Table Of ContentSoil Biology
11
Series Editor: Ajit Varma
Volumes published in the series
Volume 1
Applied Bioremediation and Phytoremediation (2004)
A. Singh, O.P. Ward (Editors)
Volume 2
Biodegradation and Bioremediation (2004)
A. Singh, O.P. Ward (Editors)
Volume 3
Microorganisms in Soils: Roles in Genesis and Functions (2005)
F. Buscot, A. Varma (Editors)
Volume 4
In Vitro Culture of Mycorrhizas (2005)
S. Declerck, D.-G. Strullu, J.A. Fortin (Editors)
Volume 5
Manual for Soil Analysis – Monitoring and Assessing Soil Bioremediation
(2005)
R. Margesin, F. Schinner (Editors)
Volume 6
Intestinal Microorganisms of Termites and Other Invertebrates (2006)
H. König, A. Varma (Editors)
Volume 7
Microbial Activity in the Rhizosphere (2006)
K.G. Mukerji, C. Manoharachary, J. Singh (Editors)
Volume 8
Nucleic Acids and Proteins in Soil (2006)
P. Nannipieri, K. Smalla (Editors)
Volume 9
Microbial Root Endophytes (2006)
B.J.E. Schulz, C.J.C. Boyle, T.N. Sieber (Editors)
Volume 10
Nutrient Cycling in Terrestrial Ecosystems (2007)
P. Marschner, Z. Rengel (Editors)
Ajit Varma
Ralf Oelmüller (Eds.)
Advanced
Techniques
in Soil Microbiology
With 94 Figures, 2 in Color
123
Prof. Dr. Ajit Varma
Director, Amity Institute of Microbial Sciences
and
Vice Chairman (International), Amity Sciences Technology Foundation
Amity University Uttar Pradesh
Sector-125, Noida 201303
India
E-mail: [email protected]
Prof. Dr. Ralf Oelmüller
Friedrich-Schiller-University Jena
Institute of General Botany and Plant Physiology
Dornburger Str. 159
07743 Jena
Germany
E-mail: [email protected]
Library of Congress Control Number: 2007921585
ISSN 1613-3382
ISBN-978-3-540-70864-3 Springer-Verlag Berlin Heidelberg New York
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Editor: Dr. Dieter Czeschlik, Heidelberg, Germany
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Preface
There is general belief and admission that important, innovative and novel ideas
emerge over a cup of ‘Indian Darjeeling tea’ or a glass of ‘German beer’. The
editors of this book were sipping a cup of tea on the lush green garden lawns
of North Maharastra University, Jalgaon, India. The weather was congenial and
most suitable for materializations of original ideas. The genesis of this book un-
derlines the concept developed in 2006.
The field of microbiology began concurrently with the discovery of micro-
organisms by two Fellows of The Royal Society, Robert Hooke and Antony van
Leeuwenhoek, during the period 1665–1683. Later, during the golden era of mi-
crobiology, noted scientists Louis Pasteur and Robert Koch laid a sound foun-
dation for the modern microbiology. The study of microorganisms has became
a valuable science in the last 100 years as it has provided both the means to
control a number of infectious diseases and the experimental systems for the
development of molecular biology. New developments in biotechnology and
environmental microbiology signify that microbiology will continue to be an
exciting field of study in the future. Various modern tools and techniques are
required for a proper understanding of the roles of microbes in the causation
of infectious diseases and the recycling of chemical elements in the biosphere.
Assorted laboratory experiments not only motivate researchers and students by
stimulating interest and enjoyment but also enhance the acquisition of scientific
knowledge along with the development of ‘scientific attitudes’, such as open-
mindedness and objectivity.
