Table Of ContentTECHNIQUES IN MYCORRHIZAL STUDIES
Techniques in Mycorrhizal
Studies
Edited by
K.G. Mukerji
Department of Botany, University ofD elhi,
Delhi, India
C.Manoharachary
Department of Botany, Osmania University,
Hyderabad, India
and
B.P. Chamola
Department of Botany, University of Delhi,
Delhi, India
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.
A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-90-481-5985-7 ISBN 978-94-017-3209-3 (eBook)
DOI 10.1007/978-94-017-3209-3
Printed on acid-free paper
AII Rights Reserved
© 2002 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2002
Softcover reprint of the hardcover 1s t edition 2002
No part of this work may be reproduced, stored in a retrieval system, or transmitted
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Preface
The importance of mycorrhiza for the improvement ofp lant growth is
increasingly being realised in Agriculture and Forestry and several
mycorrhizal fungi have been commercially recognised for the purpose.
The aim oft his book is to describe the various techniques used to study
the mycorrhizal biology. Problems with preparing such a book are many.
Mainly mailing ofm anuscripts to and from authors resulted in irregular
peer review and final editing. Every effort was made not to change the
original manuscript to ensure accuracy. Our sole aims is to communicate
to the greatest extent possible a current world need in mycorrhizal research.
Plant productivity is regulated by microbial associations established in the
plant root systems. The interactions ofm icroorganisms and plant roots
are especially important in providing nutritional requirements oft he plant
and the associated microorganisms. Plant growth and development are
controlled largely by the soil environment in the root region -rhizosphere.
This is a very complex environment in which the effects oft he plant on soil
microorganisms and the effects of microorganisms on the plant are
interacting, interdependent and highly complex. Plant root exudates and
breakdown products feed the microbes and the microbe in tum often
benefit the plant.
Mycorrhizal fungi are important tools for increasing growth, development
and yield of economically important plants, they play important role of
biofertilizer which can help establish plants in nutrient deficient soils,
particularly phosphorus deficient soils, arid, semi-arid and waste lands.
Mycorrhizal plants grow well under stress conditions and are resistant to
pathogens (bioloical control). Mycorrhizal technology, therefore, has
assumed greater relevance in forest production of exotics and artificial
regeneration of rare plants. Its potential in agriculture, social and
agroforestry programmes aimed at increasing productivity of food, fuel,
fodder and fibre for exanding popUlation is attracting serious attention in
vi Techniques in Mycorrhizal Studies
recent years. Mycorrhizaresearch thus contribute an important component
in the field ofb iotechnology, the practical application ofw hich can lead to
successful afforestation, additional timber production with the modest
increase in survival and growth rate oft rees.
Since Mycorrhizal biology has become important in Agriculture, Forstry
and Horticulture, a good book on techniuqes in mycorrhizal studies was
needed. There is already a book on molecular aspects of mycorrhizal
research, there was great demand for a book on fundamental and basic
aspects of mycorrhiza, without knowledge of which no research on
mycorrhiza can be initiated. It is with this aim that this book has been
compiled covering all major aspects ofm ycorrhizal biology. An entire
gneration ofm ycorrhizologists have been trained primarily in molecular
biology, and more specifically in cloning and sequencing. Although such
tools will remain fascination ofi nvestigators for some time to come, there
is a critical need to carry out multidisciplinary investigations. It was set
against this back ground that we conceived the idea for this book. We
wanted to bring together the diversity oftoolsltechniques that are necessary
for contemporary study of mycorrhizal biology into a single volume.
Although assimilating the theoretical background and wealth ofl iterature
in all oft he necessary disciplines would have been an impossible job, we
considered that this was ofless importance than providing, simple, well
explained laboratory protocols oft he relevant procedures. After all, it is
at the front line ofm ycorrhizal research that we want this book to be used
by students and learners at all stages of their growth from graduate to
post-doctoral level, nursery workers to field reserachers.
The book contains twenty four Chapters and starts with an overview of
Mycorrhizal Studies and its scope. The Chapters 1 to 5 deal about basic
knowledge of soil and root microflora, their ecology, techniques used in
their isolation and identification. Emphasis has been given on mycorrhizal
fungi in relation to soil factors and root exudation. Methods used to collect
and analyse root exudate has also been given. Chapters 6 to 8 deal with
ectomycorrhizal fungi, their isolation, mass culture and identification.
Chapters 9 to 17 deal with vesicular-arbuscu1armycorrhiza with particular
emphasis on techniques to isolate, mass-culture and identifying VA M fungi.
Chapter 17 is slightly drifted form the main theme oft he book but significant
Vll
as it pertains to degradation of(VA ) mycorrhizal root leading to soil fertility.
