EFFECTS OF INTERACTIONS BETWEEN ARBUSCULAR
MYCORRHIZAL FUNGI AND RHIZOBIUM LEGUMINOSARUM ON
PEA AND LENTE
A Thesis Submitted to the College of
Graduate Studies and Recvarch
in Partial Fulnllment of the Requirements
for the Degree of Doctor of Philosophy
in the Department of Applied Microbioloa and Food Science
University of S&atchewan
Saskatoon
Liset Johnny
Spnng, 1999
@Copyright Liset Johnny. 1999. AU rights reserved.
I*m
Bibliothèque nationale
National Library
of Canada
du Canada
Acquisitions and
Bibliographie Services
Acquisitions et
services bibliographiques
395 Wellington Street
OttawaON K1A O N 4
395. rue Wellington
Ottawa ON K1A ON4
Canada
Canada
Your me vorm rélërenccr
Our & Alorre r8fdrence
The author has granted a nonexclusive licence allowing the
National Library of Canada to
reproduce, loan, distribute or sell
copies of this thesis in microform,
paper or electronic formats.
L'auteur a accordé unie licence non
exclusive permettant à la
Bibliothèque nationale du Canada de
reproduire, preter, distribuer ou
vendre des copies de cette thèse sous
la forme de microfiche/fh, de
reproduction sur papier ou sur format
électronique.
The author retains ownership of the
copyright in this thesis. Neither the
thesis nor substantial extracts fiom it
may be printed or otherwise
reproduced without the author's
permission.
L'auteur conserve la propriété du
droit d'auteur qui protège cette thése.
Ni la thèse ni des extraits substantiels
de celle-ci ne doivent être imprimés
ou autrement reproduits sans son
autorisation.
UNIVERSITY OF SASKATCHEWAN
College of Graduate Studies and Research
SUMMARY OF DISSERTATION
Subrnitted in partial fulfillment
of the requirements for the
by
Liset Johnny
Depamnent of Applied Microbiology and Food Science
Universisr of Saskatchewan
Spring 1999
Exarnining committee:
Dr. A.E.
Slinkard
Xk%XdJLWWW%DW,Dean's Designate. Chair
College of Graduate Studies and Research
Dr, R. T. Tyler
Chair of Advisory Cornmittee, Department of Applied
Microbiology and Food Science
Dr. J. J. Germida
Supervisor,
Head of the Department of Soil Science
Dr. G. G. Khachatourians
Department of Applied Microbiology and Food Science
Dr. L. M. Nelson
Agriurn Inc., Saskatoon, SK
Dr. D. R.Waterer
Department of Plant Sciences
Extemal Examiner:
Dr. F. B. Ho11
De~artmentof PImt Science
~ n h e r s i t yof British Columbia
Vancouver, B. C .
V6T 124
Effects of Interactions between Arbuscular Mycorrhizal Fungi and
Rhizobium Zeguminosarum on Pea and Lentil
Legumes form a ûipartite symbiosis with arbuscular mycorrhizal b g i (AMF)and rhizobia
which idluences plant productivity. This study assessed some-factors that might affect the
tripartite symbioses between AMF, Rhizobium legurninosamm bv. viceae and pea (Pisurn
sativurn L.) or lentil (Lens esculenta L.), and detennhed if specific combinations of AMF
and rhizobia enhanced plant productivity. A survey of soil and plant samples from 12 field
sites in Saskatchewan injicated k a t AMI? activity was higher under lentil (n=7) than pea
(n=5) crops. Soil and root traps used to isolatk AMF colonizing lent3 and pea revealed
more AMF spores and spore morphotypes in soi1 than within rootç, suggesting that lentil
and pea exhibited AMF-host selectivity.
The efficacy of commercial Rhizobium inoculants, reference strains and isolates
obtained from root nodules of field-grown pea and lentil was assessed under gnotobiotic
conditions. Effective and ineffective Rhizobium strâins were selected for CO-inoculation
L
W GZumus clarum NT4 or
studies with AMF. Pea and lentil were inoculated with the A
G. mosseae NT6 and/or effective and ineffec tive Rhizobium leguminosarztrn bv. viceae
saains for pea or lentil and grown in soi1 containhg indigenous AMF and rhizobia, under
growth chamber conditions. Lentil responded to CO-inoculationbetter than pea. Results
suggest that specific AMF+Rhizobiurn strain combinations enhanced plant growth and
yield. FurtJiemore, effective AMF altered the performance of inferior rhizobia and viceversa A subsequent study determined the influence of P levels on the tripartite symbiosis.