There are numerous textbooks and review papers dealing with state-of-the-
art of various aspects of molecular biology of microorganisms. However, the
readers get lost in initiating the experiments due to lack of suitable and easy
protocols. They have to search for diverse methods and techniques in a variety
of literature and journals and still do not obtain the complete information deal-
ing with the protocols in a concise manner. This book is an attempt to overcome
the inherent cumbersome search process. Every effort was made to present the
protocols in a very simple manner for easy understanding of undergraduate,
graduates, postgraduates, post doctorates, active scientists and researchers.
Soil, the main contributor to plant nourishment, is the top layer of the Earth’s
surface and consists of rock and mineral particles mixed with organic matter.
Soil microbiology is the study of the microorganisms in soil, their functions,
VI Preface
and the consequences of their activities on the nature of the soil and the effect
on the growth and health of plant life. Just a few grams of soil, less than a tea-
spoonful, may contain hundreds of millions to billions of microbes. Not only
is the total number of microorganisms in fertile soil quite high, but also, to-
gether, they weigh a lot. Soil microbial biomass can range from several hundred
to thousands of pounds per acre.
The most plentiful microbes in soil are one-celled bacteria and fungi, which
produce long, slender strings of cells called filaments or hyphae. The actinomy-
cetes come between these two organisms. It is the actinomycetes that give soil its
characteristic earthy smell. In this volume, the editors have accumulated various
advanced molecular approaches for studying the different soil microorganisms
for the benefit of humankind. Different techniques for measuring microbial
biomass and activity in soil have been developed. Primers in Random Ampli-
fied Polymorphic DNA (RAPD) techniques for species identification and other
forgotten tools like quantitative histochemistry are discussed in details in this
book with the hope that this would promote the understanding of microbes by
students and advanced researchers alike.
The editors have brought together the diverse topics related to various aspects
of molecular approaches to the detection of soil microbes, namely assessing and
detecting soil micro-fungal diversity and providing insight into their feasibil-
ity. Various problems associated with the dilution plating technique, impor-
tance of the rDNA gene in fungal systematics, the reliability of other molecu-
lar approaches (especially Denaturing Gradient Gel Electrophoresis) and their
drawbacks are discussed. Various modern tools and techniques like automated
fluorescent DNA sequencing strategy, mRNA quantitation using real time PCR,
RNAi technology, transcriptome analysis and immuno-techniques are handled
by subject experts of these specific fields for clear and easy understanding for
all. Different widely used methods like fatty acid methylester (FAME), phos-
pholipid fatty acid (PLFA) analyses and denaturing gradient gel electrophoresis
(DGGE) are elucidated with their advantages and limitations outlined. DGGE
and RISA protocols for microbial community analysis in soil are also one of the
highlights of this book.
The soil zone located in and around the active roots is called the rhizosphere.
This zone has high microbial activity. Materials released from roots, called exu-
dates, create a food-rich environment for the growth of microorganisms. Rhi-
zosphere microorganisms in turn help plants by fixing nitrogen from the soil
air, dissolving soil minerals and decomposing organic matter, all of which al-
low roots to obtain essential nutrients. Plant-Growth-Promoting Rhizobacteria
(PGPRs) generate a variety of chemicals that stimulate plant growth. The bacte-
ria grow and persist in the rhizosphere of non-woody roots. Various screening
methods for PGPRs are described in this book.
A special kind of fungus called mycorrhizae also associates with higher plants.
By colonizing large areas of roots and reaching out into the soil, mycorrhizae as-
sist in transport of soil nutrients and water into the plant. The latest methods
for conducting experiments and research in mycorrhiza have been described.
Preface VII
Cultivation of a group of mycorrhiza-like fungi belonging to family Sebacinales
is enumerated. One of the members of Sebacinales which provides stress toler-
ance activity against heavy metals and induced pathogen resistance in cereals is
discussed.
Authors have brought forth diverse approaches and methods to study the
mechanisms behind the observed pathogen resistance induced by Piriformos-
pora indica.
