudomonas isolates: Marker(M) An-1-Mag(1), Pn-
13-San(2), An-2-Mag(3), An-3-Mag(4), An-16-Kul(5),
Ar-3-Mag(6) and An-6-Mag(7)
6. Agarose gel exhibiting amplified DNA PCR products of 80-81
seven Pseudomonas isolates with random primers
OPA-05(A), OPA-07(B). OPA-08(C)
Marker(M), An-1-Mag(1) An-5-Mag(2) An-2-Mag(3),
An-3-Mag(4), An-16-Kul(5), Ar-3-Mag(6) Pn-13-San(7)
7. Effect of cyclic application of full and half concentration 90-91
of inoculum of Pseudomonas aeruginosa Pn-13-San
and An-16-Kul and their Consortia Pn-13-San + An-16-
Kul(1:1) on apple root stocks after 20 months of
plantation