Techniques in mycorrhizal studies springer netherlands

Abstract 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, thermal soil, snow covered soils and others. The dominating groups of microorganisms are bacteria, actinomycetes, fungi, nematodes and protozoans. But most of these 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 of nutrition for their survival and multiplication. Most of the fungi, bacteria and actinomycete are microscopic and show vast variation quantitatively and qualitatively in different sites of collection 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 microecological environments.

TECHNIQUES IN MYCORRHIZAL STUDIES

Techniques in Mycorrhizal Studies

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 1 st edition 2002

No part of this work may be reproduced, stored in a retrieval system, or transmitted

in any form or by any means, electronic, mechanical, photocopying, microfilming, recording

or otherwise, without written permission from the Publisher, with the exception

of any material supplied specifically for the purpose of being entered

and executed on a computer system, for exclusive use by the purchaser of the work

Preface

The importance of mycorrhiza for the improvement of plant growth is increasingly being realised in Agriculture and Forestry and several mycorrhizal fungi have been commercially recognised for the purpose

The aim of this book is to describe the various techniques used to study the mycorrhizal biology Problems with preparing such a book are many Mainly mailing of manuscripts 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 of microorganisms and plant roots are especially important in providing nutritional requirements of the 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 of the 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 of biotechnology, the practical application of which can lead to successful afforestation, additional timber production with the modest increase in survival and growth rate of trees

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 of mycorrhizal biology An entire gneration of mycorrhizologists have been trained primarily in molecular biology, and more specifically in cloning and sequencing Although such tools will remain fascination of investigators 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 of literature

in all of the necessary disciplines would have been an impossible job, we considered that this was ofless importance than providing, simple, well- explained laboratory protocols of the relevant procedures After all, it is

at the front line of mycorrhizal 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 V AM fungi Chapter 17 is slightly drifted form the main theme of the book but significant

Vll

as it pertains to degradation of(V A) mycorrhizal root leading to soil fertility Chapters 18 to 21 give current status of the other types of mycorrhiza 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 of experimental 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 of investigation 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 of a 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

17th November, 2001

K.G Mukerji C.Manoharachary 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 of ectomycorrhizaJ fungi: methods and techniques

S Kumar and T Satyanarayana 133

7 Production ofincoulum of ectomycorrhizaJ fungi

Sanjeev Kumar and T Satyanarayana 143

8 Identification of ectomycorrhizas

K Natarajan and V Mohan 167

9 Tehniques for the isolation of V AMI AM fungi in soil

Rajni Gupta and K.G Mukerji 201

10 Methods in study of viability of V AM fungal spores

M Bansal and K G Mukerji 217

11 Root-Clearing techniques and quantification of arbuscular 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 of AM fungus inoculum production

Geeta Singh and K V.B.R Tilak 273

14 Multiplication of arbuscularmycorrhizal fungi on roots

P Chellappan, S.A Anitha Christry and A Mahadevan 285

15 Biotechnological approaches for mass production of arbuscular mycorrhizal fungi : current scenario and future strategies

Abdul-Khaliq, ML Gupta and M A lam 299

16 V AM technology in establishment of plants under salinity

stress conditions

B Giri, Rupam Kapoor and K G Mukerji 313

17 Method in study of degradation of mycorrhizal 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 of monotropoid mycorrhizas

C Manoharachary, l.K Kunwar and K G Mukerji 435

22 Role of mycorrhizae in in vitro micropropagation of plant

P.S Srivastava, Nisha Bharti, Deepshikha Pande

and Sheela Srivastava 443

23 Evaluating performance of plants 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 of microorganisms 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 of nutrition for their sUlVival and multiplication Most

of the fungi, bacteria and actinomycete are microscopic and show vast variation quantitatively and qualitatively in different sites of collection 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 of root

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 if proper management is observed by deriving maximum mycorrhizal response Ectomycorrhizal fungal inoculation is a common practice and it is of immerse 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 of glomerules Members ofEricaceae develop a different type of endo-mycorrhizal association Ectendo-mycorrhiza are characterized by the presence of a 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 of ectomycorrhizal 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 of pine 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

C Manoharachary and K.G Mukerji 3

introduce new traits that can be advantageous to host The fungi that fonn mycorrhizae with vascular plants belong to the zygomycotina, ascomycotina, basidiomycotina and to the fungi imperfecti Around 5000 species of ascomycotina and basidiomycotina fonn ectomycorrhizaes Around 150 species of AM fungi belonging to zygomycotina infect 90% plants

Arbuscular mycorrhizal fungi are the naturally occurring fungal component of soil biota in most terrestrial ecosystems It also represents a obligate symbiotic group distinct from the rest of soil microbial biomass Endomycorrhizal symbiosis is a dynamic process and interaction that affects all physiological aspects of the host These fungi are unique as they are partly inside and partly outside the root The vesicles, arbuscules and hyphae that are fonned inside the root does not encounter competition and antagonism from soil micro organisms to the host rhizosphere, soil conditions and host genotype are some of the factors that affect biological interactions in that specialized ecological niche

The arbuscularmycorrhizal fungi are ubiquitous in distribution and occur abundantly Ninety percent plants ranging from non flowering to flowering plants have the dynamic association of AM fungi It is easy to identify and count plants which are not mycorrhizal than the plants which are mycorrhizal Unlike the ectomycorrhizal fungi, Arbuscularmycorrhizal fungi belong to Zygomycotina which are obligate biotrophs that can not

be cultured The arbuscular mycorrhizal fungi are the most complex group

of mycorrhizae which fonn intradical structures (i) vesicleslarbuscules inter and intra cellular hyphae inside the root tissue (ii) spores/sporocarp and branched hyphae outside the root and fonned in soil

Arbuscules are the key sites for nutrient exchange and remain active upto fifteen days depending upon host response There are six valid genera namely - Acaulospora, Entrophospora, Glomus, Gigaspora, Sclerocystis and Scutellospora Vesicles are not reported in Gigaspora

and Scutellospora, but extramatrical vesicles were reported

The fossil evidence clearly indicates that the invasion ofland by the ancestors of the present day vascular plants clearly seems to have been facilitated by the origin of symbiotic association between the plants and mycorrhizal fungi Fossil data demonstrates that ectomycorrhizal association was established from the later Ordovician and Silurian period There are about 150 AM fungal species reported colonizing 30,000 receptive hosts