Model organism A. thaliana was used as the plant partner to understand the
molecular basis for beneficial plant/microbe interactions and this is also dis-
cussed in this edition. Several other techniques like ion cyclotron resonance
Fourier transform mass spectrometry (ICR-FT/MS) for non-targeted metabo-
lomics of molecular interactions in the rhizosphere are presented. Immuno-
technology for the localization of acid phosphatase using native gel bands in
P. indica and other soil microorganism are elaborated in this volume of the Soil
Biology series.
We are grateful to the many people who helped to bring this volume to light.
We wish to thank Dr. Dieter Czeschlik and Dr. Jutta Lindenborn, Springer
Heidelberg, for generous assistance and patience in finalizing the volume. Fi-
nally, specific thanks go to our families, immediate, and extended, not forget-
ting those who have passed away, for their support or their incentives in putting
everything together. Ajit Varma in particular is very thankful to Dr. Ashok K.
Chauhan, Founder President of the Ritnand Balved Education Foundation (an
umbrella organization of Amity Institutions), New Delhi, for the kind support
and constant encouragement received. Special thanks are due to my esteemed
friend and well-wisher Professor Dr. Sunil Saran, Director General, Amity In-
stitute of Biotechnology and Adviser to Founder President, Amity Universe,
all faculty colleagues Drs. Amit C. Kharkwal, Harsha Kharkwal, Shwet Kamal,
Neeraj Verma, Atimanav Gaur and Debkumari Sharma and my Ph.D. students
Ms. Aparajita Das, Mr. Ram Prasad, Ms. Manisha Sharma, Ms. Sreelekha Chat-
terjee, Ms. Swati Tripathi, Mr. Vipin Mohan Dan and Ms. Geetanjali Chauhan.
The technical support received from Mr. Anil Chandra Bahukhandi is highly
appreciated.
New Delhi, India Ajit Varma
Jena, Germany Ralf Oelmüller
March 2007
Foreword
There is no doubt that biotechnology is one of the leading disciplines in mod-
ern biology. Concerning its ever-growing impact on the development of new
products, its importance cannot be overestimated; in terms of generating new
jobs and industries it is certainly that section of biology which is responsible for
the largest financial volume and the highest degree of application of biological
knowledge. Interestingly, biotechnology is also the most interdisciplinary sci-
ence as it uses efficiently the various biological disciplines which were often sep-
arated in the past and which even kept their own characteristics at the expense
of neighboring disciplines. In biotechnology the product counts more than the
origin, and the frontiers between animal, plant and bacterial cells are of minor
importance. Today, the central role of the new genes dominates a good part of
biotechnology; it creates new products; however, the cellular environment must
obey the laws of efficiency, practicability and production costs.
For the above reasons it is important to assemble the ever-improving meth-
ods of modern biotechnology in a book under these new guidelines, i.e. practi-
cal aspects and immediate use in the laboratory and beyond. These methods
involve all the essential methods of molecular biology, immunology, microbiol-
ogy and structural biology; the complexity of the systems involved ranges from
individual molecules to the eukaryotic organisms themselves, with a focus on
bacteria, fungi and higher plants. As it is extremely difficult to cover even the
most important state-of-the-art methods from the whole field, a comprehen-
sive book with selected authors and methods such as this is extremely useful: it
encourages students to look at biology in a different focus, assembling methods
with a clear aim at a product, and it tells the experienced researcher about the
leading laboratories and the most promising strategies.
The 26 chapters of this book are indeed an excellent and outstanding contri-
bution towards this end.