Pea and lentil were CO-inoculatedwith an effective or an ineffective AMF+Rhizobium strain
combination and grown in soi1 containing indigenous AMF diat was amended with
different levels of P fertilizer. In general, application of P fefllizer did not alter pea
response te either AMF+Rhizobium strain combination, but signifcantly increased the
yield and nutrition of CO-inoculatedlentil plants. Furthemore, inoculation of pea or lentil
with rhizobia or AMF+Rhizobiurn strain combinations yielded growth equival.prit to or
better than 20 pprn of P krtilizer.
Selected spore wall bacteria ( S m ) isolated from AMF spores stimulated or
inhibited the germination of NT4 spores in vitro. The ability of these SWB to influence the
tripartite syrnbiosis was evaluated using pea grown in a sterilized soil:sand mix. T'lie
stimulatory SWB enhanced gro*
and AMF root colonization of NT4-inoculated pea
plants, whereas the inhibitory SWB reduced AMF colonization and had no effect on plant
growth. However, in the presence of the Rhizobium strain this trend was reversed.
s u g g e s ~ that
g interactions between the S m and rhizobia altered the response of the
NT4tLX43-inoculated plants' to the SWB isolates.
My research dernonstrated that interactions between AMF, rhizobia and the legume
host were specific, and that the soil-P level and SWB c m alter the outcome of the pea and
lenal nipartite symbioses. Therefore, it is important to assess the effect of AMF-rhizobialegume interactions in the development of commercial inoculants for enhancing !egume
productivity.
BIOGRAPHICAL
B .Sc. (Ag). TamilNadu Agricultural University, Coimbatore, India
M.Sc. (Ag), TamilNadu Agricultural University, Coimbatore. India
M. Sc., Department of Soil Science, University of Saskatchewan
HQNORS
L. H. Hantelman Postgraduate Scholarship, 1997
Car1 Auerhamner Postgraduate Scholarship, 1996, 1997
President's Award for excellence in die presentation of a scientific paper, 1994
Maurice Hanson Sr. Postgraduate Scholarship, 1993
PERMISSION TO USE
In presenting this thesis in partial f ' e n t of the requirements for a Postgraduate
degree from the University of Saskatchewan, 1agree that the Libraries of this University
may make it freely available for inspection. 1further agree that permission for copying of
this thesis in any rnanner, in whole or in part, for scholarly purposes may be granted by
the professor or professors who s u p e ~ s e dmy thesis work or. i;l their absence, by the
Head of the Deparmient or the Dean of the College in which rny thesis work was done. It
is understood that any copying or pubGcation or use of this thesis or parts thereof for
financial gain shall not be allowed without my written permission. It is also understood
that due recognition shall be given to me and to the University of Saskatchewan in any
scholarly use which rnay be made of any material in my thesis.
Requests for permission to copy or to make other use of material in this thesis in whole or
part should be addressed to:
Head of the Deparmient of Applied Microbiology and Food Science
University of Saskatchewan
51 Campus Drive
Saskatoon, Saskatchewan
Canada S7N 5A8
ABSTRACT
Legumes form tripartite symbioses with arbuscular mycorrhizal fun$ (AMF)and rhizobia
which innuence plant productivity. This study assessed factors that influence the
tripartite symbioses between AMF, Rhizobium Zegunzinosanm bv. viceae and pea (Pisum
sativurn L.) or lentil (Lensesculenra L.), in order to determine if specifïc combinations of
AMF and rhizobia enhanced plant productivity. A survey of soil and plant samples from
12 field sites in Saskatchewan indicated that AMF activity (Le., number of spores and
root colonization) was higher under lentil (n=7) than pea (n=5) crops. Soi1 and root traps
were used to isolate AMF colonizhg lentil and pea. Soi1 traps yielded more AMF spores
and spore morphotypes than root traps and it appeared that lentil and pea exhibited AMFhost selectivity. The presence of the AMF fatty acid methyl ester biornarker 16: 1 o5c
was correlated with AMF root colonization detemined using microscopy.