Chapters 18 to 21 give current status of the other types ofm ycorrhiza i.e.
ericord, orchidoid and monotropoid. No account has been given on
arbutoid mycorrhiza as we considered them less significant. Last three
chapters i.e. 22 to 24 deal with two very important biotechnological
applications of mycorrhiza i.e. their role in in vitro establishment of
micropropagated plants from laboratory to field conditions and mycorrhiza
as 'biocontrol agents'.
All chapters are new but some (20, 22, 23, 24) have been especially
written for our book and probably no other publication has earlier dealt
with these topics. Other biotechnological applications have not been
included in this book since these are already given in earlier pUblications
by us and others.
Each chapter has been written by experienced scientists in the field. Our
hope in assembling this variety ofe xperimental approaches together, in a
single volume is that any researcher studying mycorrhiza can use this book
as their first reference for starting a new avenue ofi nvestigation. With each
procedure we have given theoretical information about the topic.
Collectively, we believe there is a powerful combination of experimental
tools here that will be broadly applicable.
Weare greateful to all the authors for their contribution to this book and
for accepting suggestions to produce the final balance ofthe book. The
articles are original and some aspects have been included for the first time
in any book. Since these chapters have been written by independent authors,
there is the possibility ofa slight overlap or repitition of certain statements,
but this is difficult to avoid in assignment like this.
It is our hope that this book will be useful to all students and researchers in
microbial biotechnology, microbial ecology, soil microbiology, applied
mycology, agriculture and forestry.
K.G. Mukerji
C.Manoharachary
17th November, 2001 B.P. Chamola
Table of Contents
Preface........................................................................................ v
Introduction: Mycorrhizal Studies
C. Manoharachary and K. G. Mukerji ............. .... ... ..... ....... 1
1. Soil microbes
K. G. Mukerji ...................................................................... 7
2. Soil microflora : isolation, enumeration and identification
Rani Gupta and H. Mohapatra .... ....... ..... ........ ......... ... ...... 15
3. Soil factors in relation to distribution and occurrence of
vesicular arbuscular mycorrhiza
Rupam Kapoor, B. Giri and K. G. Mukerji ....... ....... ... ........ 51
4. Rhizosphere biology
K. G. Mukerji ...... ............. ........... ..... ................. .... ... ..... ...... 87
5. Root exudate biology
Rajni Gupta and K. G. Mukerji ....... ............ ......... ... .... ....... 103
6. Isolation ofe ctomycorrhizaJ. fungi: methods and techniques
S. Kumar and T. Satyanarayana ............... .... ......... ..... ....... 133
7. Production ofincoulum ofe ctomycorrhizaJ. fungi
Sanjeev Kumar and T. Satyanarayana ..... ........ ....... ..... ..... 143
8. Identification ofe ctomycorrhizas
K. Natarajan and V. Mohan .... ................... ......... ....... ..... ... 167
9. Tehniques for the isolation ofV A MIA M fungi in soil
Rajni Gupta and K.G. Mukerji .......................................... 201
10. Methods in study of viability ofV A M fungal spores
M Bansal and K. G. Mukerji .... ............ .... ........ ..... ....... ...... 217
11. Root-Clearing techniques and quantification ofa rbuscular ..... .
myconhizal fimgi
C. Manoharachary and IK. Kunwar .. ................ ....... ..... ... 231
x
12. Arbuscular mycorrhizal fungi -identification, taxonomic
criteria, classification, controversies and tenninology
C. Manoharachary, l.K. Kunwar and K. G. Mukerji ..... ..... 249
13. Tebniques ofA M fungus inoculum production
Geeta Singh and K. V.B.R. Tilak .......................................... 273
14. Multiplication ofa rbuscularmycorrhizal fungi on roots
P. Chellappan, S.A. Anitha Christry and A. Mahadevan ... 285
15. Biotechnological approaches for mass production ofa rbuscular
mycorrhizal fungi : current scenario and future strategies
Abdul-Khaliq, ML. Gupta and M. A lam .. ................ .......... 299
16. VA M technology in establishment ofp lants under salinity
stress conditions
B. Giri, Rupam Kapoor and K. G. Mukerji .. ..... ........ .......... 313
17. Method in study ofd egradation ofm ycorrhizal roots
M Bansal and K.G. Mukerji .............................................. 329
18. Ericoid mycorrhiza -isolation and identification
Sumeet and K. G. Mukerji ................. ........ ......... ................ 345
19. Ericoid mycorrhizae -current status
Geeta Singh and K. G. Mukerji ............. ................... ... ... .... 365
20. Orchidoid mycorrhiza and techniques to investigate
S.P. Vij, T.N. Lakhanpal and Ashish Gupta ....................... 385
21. Some aspects ofm onotropoid mycorrhizas
C. Manoharachary, l.K. Kunwar and K. G. Mukerji .......... 435
22. Role ofm ycorrhizae in in vitro micropropagation ofp lant
P.S. Srivastava, Nisha Bharti, Deepshikha Pande
and Sheela Srivastava ................ .......... ...... ..... ...... ............. 443
23. Evaluating performance ofp lants infected with vescicular
arbuscular mycorrhizal fungi for alleviating abiotic stresses
P. Sharmila and P. Pardha Saradhi ..... ............ ............ ....... 469
24. Mycorrhizae as biocontrol agents
Lisette J. C. Xavier and Susan M Boyetchko ..... ....... ... ...... 493
Index ........................................................................................... 537
List ofContributers ...................................................................... 551
Introduction: Mycorrhizal Studies
C. MANOHARACHARY and K.G. MUKERJI
There is a vast microbial flora, inheriting the earth and they are found in all
types of soils which are virgin or cultivated, sands, deserts, thennal soil,
snow covered soils and others. The dominating groups ofm icroorganisms
are bacteria, actinomycetes, fungi, nematodes and protozoans. But most
ofthese organisms share a common character in being heterotrophic in
their nutrition and there by depend on other organic, dead or living organism
and inorganic source ofn utrition for their sUlVival and multiplication. Most
of the fungi, bacteria and actinomycete are microscopic and show vast
variation quantitatively and qualitatively in different sites ofc ollection and
at different depths, considerable variation occurs even between soil samples
taken a few inches apart. Biotrophic, saprobic and symbiotic microbes
are found in soil. The microbial population of soil is, therefore, dependent
on composite micro-ecological environments.