4 Techniques in Mycorrhizal Studies

Mycorrhizal fungi are abundant in soils which are deficient in phosphorus and other mineral elements Mycorrhizal fungi play significant role in phosphorus mobilization In fact mycorrhizal effect decreases with increased supply of soluble phosphate Therefore non-mycorrhizal plants

or non-inoculated plants show greater response towards fertilizer application The important aspect in mycorrhizal biotechnology seems to

be the utilization of unprocessed phosphate that contain sparingly soluble phosphorus Mycorrhizal roots provide grater exploration of"P" absorbing area resulting in increased flow of"P" into the plant The unprocessed phosphorus gets solubilized by phosphorus solubilizing fungi and other microbes, thus making soluble phosphorus available to the plant Efficient mobilization of phosphorus is possible through mycorrhizal root over non- mycorrhizal plants

Harley (2) suggested that production of phosphotases by ectomycorrhizal fungi is important in the solubilization of inorganic phytates, which constitute a large fraction of total phosphate in humic soils Phosphotases are many times more active in mycorrhizal plants than those

on non-mycorrhizal plants Experiments using 32p, it was found that mycorrhizal plants accumulate and stores "P" This accumulate remains consistent unless there is a dramatic change in the plant "P" status The phosphorus gets accumulated as polyphosphate granules and later it is released into host tissue through arbuscules or by other means

Arbuscular mycorrhizal fungal taxonomy seems to be more complicated The motpho-taxonomic criteria, wall layers, ornamentation, subtending hyphae and other character help to segregate genera and species Presence or absence of the sporacatp is also an important criteria for identifying AM fungi It has been suggested by Mosse, an eminent mycorrhizologist that arbuscule, vesicles and hyphal formation have to be established in the concerned host root tissue on inoculation so that the

AM fungal species entity can be ascertained

A complete and accurate description and identification of each

AM fungus is necessary besides fixing its taxonomic status, Motphotaxonomic criteria no doubt will help in segregating the genera and species of AM fungi but much more authenticity and scientific accuracy will be established following molecular techniques Molecular techniques are a major impetus in mycorrhizal research Molecular and biochemical parameters such as cell wall composition, protein immunogenicity, isozyme

C Manoharachary and K G Mukerji 5

electrophoretic patterns, fatty and methyl ester profiles, direct sequencing

of I8S RNA gene and also use ofRAPD approach to detect polymorphism and others have been proved more useful With the development ofPCR,

it is now possible to analyse and characterize a species at the DNA level PCR - RFLP can be used for quantitative comparison oflevels of genetic variation and interpretation of genetic similarities or differences among isolates of AM fungi Immunological studies have also added new strength for the taxonomy of AM fungi particularly in physiological and ultrastructural features during infection process or the ability of foreign AM fungi to compete with local endophytes In addition to the pAbs, mAbs are being developed or adapted from heterologous sources Although the advantages

of pAbs include their easy production with considerably less apparative expenditure as well as a higher possible titer and higher affmity for most antigens a number of problems are encountered with pAbs These can be overcome by the use of hybrid om a technology The benefits derived from this technology include the virtually unlimited supply of specified Abs, fewer problems with unspecific binding and a system that with in limits, allows a selective screening for those mAbs that have the desired specificities and affinities towards a given antigen

AM fungi can influence plant community composition by differentially affecting the growth of different plant species AM fungal symbiosis is highly dynamic interaction that affects nearly all physiological aspects of the host Restoration of disturbed land has prompted the urgent need for understanding mycorrhizas Soils which are disturbed and turned into wastelands I unproductive land were found to have reduced

mycorrhizas and that many plant species dependent of mycorrhizas failed

to establish or survive in these regions Therefore greening of such areas

is possible only with AM fungal symbiontc;

Desert, arid and semiarid soils suffer most and pose technical problems with discrete patches of vegetation Therefore mycorrhizal fungi can be important in the primary production and nutrient flow in these specialised ecosystems Mycorrhizas are of immense utility in the establishment of forest seedlings, commercial crops, economically viable plants, medicinal and aromatic plants, horticultural and ornamental plants

P uptake which is an essential process gets meticulously monitored by

AM fungi This process is of great help in increasing N, P and K status of host tissues Mycorrhizal fungal net work enhances soil binding capacity and soil fertility

6 Techniques in Mycorrhizal Studies

The benefits that are derived by plants colonized by mycorrhizae are:-

1 Improvement in absorbance area followed by increased water

and nutrient uptake

2 Efficient and greater accumulation of phosphorus and other

elements and their speedy mobilization to the host tissue

3 Degradation of complex and organic materials

4 Mycorrhizal plants offer protection against pathogens

5 Provide host plant with growth honnones like auxins, cytokinins,

gibberellins and growth regulators

6 Improves plant defense mechanism and offer tolerance to abiotic

and biotic stress conditions

7 Help in nutrient cycling and forest biomass increase

8 Increases plant growth, biomass, productively and yield

9 Provide host plant with more survival abilities

10 Increases soil binding capacity and soil fertility, mycorrhizal fungi

are non pollutants

11 Waste land development and reclamation of non productive

soils

References

Baume durch Unterisdischr Pilze Berichte der Deutschen Botanischen Gesellschaft,3: 128-145

Publishers, Dordrecht, Boston, London p 374

Kluwer Academic / Plenum Publishers, New York p.336

Press, London, New York p 605

1

Soil Microbes

K.G MUKERJI

237 DDAISFS Mukerji Apartments, East Mukerji Nagar, Delhi-ll0009, India

ABSTRACT: Different techniques for isolation of soil microorganisms have been described Since fungi form the bulk of soil micro biota, techniques to isolate these are emphasized Dilution Plate technique is probably the best for quantitative determination of microbes