The efficacy of commercial rhizobia inoculants, reference saains and isolates
~btainedfrom mot nodules of field-grown pea and lentil was assessed under gnotobiotic
conditions. Some seainç significantly increased the shoot dry weight and shoot N
content of plants, whereas other strains varied in their effectiveness. Effective and
ineffective rhizobia straids were selected for CO-inoculationstudies with AMF in a growth
chamber. Pea and lentil were inoculated with the AMF Glomus clarum NT4 or G.
mosseae NT6 and/or effective and ineffective Rhizobium Zeguminosarum bv. viceae
suaios for pea or lentii and grown in soil containing indigenous AMF and rhizobia.
Some rhizobia strains increased the growth and yield of pea and lentil, whereas others
had no effect. The AMF NT6 inoculant was generdy more effective on pea and lentii
thaa NT4. Furthemore, CO-inoculationwith rhizobia and the AMF NT4 or NT6 had
different effects on plant gowth, yield and N and P nutrition. Lentil responded to coinoculation better than pea. The most and least effective AMFcrhizobia combinations for
pea and lentil were NT4tLX43 and NT4+175P4,and NT4tLX77 and NT4+PB 101,
respectively. Thus. results suggest that specific AMF+rhizobia combinations enhanced
plant growth and yield. Furthemore, eftective AMF can enhance the performance of
inferior rhizobia and vice versa.
A subsequent smdy determined the influence of soil-P levels on the tripartite
symbiosis. Pea and lentil were CO-inocuIatedwith NT4+LX43 or NT4t175P4. and
NT4+LX77 or NT4+PB 101 respectively. and grown in soi1 that contained indigenous
AMF and was amended with different levels of P fertilizer. AppLication of P fertilizer did
not alter pea response to either AMF+rhizobia combination. but significantly increased the
yield and nutrition of CG-inoculatedlentil plants. Furthemore. inoculation of pea or lentil
with rhizobia or rhizobiatAMF combinations yielded growth equivalent to or bener than
20 ppm of P ferslizer.
Selected spore wall bacteria (SWB) isolated from AMF spores stimulated or
inhibited the germination of NT4 spores in vitro. The ability of these SWB to influence
the tripartite symbiosis was evaluated using NT4+LX43-inoculated pea grown in a
sterilized soi1:sand mix. The stimulatory S m enhanced growth and AMF root
colonization of NT4-inoculated pea plants, whereas the inhibitory SWB had no effect.
However, in the presence of the rhizobia strâin LX43, this trend was reversed,
s u g g e s ~ that
g interactions between the S M and rhizobia altered the response of the
NT4+LX43-inocuiated plants to the SWB isolates.
My research showed that interactions between AMF, rhizobia and the legume host
were specific, and that microsyrnbiont efficacy, soil-P level, and SWB can alter the
outcome of the tripartite symbioses. Therefore, it is important to assess the effect of
AMF-rhizobia-iegume interactions in the development of commercial inoculants for
enhancing legume productivity.
ACKNO WLEDGMENTS
I wish to express my sincere appreciation and gratitude to my s u p e ~ s o("Guru")
r
Professor James Germida for his training. guidance. valued criticisms and support
throughout my program. By example, he has taught me persistence. patience and
diligence. and has heightened my sense of scientific curïosity.
1acknowledge with gratitude the contributions made by members of my Advisory
cornmittee, Drs. Robert Tyler, George Khachatourians. Louise Nelson. Douglas Waterer
and Michael Ingledew, durhg the course of this study.
My sincere thanks to Dr. F. B. Ho11 for serving as external examiner and for his
valued suggestions and contributions to my thesis.
My special thanks tc Drs. Robert Baker, Renato de Freitas and Craig Stevenson
for their professional and personal help during my program. My thanks to Cindy Theoret
for her love and support and the many hours of cheemil and voluntag assistarce she
provided d u h g rny program. I also wish to thank past and present members of the Soi1
Microbiology group. especially Mrs. Arlette Seib. for their camaraderie and assistance.
1am indebted to the Departrnent of Applied Microbiology and Food Science and
Deparmient of Soi1 Science, the staff and my fnends in both Departments for their
friendship and support.
The financial support provided by me Western Grains Research Foundation. the
NSERC - Agriculture Agn-Food Canada Research Partnership Program and the
Agriculture Development Fund is gratefully acknowledged.