Mattthias Rögner Ruhr Universität Bochum, Germany
Govindjee University of Illinois at Urbana-Champaign, USA
Contents
1 DetectionandDiversityofFungifromEnvironmentalSamples:
TraditionalVersusMolecularApproaches ........................ 1
R. Jeewon and K.D. Hyde
1.1 Introduction ............................................ 1
1.2 Microscopy and Culture-Based Methods .................... 2
1.3 Molecular-Based Methods ................................ 4
1.4 The Nuclear-Encoded Ribosomal DNA Gene:
Phylogenetic and Systematic Value ......................... 5
1.5 Denaturing Gradient Gel Electrophoresis:
Applicability, Usefulness and Bias .......................... 7
1.6 Conclusions and Future Directions ......................... 11
References .................................................... 11
2 FunctionalGenomicApproachesforMycorrhizalResearch ........ 17
A. K. Pandey, H. White, and G.K. Podila
2.1 Introduction ............................................ 17
2.2 Yeast Two Hybrid: An Approach
for Understanding Signaling Pathways ...................... 18
2.3 Agrobacterium-Mediated Transformation in Laccaria bicolor ... 22
2.4 Materials and Methods ................................... 24
2.4.1 Interaction Studies of Laccaria bicolor with Aspen
(Populus tremuloides) Seedlings ..................... 24
2.4.2 Yeast Two-Hybrid Protocol ......................... 26
2.4.3 Agrobacterium-Mediated Transformation in Laccaria
bicolor ........................................... 28
References .................................................... 30
3 AutomatedFluoroscenceSequencingandTroubleshooting ......... 35
S. Gochhait, D. Malhotra, E. Rai, and R.N.K. Bamezai
3.1 Introduction ............................................ 35
3.2 Evolution of the Method .................................. 36
3.2.1 Manual Sequencing ............................... 36
XII Contents
3.2.2 Automated Sequencing ............................ 37
3.2.3 Pyrosequencing ................................... 39
3.3 Methods ................................................ 39
3.3.1 Template Preparation .............................. 39
3.3.2. Reaction Setup (BigDye Terminator Cycle Sequencing) 40
3.3.3 Performing Cycle Sequencing ....................... 41
3.3.4 Preparing Extension Products for Electrophoresis ..... 42
3.4 Trouble Shooting ........................................ 43
3.4.1 Problem: Flat Line or “Dead On Analysis” ............ 43
3.4.2 Problem: Noisy Data (Background) .................. 45
3.4.3 Problem: Reading Near the Primer .................. 46
3.4.4 Problem: Strong Terminator Peaks .................. 46
3.4.5 Problem: Low Intensity of Shorter Products .......... 48
3.4.6 Problem: Longer Fragments Missing ................. 49
3.4.7 Problem: Presence of Spikes ........................ 49
3.4.8 Problem: Weaker Signals ........................... 49
References .................................................... 50
4 mRNAQuantitationUsingRealTimePCR ....................... 53
S. Gochhait, S.I. Bukhari, and R.N.K. Bamezai
4.1 Introduction ............................................ 53
4.2 Methods ................................................ 54
4.2.1 Chemistry and Primer/Probe Design ................ 54
4.2.2 RNA Isolation from the Sample ..................... 57
4.2.3 Reverse Transcription ............................. 58
4.2.4 Real Time PCR Set Up ............................. 59
4.2.5 Instrumentation .................................. 61
4.2.6 Data Analysis .................................... 67
4.3 Notes .................................................. 68
References .................................................... 71
5 LaboratoryPracticefortheProductionofPolyclonal
andMonoclonalAntibodies .................................... 73
S. Khurana, S. Bhaskar, and A. Mukhopadhyay
5.1 Introduction ............................................ 73
5.2 Production of Polyclonal Antibodies ....................... 74
5.2.1 Choice of Animal and Method of Immunization ...... 74
5.2.2 Preparation for Immunization ...................... 75
5.2.3 Production of Polyclonal Antibodies ................. 76
5.2.4 Materials and Equipment .......................... 77
5.3 Production of Monoclonal Antibodies ...................... 77
5.3.1 Immunization of Mice or Rats ...................... 77
5.3.2 Myeloma Cell Culture ............................. 78
5.3.3 Setup for Fusion of Myeloma with Spleen Cells ........ 79
5.3.4 Selection and Cloning of Hybridoma ................ 79