1 Introduction

Natural environments are extremely diverse and the majority contain a wide range of microorgnaisms which reflect the nature of the habitat and the ability of individual members to compete successfully and coexist within the given ecosystem In general terms the greater the heterogenecity

of the environment, the more diverse and complex will be the microflora (21) For example in garden soil with numerous microenvironments, the microbial flora is extremely complex whereas in thermophilic or hypersaline environments where one physical or chemical characteristic dominates over all others, only a few specialised species can grow under such extreme ecological conditions (12) Some organisms are present in very low number and isolation of such microbes is possible using some form

of enrichment of the medium The particular microorganism which develops

in enrichment culture clearly depends upon the chemical composition of the medium used, along with other factors like temperature, pH, presence

of selective inhibitors, light, gas phase and others (13) The microbes include bacteria, algae, fungi (including mycorrhizal fungi), actinomycetes, protozoa and nemtodes etc

7

K G Mukerji et al (eds.), Techniques in Mycorrhizal Studies, 7-13

© 2002 Kluwer Academic Publishers

8 Techniques in Mycorrhizal Studies

2 Soil Fungi

As heterotrophic organisms the fungi in soil are saprophytes, symbionts

or parasites Thus they play important roles in terrestrial and aquatic ecoystems as decomposers of organic matter (in nutrient cycling), as pathogens, or as symbionts with terrestrial plants The ecologically important characteristics of terrestrial fungi (11) are as follows:

1) The fungi present in soil are influenced by the amount of organic matter

entering the soil through different types of vegetaion growing on it or near it, soil-moisture, soil-temperature, soil-pH etc and other environmental factors (22) Different groups of fungi have been isolated (2,3)

2) The quality and quantity of fungi is governed by the microhabitats like

decomposing organic matter and living roots

3) The fungi exists in various morphological forms and physiological states,

e.g spores, resting structures and hyphae (6)

4) Methods which are known for the isolation of fungi from soil are all

selective (8) and therefore are incomplete for assessing quantitative nature of soil mycoflora

There are enormous reports on fresh water and marine fungi They are equally important like soil fungi as decoposers of organic matter, as pathogens and also serve as food sources for aquatic invertebrates

3.2 Soil Immersion Technique

The fungi present in soil as actively growing hyphae are isolated by Immersion Technique This was originally reported by Chesters (7) which

In Mueller and Durrell's (21) technique autoclavable centrifuge tubes are used Holes are bored at equal distance on walls of each centrifuge tube These holes are wrapped (closed) with cellotapelscotch tape so as to temporarily closing the holes Isolating medium is poured

in each tube leaving enough space at the top The tubes are cotton plugged and auotclaved In the field the plastic tape is pierced through the holes in the tube using pre-sterilised needles The sterilsed tubes containg medium are inserted in soil and kept there for 5-8 days after which they are taken to laboratory The fungi are isolated by taking out the agar core from each tube and cutting the core into small pieces where the hole was in contact with the medium Individual pieces are plated

3.3 Warcup's Soil Plate Method:

Small volumes of soil are dispersed in known volume of nutrient agar, using sterile needles with flat tips 5-15mg of soil sample is plated in a sterile petridish, a drop of sterile water is added and soil sample thoroughly broken up and dispersed over the base of the dish 10mI of molten

10 Techniques in Mycorrhizal Studies

(c 45°C) agar is poured over the soil in each petridish and slowly roated

to disperse the soil into the agar medium After setting, the plates are incubated at 22-28°C Fungi appearing in these plates are isolated and pure cultures grown are used for identification This is simple method but like Dilution Plate Method it is disadvantageous as this method is selective for fungi present as spores

Warcup (33,34) devised a more precise technique for isolation offungi present in active state in soil Small amounts of soil are saturated with sterile water and dispersed with a fine jet of sterile water The coarse soil particles are sedimented and the finer particles decanted off The supematent is resuspended and procedure repeated several times The coarser particles are spread in a film of water on plate The plates are observed under dissectinglbinocular microscope and hyphal bits are picked with fine sterile forceps or a needle The hyphae are carefully drawn through semi-solid agar to trap other fungal spores, bacteria or organic debris The cleared hyphae are then plated on nutrient agar plates The position of each hyphal bit is marked at the back ofthe plate The plates are incubated at 22-28°C for identification This is the most ideal method for isolation of actively growing fungal hyphae in soil (4) 3.4 Dilution Plate Method (14,31)

The soil samples are made into suspensions in a dilution series (Fig 1) i.e 1: 100; 1: 1000, 1: 10,000 (27) The suitable dilutions are plated onl

in an appropriate nutrient medium The soil suspensions are made of known amount (by weight and volume) and type of soil sample This method is not useful for study of fungi which occur in hyphal state The hyphal propagules can be investigated using Warcup's (32,33,34) soil plate method as described earlier This method, however gives a near total count of spores in a particular soil sample The number offungil

gram dry weight of soil is calculated by multiplying count with the dilution (31) To get a complete picture of the soil mycoflora Dilution Plate Method and Warcup's Soil Plate Method should be used together (14)

Aquatic fungi can be isolated using Baiting technique (5,17), Dilution plate technique (24), Particle Plating method (26) and Concentration - centrifugation, Filtration technique (9,10,20)'

Weigh Ig soil sample

K.G Mukerji 11

' ;;::";;;=-=~ 1 Oml 10-' 1

E§3 10- 7

Fig J Preparation of Soil Dilution

Detailed account of isolation of soil microflora is described elsewhere therefore it is not repeated here (23)

References :

soil fungi and studying their activity in the vicinity of roots Phytopathology,

!'!!'!: 592-594

microfungi in forest soils of Delhi Folia Geobotanica et Phytotaxonomica, 20:

291-311

12 Techniques in Mycorrhizal Studies

3 Behera, N and Mukerji, K G 1985b Ecology of micromycetes in forest soils of Delh Acta Mycologica, 21: 101-108

apparatus for removing fungal spores form soil Canadian Journal of Microbiology, 18: 1399-1409

5 Booth, C 1971 Fungal culture media In, "Methods in Microbiology" (ed Booth, C.) 4: 49-94 Academic Press London

Fungi" (eds Parkinson, D and Waid, J.S.) Liverpool University Press, Liverpool.pp.185-191

British Mycological Society 24: 352-355

Differential Media In, "Source Book of Laboratory Exercises in Plant

Londonpp 19-21

aquatic fungi Bioscience, 14: 45-46

10 Gams, W; Vander Aa HA, Vander- Platts-Niterink, AJ., Samson, RA and Stalpers, J.S 1975 C.B.S course of mycology, Centralbureau voor schimelcultures, Baarn, Netherlands