1am grateful to my mom for bearing rny absence from home for the last seven
years. for loving me despite all rny shortcomings. and for supporàng me in my pursuit
for higher education.
I dedicate this thesis to my husband, Dr. Ilungo J. Xavier and to the "little
person" who has been a part of Our lives for the last few months. My husband's
unconditional love, support and patience were my inspiration.
TABLE OF CONTENTS
PERMISSION TO USE
ACKNOWLEDGMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
1.0
INTRODUCTION
2.0
LITERATURE REmw
2.1
The rhizosphere
2.2
Rhizobia
2.3
Arbuscular mycorrhizal fun@
2.3.1 AMF-host specificity
2.4
Interactions between AMF and beneficial bacteria
2.4.1 Interactions between AMF and non-nodulating bacteria
2.4.2 Interactions between AMF and rhizobia
2.4.2.1
Nutritional benefits
2.4.2.2
Non-nutritional benefits
2.4.2.3
Effects of inter-endophyte
compatibility on legume growth
2.4.2.4
Factors influencing interactions between
AMF and rhizobia
Host species
2.4.2.4.1
Efficacy of the microsymbionts
2 -4.2 -4.2
2.4-2.4.3
Nutrient levels
2.5
Conclusions
3.0
4.0
OCCURRENCE AND ACTNITY OF ARBUSCULAR
MYCORWHIZAL FUNGI IN THE 3WIZOSPHERE AND
ENDORHIZOSPHERE OF LENTIL A h ! PEA IN
SASKATCHEWAN SOILS
3.1
Introduction
3.2
Materials and methods
3 -2.1 Sample collection and soil nutrient analyses
3 2.2 Trap culture generation using field soil or plant roots
3.2.3 Parameters
3-2.3.1
Spore enurneration
3-2.3.2
Mycorrhizal colonization
3.2.4 Statistics
3.3
Results
3.3.1 Field
3.3 -2 Trap cultures
3.3.2.1
Soil trap cultures
3.3.2-2
Root trap cultures
3.3.3 CorreIation of AMF root colonization with the FAME
16: 1 o 5 c withùi roots of trap hosts
3-4
Discussion
ISOLATION AND IDENTIFICATION OF RHIZOBIA AND
EVALUATION OF THEIR EFFICACY ON PEA AND
LENTIL
4.1
Introduction
4.2
Materials and methods
4.2.1 Isolation. purification and maintenance of Rhizobium
cultures
4.2.2 Commercial inoculant and reference Rhizobium strains
4.2.3 Evaluation of rhizobia on pea and lentil
4.2.4 Parameters
4.2.5 Statistics
5-0
4.3
Results
4.3.1 Effect of rhizobia on pea
4.3 -2 Effect of rhizobia on lentil
4.4
Discussion
EFF'ECTS OF INTERACTIONS BETWEEN TWO GLOMUS
SPECIES AND RHIZOBIUM LEGUMINOSARUM BV.