Mycorrhizae are the structures fonned by the association ofr oot
in plant with fungi. Such root fungus associations were found to be nonnal
on the root system. The studies ofGennan Botanist Frank (1) had initiated
worldwide interest on myconbizae. Myconhizae fonn symbiotic association
with many plants under natural conditions. Fungi receive carbohydrates
from host plant while plants receive fastly mobilized phosphorus and other
nutrients throughmyconhizal roots. Seven types of mycorrhizal associations
are known namely - ectomycorrhizae, arbuscular mycorrhizae, ect-endo
mycorrhiza, arbutoid mycorrhiza, monotropoid, ericoid and orchidoid
mycorrhizae (3,4,5). Mycorrhizal plants suggest saving of 50% of
phosphorus ifp roper management is observed by deriving maximum
mycorrhizal response. Ectomycorrhizal fungal inoculation is a common
practice and it is ofi mmerse utility in forestry, silvicuture and agroforestry
operations.
K. G. Mu'kerji et al."( eels.), Techniques in Mycorrhizal Studies, 1"'{).
© 2002 Kluwer Academic Publishers.
2 Techniques in Mycorrhizal Studies
The endomycorrhizal associations produce minor changes in the
root morphology. The mantle of the root is absent. Orchids have
endomycorrhiza which is called glomerate endomycorrhiza because of
the presence ofg lomerules. Members ofEricaceae develop a different
type of endo-mycorrhizal association. Ectendo-mycorrhiza are
characterized by the presence ofa small mantle and mycelium penetrating
the root cells. AM fungi are found associated with crop plants, forest
plants, horticultural plants, ornamental plants and other plants. These fungi
are not host specific but differ in their characteristic association with host
rhizosphere. AM fungi produce azygospore, chlamydospore. Most AM
fungi sporulate outside the plant root in soil but for Glomus tenuis and
Glomus intraradices. However there are around 10 species of Glomus
which can be multiplied in soil pot culture. Endomycorrhizae can not be
cultured on agar media or in axenic culture as AM fungi are obligate
symbionts.
Ectomycorrhizae can be distinguished from endomycorrhizae
because in them the root system is heterorhizic, comprising long and short
roots. The ectomycorrhizal plants are characterized by the presence of
mantle and the hartig net. Morpho-anatomical features, sporophore
morphology, basidiospore structure and other characters fonn important
criteria in recognizing ectomycorrhizal fungi. Various techniques have been
used for establishing the mycorrhizal association of trees and fungal fruit
bodies. Taxonomically the ectomyconhizal fungi belong to Basidiomycotina
and few are also members of AscomycotinalDeuteromycotina. Around
2000 wooden plant species are colonized by 5000 fungal species/strains.
These fungi can be easily identified from their fruit bodies. The pure culture
isolations ofe ctomycorrhizal fungi have been tried from the fruiting bodies,
surface sterilized mycorrhizal roots and sclerotia. Pisolithus and
Thelephora have proved as the most efficient and beneficial bioinoculants
as they have shown dramatic improvements in survival and growth ofp ine
and other seedlings. Ectomycorrhizal fonnations are confined to forest
and ornamental tree species belonging to Pinaceae, Salicaceae, Betulaceae,
some members of Rosaceae, Leguminosae, Ericaceae, Juglandaceae and
Dipterocarpaceae.
Experimental synthesis of ectomycorrhizae for field inoculation
has undergone change so as to adopt to desired soil and environmental
conditions. With the discovery of molecular tools it has become easy to