11 Harley, J.L 1971 Fungi in ecosystem Journal of Ecology, 59: 653-668

12 Herbert, RA 1982 Procedures for the isolation, cultivation and identification ofbacteria In, "Experimental Microbial Ecology" (eds Burns, R G and Slatter, J.H.) Blackwell Scientific Publication Oxford, London pp 3-21

Archives flir Microbiologie 59: 165-173

14 Johnson, L.F and Curl, E.A 1972 Methods for Research on the Ecology of Soil-borne Plant Pathogens Burges Publishing Co Minnessota, U.S.A

15 Jones, F.B.G 1971 Aquatic fungi In "Methods in Microbiology" (ed Booth,

C ) Vol 4, Academic Press, London, pp 335-365

16 Jones, F.B G 1974 Aquatic Fungi, fresh water and Marine, In., "Biology of Plant Litter Decoposition" (eds Dickinson, C.H and Pugh, GJ.F.), Vol 2 Academic Press, London, pp 337-383

17 Jones, F.B.G 1976 Recent advances in aquatic mycology Elek Science, London

Academic Press, London

19 Luttrell, E.S 1967 A strip bait for studying the growth of fungi in soil and aerial habitats Phytopathology, 57: 1266-1267

20 Miller, C.E 1967 Isolation and pure culture of aquatic phycomycetes by membrane filtration Mycologia, 59: 524-527

21 Mueller, K.E and Durrell, L.W 1957 Sampling tubes for soil fungi Phytopathology, 47: 243

K G Mukerji 13

22 Mukerji, K G 1966 Ecological studies on the microorganic population of usar

microorganisms: Screening and Evaluation: In, "Mycorrhiza Mannual" (ed Vcmna, A) Springer, Berlin, pp 85-97

24 Park, D 1972, Methods of Detecting fungi in organic detritus in water Transactions of the British Mycological Society, 58: 281-290

25 Parkinson, D 1957 New methods for qualitative and quantitative study of fungi in therhizosphere Pedologia Gand, 7: 146-154

26 Parkinson, D 1982 Procedures for the isolation, cultivation and identification offungi In, "Experimental Microbial Ecology" (eds Burns, RJ and Slatter, J.H.) Blackwell Scientific Publications, Oxford, Lodon, pp 22-30

27 Piper, C.S 1944 Soil and Plant Analysis University Adelaide Press, Adelaide, Australia

28 Sparrow, F.K 1968 Ecology of fresh water fungi In "The fungi" (eds Ainsworth, G.C and Sussman, A.S.) Vol 3 Academic Press, London, pp 41-

93

29 Starkey, RL 1938 Some influences of the development of higher plants upon the microorganisms in the soil Iv Microcopic examination of the rhizosphere Soil Science, 45: 207-249

30 Thornton, RH 1952 The sereened immersion plate A method for isolating soil microrganisms Research, London 5: 190-191

31 Waksman, S.A 1922 A tentative outline of plate method of determining the number of microorganisms in soil Soil Science, 14: 27-28

32 Warcup, J.H 1950 The soil plate method for isolation of fungi from soil Nature, London 116: 117

33 Warcup, J.H 1955 Isolation of fungi from hyphae present in soil Nature, London 175: 953-954

34 Warcup, J H 1957 Fungi in soil In, "Soil Microbiology" (eds Burges, N.A

isolating soil fungi Plant Disease Reporter, 44: 594

2

Soil Microflora : Isolation, Enumeration and Identification

RANI GUPTA and H MOHAPATRA

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi - no 021

ABSTRACT: The soil and rhizosphere microorganic population is very varied consisting of different groups of microbes including actinomycetes, bacteria, fungi and micro-algae They are essential for the healthy growth and development

of plants Various techniques of isolation and identification of these microbes have been described Modern methods of microbial identification have also been described

1 Introduction

There is considerable diversity in the population of rhizosphere microflora and their effects on plant growth Hiltner was the first to recognize the importance of microbial activity associated with root systems in plant nutrition and used the term rhizosphere to desbcribe the zone of intense activity around the roots of the leguminaceae (Fabaceae) The rhizosphere

is a zone of predominantly commensal and mutualisitc interactions between the plant and microbes Interactions between soil microorganisms and plant roots satisfy important nutritional requirements for both plant and the associated microorganisms (9, 14,26) This interaction of plant roots and rhizosphere microorganism is based largely on interactive modification of the soil environment by processes such as release of organic chemicals of the soil by roots, water uptake by plants, microbial production of plant growth factors and microbially mediated availability of mineral nutrients The plant roots directly affect the density and microbial community of the rhizosphere This rhizosphere effect can be measured as a ratio of the

15

K O Mukerji et al (eds.), Techniques in Mycorrhizal Studies, 15-50

© 2002 Kluwer Academic Publishers

16 Techniques in Mycorrhizal Studies

number of microorganisms in the rhizosphere soil (R) to the number of corresponding microorganisms in soil away from the root (S)-the RlS ratio (3) Generally RlS ratios range from 5-20 but it may reach upto 100

in certain cases Such an increase represents the direct influence of the plant root exudates on soil microorganisms, which favour organisms with high intrinsic growth rates (4)

Just as the plant roots have a direct effect on the surrounding microbial populations, rhizosphere microbial community also has a marked influence

on the growth of the plants The rhizobial microbial community benefit the plant in a variety of ways including recycling and solubilization of mineral nutrients, synthesis of vitamins, amino acids, auxins, cytokinins and gibberellins, which stimulate plant growth and antagonism with potenitial plant pathogens through competition and development of amensal relationships based on production of antibiotics (2) Among the various types of plant-microbial associations, the myconhizal association and nitrogen fixing ability are by far the most important ones (see pp 88-101 this book)

Some fungi enter into mutualistic relationship with plant roots called 'mycorrhizae' in which the fimgi actually become integrated into the physical structure of the roots The fungus derives nutrition (photosynthates) from the plant and in return contributes to its nutrition without causing disease The mycorrhizal associations are more specific and organized than the rhizosphere association In this association the fungi becomes integrated with the plant roots forming distinct morphological units