VICEAE ON THE GROWTH, Y E L D AND NUTRITION OF
PEA AND LENTIL IN NON-STERILE SOIL CONTAINING
INDIGENOUS AMF AND lPHIZ0BI.A
5- 1 Introduction
5.2
Materials and rnethods
5 -2.1 Effect of AMF-rhizobial interactions on pea
5.2.1.1
Treatments set-up for pea
5.2.1.2
AMF inocula
5-2.1.3
Rhizobium leguminosarurn bv. viceae
strains
5.2.1.4
Soil
5.2.1.5
Inoculation and plaît gowth
5.2.1.6
Pararneters
5.2.2 Effect of AMF-rhizobial interactions on lentil
5.2.3 Statistics
5.3
Results
5 3 . 1 EffectofAMF-rhizobialinteractions onpea
5.3.1.1
Dry matter production
5.3.1.2
Nutrient parameters
5.3.1.3
AMF colonization and nodulation
5 -3-2 Effect of AMF-rhizobial interactions on lentil
5.3.2.1
Dry matter production
5.3.2.2
Nutrient parameters
5.3.2.3
AMF colonization and nodulation
5.4
Discussion
vii
6.0
THE INFLUENCE OF PHOSPHORUS ON THE TRIPARTITE
SYMBIOSE BETWEEN AMF, RHIZOBIA AND LEGUiMES
6.1
introduction
115
6.2
Materials and methods
6.2.1 Experimental design for pea
6.2.1.1
M inoculum
6.2.1.2
Rhizobitim legwninosanim bv- vtceae
strains
6.2- 1.3
Soi1
Inoculation and plant growth
6.2- 1.4
6.3.1.5
Parameters
6.2.2 Experimental design for lentil
6-2.3 Statistics
6.3
Results
6.3.1 Effect of P on the growth and yield of pea
6.3 -2 Effect of P on the nutrient content of pea
6.3 -3 Effect of P on the AMF colonization and nodulation of pea
6.3.4 Effect of P on the growth and yield of lentil
6.3 -5 EfYect of P on the nument content of lentil
6.3.6 EEect of P on the AMF colonization and nodulation of
lentil
120
120
126
132
Discussion
149
6.4
7.0
Il5
134
139
147
EFFECTS OF BACTEIUA ASSOCIATED WITH ARBUSCULAR
MYCORRHIZAL FUNGAL SPORES ON THE GLOMUSRHIZOBIUM-PISUM SYMBIOSE
154
7.1
Introduction
154
7.2
Materialsandmethods
7.2.1 AMF spores
7.2 -2 AMF spore decontamination
7 -2-3 Isolation of SVVB
7.2.4 identification of SWl3
7 -2.5 In v i n 0 bioassays
7.2.5.1
Response of rhizobia to SWB
7.2.5.2
Response of AMF spores to SWB
7.2.6 Plantassay
7.2-7 Statistics
Results
7.3.1
AMF spore decontamination
7.3.2 Identification of SWB
7.3 -3 Response of rhizobia to SWB
7.3 -4 Response of AMF spores to SWB
7.3.5 Effect of SWB on the bripartitesymbiosis
Discussion
8.0
GENERAL DISCUSSION AND CONCLUSIONS
9.0
REFERENCES
10.0
APPENDICES
LIST OF TABLES
3.2.1
Field site location, number, and select characteristics of the
Saskatchewan field sites cropped to lentil and pea.
3.2.2
Chemical characteristics of soils collected from different
Saskatchewan field sites cropped to lentil and pea
Mean (n=2) &SE number of A M F spores recovered from soiI in soi1
trap cultures, and first and second cycle root trap cultures. and percentage
AMF colonization of sorghum sudanpass (soil trap and first cycle root
trap) and maize (second cycle root Uap) roots.
3.3.1
3.3.2
Mean (n=2) number of AMF morphotypes recovered from soi1 in the soi1
trap (ST) cultures produced using sorghum sudangrass plants (soil trap)
and second cycle root aap (2ORT) cultures produced using maize plants.
4.2.1
Bacterial isolates cbtained from root nodules of pea and lentil grown at
various field sites in Saskatchewan during 1995.
4.3.1
Mean (n=3) shoot dry weight, percentage and total N content, and
nodulation of pea (cv. Trapper) inoculated with commercial inoculants.
reference RhL;obium strains, or field isolates (LX) and grown for 6 weeks
in a Leonard jar.
4.3.2
5.2.1
5.2.2
Mean (n=3) shoot dry weight, percentage and total N content, and
nodulation of lent. (cv. Laird) inoculated with commercial inocdants.
reference Rhizobium strains, or ficld isolates (LX) and growo for 6 weeks
in a Leonard jar.
71
Sources of the R. leguminosarum bv. viceae strains and isolates used. and
their effectiveness on pea.
81
Sources of the R leguminosanrm bv. viceae strains and isotates used, and
the& effectiveness on lzntil.
85
5.3.1
Mean (n=4) shoot and total root dry weight (nodule+root) of pea plants
inoculated with the AMF species Glomus clanrrn NT4 or G.rnosseae NT6
and/or 10 Rhizobium leguminosanrm bv. viceae strains and grown for 90
d in soi1 containhg indigenou AMF and rhizobia
5.3.2
Mean (n=4) grain yield and harvest index (grain yieldtotal aboveground
dry matter x 100) of pea plants inoculated with the AMF species Glomus
clanun hT4 or G. mosseae NT6 and/or 10 Rhizobirim leguminosanirn bv.
viceae strains and grown for 90 d in soil containing indigenous AMF and
rhizo bia,
5.3.3
Mean ( n d ) shoot N and P content of pea plants inoculated with the AMF
species Glomus clarum NT4 or G. mosseae NT6 andor 10 Rhizobium
leguminosarurn bv. viceae saains and grown for 90 d in soil containing
indigenous -4MFand rhizobia.