One of the most important and technologically explored mutualistic relationships of the plant and microorganisms is that of the 'nitrogen fixing bacteria' and plant roots These bacteria infect the host plant and form 'nodules' where the conversion of atmospheric nitrogen to ammonia takes place The nodulating bacteria associated with the leguminous plants were edier placed into a single genus, Rhizobium but recently two new genera Azorhizobium and Bradyrhizobium have been recognized Table 1 lists

the characterstics of all the above three genera

The conversion efficiency of atmospheric nitrogen to ammonia depends upon the nitrogenase enzyme system Nitrogenase being oxygen sensitive is restricted to habitats, which are aerobic In addition to nitrogen

and can also reduce other substrates such as acetylene to ethylene Only

a few strains of Rhizobium and Bradyrhizobium possess hydrogenase

Rani Gupta and H Mohapatra 17

TABLE 1

Characteristics of Rhizobium, Bradyrhizobium and Azorhizobium

medium

of strains outside host

plant

identified so far

cro~s

activity and can utlize hydrogen The infection of Rhizobium and

Bradyrhizobium is greatly host specific The association between the

leguminous host and the bacteria is based on specific chemostatic response and specific binding to the root hair prior to invasion and establishment of the root nodule respectively Apart from the leguminous plants nitrogen

fixation also takes place in non-legumes by Rhizobium, cyanobacteria and actinomycetes To cite a few examples, Rhizobium can fix nitrogen in association with Trema, a tree found in tropical and sub-tropical regions Similarly, actinomycete (Franlda) symbiosis is more frequent in various woody shrubs, small trees as Alnus, Myrica, Hippophae, Comptonia,

Casuarina and Dryas These trees are common in the sub-tropical and

tropical regions The liverworts, mosses, pteridophytes, gymnosperm and angiosperms are able to establish mutualistic relationships with nitrogen

fixing cyanobacteria Nostoc or Anabaena The gymnosperm Cycas develops corolloid roots while the angiosperm Gunera has stem nodules

Inspite of all the above beneficial effects tapping of these microbial resources still pose a big challenge

Measurement of microbial biomass or population size in soil is difficult and the techniques commonly used have limited accuracy For example, direct microscopic observation of the soil samples tends to over estimate the number of viable cell propagules because non-viable cells are included

in counts Alternatively, the indirect plate count technique tend to under estimate the number of soil micro-organisms, due to factors such as incompatibility between microbes in the soil sample and the nutrient medium

18 Techniques in Mycorrhizal Studies

used for incubation or incomplte suspension of microbial propagules in

original soil dilution (see pp 7-13 this book) Recent molecular techniques

based on the analysis of ribosomal RNA (rRNA) offers a culture independent method for microbial identification and should result in a more objective assessment of variability and population size in natural mocrobial community

This chapter deals with the isolation, purification and identification of the naturally occurring microbes and envisages the importance of modem molecular taxonomy in the flawless positioning of these microbes in the taxonomical hierarchy The isolation, purification and identification of bacteria and fungi have been dealt with at the beginning of the chapter and

it concludes with an account of procedures for isolation, and identification

of actinomycetes as a separate section

2 Isolation

The preparation of a pure culture involves the isolation of a given microorganism from a mixed natural microbial population Pure cultures may simply be obtained by pour or spread plate method This involves the separation and immobilization of individual organism on solidified nutrient medium

Prior to isolation, the soil sample should be homogenized to produce

a well-dispersed suspension of soil or sediment that can be diluted readily for inoculation The method also serves to release microbial cells from particle surfaces, but the extnet to which it does so, depends on the physical and chemical nature ofthe sample being processed

Sample preparation

Procedure

Note: Metal "semimicro" (25-250ml) blender heads are recommended because

with most blenders

the sample in the blender This produces a I: 10 suspension, assuming approximately 50% pre space in the sample

d Rapidly transfer the blended material to a sterile container that can be stoppered tightly and shaken (e.g a flask or bottle)

Rani Gupta and H Mohapatra 19

Note: It may be necessary to pour the liquid back and forth rapidly between the blender head and sterile container in order to transfer as much of the solid material to the container as possible

2.1 Strain isolation

Isolation of the stains and pure colonies of bacteria can be obtained by streak plating or spread plating method In such cases the specific plating media may be used for faciliating isolation of a specific group Similarly enriclunent cultures in liquid broths can also be carried out to isolate groups with one or more specific attributes

2.1.1 Streak plating technique

Streak plating is accomplished by dipping a sterillzed bent wire into a suitably diluted suspension of organisms (mixed culture) and then using the same to make a series of parallel, non-overlapping streaks on the surface of an already solidified agar plate (Figure 1) The inoculum is thus progressively diluted with each successive streak and well isolated colonies develop along the lines oflate streaks

Fig 1 Methods used for isolation and enumeration of bacterial and fungal colonies

20 Techniques in Mycorrhizal Studies

Subsequently, using a sterilized inoculation loop or a bent wire a visible colony is then removed and suspended in a physiological solution (e.g 0.9% NaCI) An inoculum from the resulting suspension is then used

to make series of streaks on the surface of a new agar plate This procedure can be repeated several times When a pure culture identified by having one type of identical cells is obtained, then a colony from the last plate should be removed and used to inoculate a sterilized agar or liquid media

2.1.2 Soil Plate Method

Fungi can be isolated into pure culture directly from pieces of mycelium / mycelial aggregate or by allowing mycelium to grow out of organic sources

by plating directly soil on the nutrient agar Soil plate method overcomes the major limitations of dilution plate methods, which is not only time consuming but also favours the growth of sporulating fungi Moreover, the method does not provide accurate information on the numbers of fungal biomass Hence the soil plate method is a better alternative (Figure 1) It

can be carried out in the following steps:

(i) Disperse less than 1 mg of soil over the bottom of a petridish

(ii) Cover with cool molten agar

(iii) Incubate agar plates under conditions that mimic as far as possible of natural physcial environment

(iv) Subculture the fungi that develop by transferring a small amount (say

2 x 2 x 2) of agar plus mycelium into a fresh medium To give the target fungi a chance to develop, remove isolates that are obviously contaminants (e.g those with very rapid growth) before they sporulate

or overgrow the original isolation plates

Sewell's Slide Trap also permits the isolation of a wide range of fungi It consists of shallow chamber made from a Perspex strip (75 X 25

X 2.5 mm) as the base and narrow length (3 X 2.2 mm) to form the walls The chamber is divided into two unequal parts by a crosspiece The Perspex pieces are joined with chloroform The chamber is stored in alcohol and sterilized by flaming when required Sterile molten agar is pipetted into the large section of the chamber After solidification the chamber is covered with sterile glass slide held in place by two wire paper clips (Figure 2) The trap is then immersed vertically into the soil such that the large chamber is buried completely

Rani Gupta and H Mohapatra 21

A

B

Fig 2 Sewell's slide trap for isolating fungi from soil A Top view;

B Longitudinal section; C Trausverse section

c

It is important to stress on the fact that individual hyphae can also be picked from soil particles and plated into agar, but the success rate is extremely low

3 Enumeration and Counting

The most common counting techniques used for and counting of the culturable microorganisms in the soil are the pour plate count method and the spread plate count method

3.1 Pour plate counting method

Procedure

in the petri dish

22 Techniques in Mycorrhizal Studies

on the table surface (about three times clockwise and counter clockwise)

illuminated counting chamber at those dilutions that have developed 20-100 colonies after 5-7 days

Note: (i) If mould are spreading and covering more than 15-20% of the agar surface, the plates should be rejected because the development of bacteria may have been inhibited (25) (ii) Adding mycostatin (2mglml) can reduce the development offungi prior to pouring the plates (33)

One major drawback faced by the pour count method is that it prevents the growth of aerobic bacteria Thus to overcome this flaw the spread plate counting technique has been suggested Spread plating is commonly used for determining total viable counts (TVC) of pure cultures

or specifically coloured (by pigments or dyes in the agar) aerobic bacteria that are both selectively identified and counted

The success of this spread plating technique depends on the organism

in question, the medium, the accuracy of dilution and the care used in spreading the inoculum on the surface of plates (18) Table 2 lists some common media used for enumeration of bacteria

TABLE 2 Some common media used for enumertion of bacteria

preparation

Rani Gupta and H Mohapatra 23

3.2 Spread Plate Technique

This technique is very much suitable for the estimation ofbacterial colony forming units on the surface ofthe agar plates (Figure 1) Spread plate each dilution on the desired nutrient medium in triplicate as follows:

a Thoroughly shake the dilution and immediately pipette 0.1 m1 of the shaken dilution (as an inoculum) to each of three nutrient plates

errors by the settling of the sands and other heavy solids in the pipette (ii)

The use ofO.lml inoculum increases the effective dilution on the plate by a factor of 10 e.g A plate that is inoculated with 0.1 ml from a 10.2 dilution is actually counted as a 10.3 dilution plate

b Using a sterile glass spreading rod, spread the inoculum over the surface of each plate as evenly as possible

c Repeat steps 1 and 2 until all ofthe diultions have been plated

d Check the plates for development of microbial colonies; incubate until new colonies cease to appear

Note: (i) Most colonies develop on concentrated or nutrient rich media within

3 to 7 days (ii) Full colony development on dilute or nutrient poor media may

require 2-8 weeks (iii) Extended incubation time may require that the plates are wrapped in parafilm or stored in plastic petridish sleeves to prevent excess drying of the agar

e Count the number of colonies on the plates

f Calculate the number of culturable microorganisms in the original sample by multiplying the average number of colonies on the plates from the "countable" dilution, by dilution factor The countable dilution

is the dilution that produces an average of30 to 300 colonies per plate (Table 3)

TABLE 3 Average count of colonies per plate in each dilution

Note: TNTC-Too Numerous to Count

Correct count = 36 x lOS = 3.6 X 106 colony forming units (CFU)! gram wet weight

24 Techniques in Mycorrhizal Studies

At this stage it is important to bear in mind that the result should be reported as CFU/g of soil sample, rather than the number of cells per gram, because one cannot be certain that each colony on the plate arose from a single cell

3.3 Fungal Plate Count

The plate cont method is less suitable for enumeration offungal populations

or densities because spores as well as fragments of one and the same mycelium fragmented by dilution agitation will develop as single colonies, which are counted If used the results should be expressed as colony forming units per gram of dry soil (CFU/g) Agar plates are more appropriate for qualitative examinations and isolation purposes Media used to enumerate and isolate fungi should be supplemented with streptomycin (30~Wml), aureomycin(20~Wml) or chloramphenicol (1 OO~W

ml) after filtration in order to suppress the development of rapidly growing bacteria Some agar media for the enumeration offungal CFU are given in Table 4

TABLE 4 Common agar media used for counting and isolating fungi from soil and

other natural substrates

agar, pH 7.0 ± 0.2

7.0±O.2

Peptone dextrose-rose bengal, streptomycin, aureomycin agar (20)

plate method because bacteria and actinomycetes are essentially suppressed

Rani Gupta and H Mohapatra 25

However the following points have to be borne in

mind:-1 Colony morphologies are highly dependent on the growth substrate

2 Colony characteristics observed for a strain on one medium may be quite different on another medium, This distinction becomes clearly visible when one is comparing colony morphologies on rich versus dilute media

3 Groupings of isolates into similar morphotypes should be done among colonies of similar age and from the same batch of media

4 There may also be variation in morphologies of colonies ofthe same strain Hence categorization if isolates based on specific colony characteristics should be broad and based on several aspects of morphology rather than on only one or two characteristics

Similarly in case offungal taxonomy the pattern of mycelial growth and the type of outline playa significant role in the primary classification of the organism to the generic level

4.1.2 Characterization based on growth substrates

The ability of the organisms to utilize specific substrates for growth and their fennentative ability provide an important key towards their taxonomic placement (17) This type of analysis is preceded by initial characterization reaction ofthe isolates towards Gram's stain followed by cell shape, size, oxidase and catalase reactions A variety of ready to use fermentative media are available for carrying out such reactions

26 Techniques in Mycorrhizal Studies

4.l.3 Colony hybridization

Hybridization of the isolated colonies with gene probes or cloned versions

of the genes of interest can detennine the presence or absence of particular genetic sequences within the cu1turable isolates This provides an additional tool in the identification of the isolates This method provides advantage of being very specific towards enumerating individual genotypes However its application is limited by the lack of knowledge concerning the molecular genetics of the soil microorganisms It requires a prior knowledge concerning the molecular genetics of both the gene probe and the target orgamsm