5.3.4
Mean (n=4)shoot and grain P use efficiency of pea plants inoculated with
the A M F species Glomus clarum NT4 or G. rnosseae NT6 and/or 10
Rhizobium leguminosarum bv. viceae strains and g r o m for 90 d in soi!
containing indigenous AMF and rhizobia.
5.3 -5
Mean (n=4)percentaze of AMF-colonized root Iength and nodulation of
pea plants inoculated with the AMF species Glomus clarum NT4 or G.
mosseae h i 6 andfor 10 Rhizobium leguminosarurn bv. viceae suains and
grown for 90 d in soil containing indigenous AMF and rhizobia
5.3.6
Mean (n=4) shoot and total root dry weight (r?odule+root) of lentil plants
inoculated with the AMF species Glomus clarum NT4 or G. mosseae NT6
a n d o r 9 Rhizobium leguminosarum bv. viceae strains and grown for 110
d in soi1 containing indigenous AMF and rhizobia
99
5.3 -7
Mean (n=4) grain yield and harvest index (grain yieldtotal abovegound
dry matter x 100) of lentil plants inoculated with the AMF species Glomus
clarum NT4 or G. mosseae NT6 and/or 9 Rhizobium legztrninosanrm bv.
viceae strains and grown for 110 d in soil containing indigenous AMI? and
rhizobia.
5.3.8
Mean (n=4) shoot N and P content of lenal plants inoculated with the
*L\MFspecies Glormts clanon NTLF or G. mosseae NT6 andor 9
Rhizobium leguminosantrn bv. viceae strains and grown for I l 0 d in soil
containing indigenous AMF and rhizobia.
5.3 -9
Mean (n=4) shcot and grain P use efficiency of lentil plants inoculated
with the AMF species Glomcis clarum NT4 or G. mosseae NT6 andfor 9
Rhizobilmz leguminosantrn bv. viceae strains and grown for 110 d in soil
containing indigenous AMF and rhizobia.
5.3.10
Mean ( n 4 ) percentage of AMF-colonized root length and nodulation of
lentil plants inoculated with the AMF species Glomus clarum NT4 or G.
rnosseae NT6 and/or 9 Rhizobium leguminosarum bv. viceae strahs and
grown for 110 d in soi1 containing indigenous AMF and rhizobia
6.3.1
Mean (n=5) shoot and total root dry weight of pea inoculated with the
AMF species Glomus clarum NT4 andfor the Rhizobium leguminosarum
bv. viceae strains 175P4 and LX43 and grown for 95 d in soi1 amended
with 0, 10 or 20 mg kg-1 of P and containing indigenous AMF. -
6.3.2
Mean (n=5) grain yield and harvest index (grain yieldtotal aboveground
dry matter x 100) of pea inoculated with the AMF species Glomus clarum
NT4 and/or the Rhizobium leguminosarwn bv. viceae strains 175P4 and
LX43 and grown for 95 d in soi1 amended wiih 0,10 or 20 mg kg-' of P
and containing indigenous AMF.
101
6.3.3
6.3 -4
Mean (n=5)shoot and grain P use effkiency (PUE) of pea inoculated with
the AMF species Glorn~sclanun hT4 andfor the Rhizobium
legrrminosarum bv. viceae strains 275P4 and LX43 and grown for 95 d in
soil amended with 0. 10 or 20 mg kg-1 of P and containing indigenous
AMF,
131
Mean (n=5)percentage AMF-colonized root length and nodukition of pea
inoculated with the AMF species Glomus clarum NT4 andor the
Rhizobium legurninosamm bv. viceae strains 175P4 and LX43 and grown
for 95 d in soil amended with 0, 10 or 20 mg k g 1 of P and containing
indigenous AMF.
133
6.3.5
Me:m(n=5)shootandtotalrootdrywei~toflentilinoculatedwiththe
AMF species Glomus clarum NT4 andor the Rhizo biurn leg uminosarum
bv. viceae strains PB 1O l and LX77 and grown for 2 10 d in soil amended
with 0. 10 or 20 mg k g 1 of P and containing indigenous M .