The steps involved in enumerating CPU having a particular genotype include the following:

1 Incubate quantitatively the cells in appropriate growth medium unto

2 weeks

2 Incubate appropriate positive and negative control strains The positive control strains are those cells from which the gene probe was originally cloned and the negative control strain are the ones containing the cloning vectors and strains harboring the genes that are related to but different from the gene ofinterest

3 After the growth period the colonies are transferred to a DNA binding membrane and lysed with a detergent such as sodium dodecyl sulfate and hydroxide The released DNA is fixed to the membrane according to standard procedures (29)

Note: The membrane has to be examined at this stage to ensure that no cell debris that might participate in non-specific binding of the probe remains on

Note: Salt content and temperature are critical for hybridization The lower the

salt content, the higher the washing temperature and greater the fidelity of

hybrids These values are determined by empirical calculations to avoid

hybridization with negative controls

6 Detect the presence of genes by autoradiography

Rani Gupta and H Mohapatra 27

4.1.4 Lipid analysis

The presence or absence of several classes and sub-classes of lipids viz

free acids, hydrocarbons, fatty alcohols or other compounds of bound fatty acids (phospholipids, peptidolipids, glycolipids etc.) prove to be valuable guidelines in the identification ofthe bacterial strains at the species level (32)

Fatty acids can be extracted and esterified with methanol to form fatty and acid methyl esters (FAME's) (5) and phospholipids can be purified from mixtures oflipids by solid phase extraction (3 5) and from phospholipid fatty acid methyl esters (PLFA's) These are analyzed qualitatively and quantitatively by high resolution fused silica capillary gas chromatography (21) This process is being practiced widely and can be automated The pattern obtained from an unknown isolate is compared with those of reference strain in a computer database to identify the unknown isolate

Precautions:

(i) Purity of the isolate is very important

(ii) The selection of the reference strain is critical to the success of the identification

isolates are needed for the detemination of taxonomic relationships, because fatty acid patterns are influenced by many factors including growth medium,

(11,31)

(iv) The database of the reference strains is limited and species identification may

4.1.5 rRNA characterization

The characterization ofrRNAgenes (the DNA sequences coding for rRNA) has become a powerful tool for the identification of the environmental isolates upto the phylogenetic levels (34) The basic concept behind this

technique is the fact that certain specific rRNA molecules (viz 16S and

23S) ofthe prokaryotes are highly conserved throughout evolution and can thus be used as specific indicators ofthe phylogenetic levels of the unknown strains The nucleotide sequences of the portions of the 16S and

23 S RNA molecules change at a faster rate over evolutionary time than other portions ofthe same molecule The regions of the gene that change

at very slow rate over evolutionary time are characteristic of broad

28 Techniques in Mycorrhizal Studies

phylogenetic group while those regions that change at a faster rate are characteristic of narrower phylogenetic groups such as high G + C content

in gram positive bacteria The regions ofthese molecules that change most rapidly over evolutionary time (so called hypervariable regions) are characteristic of individual species (19)

There are two methods for rRNA characterization:

L Colony hydridization with synthetic oligonucleotide probes directed toward specific phylogenetic groups

n Analysis ofthe nucleotide sequences ofthe 16S rRNA genes This helps to quickly categorize large number of isolates broadly upto the phylogenetic groupings and can be quantitative if quantitative procedures are used in cultivation of the colonies

However, this approach is limited by the lack of knowledge of the rRNA sequences of most environmental mocroorganisms The available synthetic oligonucleotide rRNA probes (maintained by the ribosomal database project - RDP, at University oflllinois) are not able to hybridize with the RNA molecules ofthe environmental isolates This is due to the fact that majority ofthe soil microorganisms have never been sequenced Secondly, this procedure is much more informative than the colony hybridization with synthetic oligonucleotide probe but is much more time consuming and technically demanding

Procedure:

of the amplification product depends on the primer selected but in general varies between I and 1.5 kb

suited for the purpose

Note: A number of such vectors are available in the market A few to mention are 'TA cloning vectors' (Invitrogen San Diego, California) and the 'Choneamp vectors' (GmCO BRL, Bethesda, Madison)

Note: The presence of strong secondary structures makes the denaturation

of the template difficult and thus makes the sequencing of 16S rRNA genes

from some speices may require alternative strategies

Rani Gupta and H Mohapatra 29

4 1.6 DNA fingerprinting: RFLP, AP-PCR and REP-PCR

The restrictionfragrnent length polymorphism analysis, the arbitrarily primed PCR (AP-PCR) and the repetitive extragenic palindromic PCR (REP- PCR) are used to differentiate between closely related environmental strains

It should be borne in mind, while going in for such approaches, that the fingerprinting techniques are useful in revealing small genetic differences

or similarities between closely related species or strains Thus these are more helpful in categorizing the isolates to a finer species level and are not meant to be used for categorizing the isolates to a broad phylogenetic levels:

(A) RFLP

This is the oldest of the approaches used for identification of isolates

Procedure:

endonucleases in order to identify polymorphism's within individual genes

(iJ.) Separate fragments of different sizes within the digested DNA by agarose gel electrophoresis

(iiI) Transfer to a DNA binding membrane by standard procedures

(iv) Hybridize with labeled probe specific for the gene of interest

(v) See for differences or similarities between different isolates

Precautions:

enzyme digestion, thereby yielding partial digestion products that interfere in the analysis

(ii) Chromosomal DNA should be subjected to multiple rounds of chloroform extraction and then atleast two rounds of chloroform extraction

phenol-(iii) The amplification of genes by PCR proves to be the easiest for obtaining DNA suitable for RFLP, as the DNA is amplified without any contaminants The amplification products are digested with enzymes of choice and visualized

by gel electrophoresis

(iv) The enzymes used for RFLP analysis are typically 6bp recognition restriction endonucleases such as EcoRI, Hind III, Bam III, however for relatively small (less than or equal to SOObp) segments of DNA or for highly conserved genes such as rRNA genes, restriction endonucleases that recognize 4 bp and generate a greater number offragments such as Taq, I Hae III and Sau 3A are used