6.3.6
Mean (n=5)g a i n yield and harvest index (grain yieldhotal aboveground
dry matter x 100) of lentil inoculated with the AMF species Glomus
clururn NT4 andor the Rhizobium leguminosarum bv. viceae strains
PB101 and LX77 and grown for 110 d in soil amended with 0,10 or 20
mg k g 1 of P and coniaining indigenous AMF.
6.3 -7
Mean (n=5) shoot and grain P use efficiency (PUE) of lentil inoculated
with the AMF species Glomus clarum NT4 and/or the Rhizobium
legurninosamm bv. viceae strains PB 101 and LX77 and grown for 110 d
in soil amended with 0, 10 or 20 mg kg1 of P and containing indigenous
AMF.
6.3 -8
Mean (n=5)percentage AMF-colonized root length and nodulation of lentil
inoculated with the AMF species Glomus clunznz NT4 andor the
Rhizobium leguminosarurn bv. viceae strains PB 101 and LX77 and
grown for 110 d in soil amended with 0, 10 or 20 mg k g 1 of P and
containing indigenous AMF.
7.3.1
7 -3-2
7 -3-3
Bacterial species recovered fiom untreated and decontmuiated Glornus
clurum NT4 spores.
165
Response of Rhizobium legrninosarum bv. viceae sîrains to selected
spore wall bacteria ( S m ) recovered from untreated Glumus c l a ~ m
NT4
spores, as assessed in a cross streak assay.
167
Response of Rhizobium leg~rminosartmbv. viceae strains to selected
spore wall bacteria ( S m )recovered from Glornus clar~lrnNT4 spores
decontaminated for 60 min., as assessed in a cross streak assay.
169
7.3 -4
7-3.5
Response of Glomus clanrm NT4 spores to direct contact, and diffusible
and volade chzmicals produced by spore waU bacteria (SWB). as
assessed in a plate assay.
170
Mean (n=5) shoot and root dry weight, root:shoot ratio. number of
nodules and percentage AMF-colonized root length of pea plants coinoculated with the AMF species Glomus ciarum NT4. the Rhizobium
leguminos~rumbv. viceae strain LX43, and the AMF spore wall bacterial
isolates Bacillus chitinosporus stiâin LA6a or B. pabuli main LA3 âfter 6
weeks of growth in a sterile 2 :2 soi1:sand mix.
175
7.3 -6
Mean (n=5) shoot N and P content and P use efficiency of pea plants coinoculated with the AMF species Glomus clarum NT4, the Rhizobium
leguminosarum bv. viceae strain LX43.and the AMF spore wall bacterid
isolates Bacillus chitinospoms stïain LA6a or B. pabuli strain LA3 after 6
weeks of gowth in a sterile 1:2 soil:sand mix.
A. 1
ANOVA for the shoot and root dry weight, grain yield, harvest index.
shoot N and P content, gain N and P content, shoot and grain PUE and
% AMF-colonized root length of pea inoculated with the AMF species
Glomus clanlm NT4 or G- mosseae NT6 andor 10 Rhizobium
leguminosarum bv. viceae strains and grown for 90 d in soi1 containing
inditenous AMF and rhizobia.
Means for shoot and root dry weight. grain yield and harvest index of pea
inoculated with the AMF species Glumus clanm NT4 or G. mosseae hT6
andor 20 Rhimbirim leguniinosanrrn bv. viceae strains and grown for 90
d in soil containing indigenous AMF and rhizobia
Means for shoot and grain N and P content of pea inoculated with the
AMF species Glomus clamm N T 4 or G. mosseae NT6 and/or 10
Rhizobium leguminosarum bv. viceae strains and grown for 90 d in soil
containuig indigenous AMF and rhizobia.
Means for shoot and grain P use eficiency (PUE) and AMF-colonized
root length of pea inoculated with the AMF species Glomus clarum NT4
or G. rnosseae NT6 ancilor 10 Rhizobium leguminosanim bv. viceae
strains and grown for 90 d in soi1 containing indigenous AMF and
rhizobia.