30 Techniques in Mycorrhizal Studies

Arbitrarily primed PCR is similar to RFLP analysis as even this is used to differentiate between closely related strains However, it differs from RFLP in the fact that this is designed to screen differences betwen entire genome, Moreover, AP-PCR being PCR based is faster than RFLP and requires no digestion with restriction endonucleases Also, it uses only one primer (in contrast to two being used for most PCR's) of random base composition between 8-10 bases in length

Precautions

variables including primer sequence, primer concentration, template concentration, template quality, cycling parameters and thermocycles (n) Due to the sensitivity to AP-PCR banding patterns to so many variables, lack

of reproducibility is a frequent problem For this, three replicate amplifications should always be conducted

(C) REP-PCR

REP-PCR is based on amplification of repeated sequences that are highly conserved and widely distributed among gram-negative genomes (10) This is therefore not random and uses two primers (10)

(1) REP-PCR produces less variability problems than AP-PCR (n) Species-specific probes may be generated to identify individual species either by colony hybridization or in extracts of community

DNA (10)

4.2 Identification of Fungal Cultures and Spores

Identification ofthe fungal cultures is dependent to a great extent on the characteristics ofits fruiting bodies However all fungi do not fruit and hence fruiting is induced artifically e.g exposure to UV light (8) Since keys are available only for a limited range of species (22) researchers often need to construct their identification schemes Immunologic and genetic probes go a long way in identifying fungi

4.2.1 Identifying mycelia in culture

Procedure

(u) Isolate mycelia into culture from these fruit bodies ensuring that enouih

individuals have been obtained to cover inherent cultural variability

(iiI) Define characteristics that can discriminate between species in that habiL

Rani Gupta and H Mohapatra 31

(28) and include colour, odour, texture, tissue like structures, hyphal characteristics, diameter, wall thickness, clamp connections, branch angle and

frequency, straightness, surface deposits, inclusions and terminal structures, extension rates under different conditions and enzyme tests (30)

(iv) As some characeteristics are better discriminators than others for a particular set of fungi, reduce the range of characteristics recorded accordingly Nonetheless, many characteristics may still be necessary, especially if there are a lot of species in the habitat Also it may not always be possible or convenient to record all the characteristics of an "unknown" Further, it is important to note that characteristics of an individual may not always be the same, some may be "turned on" by physiologic switches and environmental conditions In these cases, some sort of computerized pattern recognition systems will be useful

(v) Confirm identifications or discriminate among several possibilities by determining whether the isolate can mate with "tester strains" of known identity

Direct pairing tests for somatic incompatibility is also useful in determining similar strains of ascomycetes and basidiomycetes

5 Identification ofUnculturable Soil Microflora

5.1 Bacterial Microtlora

To overcome the limitations of laboratory cultivation, alternative approaches based on analysis of indicator molecules extracted directly from samples are carried out The most commonly used molecules are DNA, RNA and phospholipids

32 Techniques in Mycorrhizal Studies

The relative concentrations of different kinds of polar lipids in each membrane is characteristic of a particular group, but the fatty acid components of the individual membrance lipids are not fixed and vary with the environmental, physiological and nutritional condition of the cell Thus, the fatty acid profile patterns especially the phospholipid fatty acids provide important biomarkers for certain microbial types (12, 13) However, a major limitation faced by this method is that the phospholipid fatty acid patterns cannot be correlated with the dynamics

of specific groups of organisms

Procedure

applied to a DNA binding membrane adjacent to the environmental samples Note: (i) Triplicate samples are advised because of the variability of the procedures and membrances (ii) The inclusion of negative control (as described

in colony hybridization) is important

(11) DNA is applied to the membrance as a grid through a vacuum manifold DNA maybe applied as circles (do blots) or as narrow ellipses (slot blots) Note: Slot blots are used for quantitative hybridization because the resulting autoradiograms are more easily analyzed by scanning densitometry

(ill) Standard procedures for labeling the probe (either with radiolabels such as

e2 P} dClP or with non-radioactive labels), pre-hybridization, hybridization and washing are followed (24, 29)

Note: The stringency of washing is kept high to avoid possible non-specific hybridization with related but non-target genes in the environmental sample (24)

(iv) The extent of hybridization is measured by exposure of the membrance to rayfilcon (autoradiography.)

X-Note: (i) X-ray film should be preflashed with a photographic strobe (29) in order to increase the sensitivity and the linear range of the autoradiogram (ii)

The membrane is wrapped in plastic wrap such as Saran wrap and the film and membrance are placed in an autoradiography cassette (iii) The cassette should

be kept at - 80°C for between 3h and I week depending on the intensity of the signal emitted from the bound probe The length of the exposure has to be determined for each application

Rani Gupta and H Mohapatra 33

(v) After appropriate time, the film is developed and the densities of the exposed

scanning densitometry

Note: The amounts of hybridization in different samples may be determined from a standard curve relatig density to mass of target generation from the dilution series of target genes

Precautions:

The regions of the probe may hybridize with the non-target genes In the sample, resulting in an over estimation of the target gene Alternatively, limited regions of the probe may hybridize with the target genes in the sample because of the evolutionary divergence of these genes, thereby underestimating the concentrations

of the target sequences For these reasons relative short probes (approximately 500bp) segments of an internal region of a well characterized gene is preferred and the washing conditions should be kept relatively stringent (i.e low salt and high temperature)

5.1.3 Comparison by percent G + C profiles

Comparison of the G + C prove useful in the analysis of microbial community structure (15) The bacterial chromosomes have G+ C contents characteristics of their taxonomic groups Thus the G + C contents compared with the available G + C profile of the community gives an authentic data with regard to the taxonomic placement of the isolate

Procedure

gradients by ultracentrifugation

(ii) The more dense G + C rich regions form a gradient of DNA of increasing G +

C contents downward through the ultracentrifuge tube

(m) The tube is sampled by taking small fractions from the bottom of the gradient

(iv) The concentration of DNA (or A200) is measured for each fraction and plotted

as a function offractiom number

5.1.4 Analysis ofrRNA and rDNA

The rRNA and rDNA hybridizing probes can also be used to identifY an isolate unto its species level The methods and the procedures are similar

to the previous ones

5.2 Fungal flora

The classical methods used for isolating fungi from their natural communities suffer from generating a distorted picture of species composition since some organisms are detected more readily than other Moreover growth