ANOVA for the shoot and root dry weight. grain yield, harvest index.
shoot N and P content, grain N and P content, shoot and grain PUE and
% AMF-colonized root length of lentil inoculated with the AMF species
Clornus clarum NT4 or G. mosseae NT6 andlor 9 Rhizobium
leguminosarum bv. viceae strains and grown for 110 d in soil containing
indigenous AMF and rhizobia.
Mems for shoot and root dry weight, grain yield and harvest index of
lentil inoculated with the AMF species Glomus clarum NT4 or G.
mosseae NT6 a n d o r 9 Rhizobium leguminosarum bv. viceae strains and
grown for 110 d in soil containing indigenous AMF and rhizobia
Means for shoot and grah N and P content of lentil inoculated with the
AMF species Glomus clarum NT4 or G. mosseae NT6 and/or 9
Rhizobium leguminosarum bv. viceae strains and grown for 110 d in soil
conta in in^ indicenous AMF and rhizobia.
A.8
Means for shoot and grain P use effficiency (PUE) and A - - c o l o n i z e d
root lengùi of lent. inoculated with the AMF species GIornrrr clamn hT4
or G. mosseae NT6 andor 9 Rhizobium legrrmiplosanrm bv. viceae strains
and grown for I l 0 d in soil containing indigenous AMF and rhizobia.
B. 1
ANOVA for the shoot and root dry weight. gain yield. harvest index,
shoot N and P content, g a i n N and P content, shoot and grain PUE and
% AMF-colonized root length of pea inoculated with the AMF species
Glumus clarum NT4 andor the Rhirobium leguminosanrm bv. viceae
strains 173P4 and LX43 and grown for 95 d in soi1 amended with 0. 10
or 20 mg k g 1 of P and contaùiing indigenous AMF.
250
3 -2
Means for shoot and root dry weight, grain yield and harvest index of pea
inoculated with the AMF species Glornus clarum NT4 and/or the
Rhizobium leguminosarum bv. viceae strains 175P4 and LX43 and g o w n
for 95 d in soil amended with 0. 10 or 20 mg k g 1 of P and containing
indigenous AMF.
B -3
Means for shoot and grain N and P content of pea inoculated with the
AMF species Glomus clarum NT4 ancüor the Rhizobium leguminosarum
bv. viceae strains 175P4 and LX43 and grown for 95 d in soi1 amended
with 0,10 or 20 mg kg-1 of P and containllig indigenous AMF.
B .4
Means for shoot and grain P use eEciency (PUE) and AMF-colonized
root length of pea inoculated with the AMF species Glornus clarum NT4
andor the Rhizobium Zegurninosarrrrn bv. viceae strains 175P4 and LX43
and grown for 95 d in soil amended with 0,10 or 20 mg k g 1 of P and
containing iodigenous AMF.
B -5
ANOVA for the shoot and root dry weight, grain yield, harvest index.
shoot N and P content, grain N and P content. shoot and grain PUE and
% AMF-colonized root length of lentil inoculated with the AMI? species
Glornus clanrm NT4 aod/or the Rhizobium leguminosarum bv. viceae
strains PB101 and tX77 and grown for 110 d in soil amended with O* 10
or 20 mg k g 1 of P and containing indigenous AME
xvi
B.6
Means for shoot and root dry weight. grain yield and harvest index of
lentil inoculated with the AMF species Glomus cLarrrm NT4 and/or the
Rhizobium leguminosnrum bv. viceae saains PB 10 1 and LX77 and
prown for 110 d in soil amended with 0, 10 or 20 mg k g 1 of P and
COntaining indigenous AMF.
B -7
Means for shoot and grain N and P content of lentil inocuiated with the
AMF species Glomus clanrrn NT4 andor the Rhizobium legurninosancm
bv. viceae strains PB 101 and LX77 and groan for 95 d in soil amended
with 0, 10 or 20 mg kgl of P and containing indigenous AMF.
B. 8
Means for shoot and grain P use efficiency (PUE) and AMF-colonized
root length of lentil inoculated with the AMF species Glomus clarum NT4
andor the Rhizobium legurninosarurn bv. viceae strains PB 10 1 and LX77
and grown for 110 d in soil amended with 0. 10 or 20 mg k g 1 of P and
containing indigenous AMF.
xvii
LIST OF FIGURES
3.3.1
3 -3.2
3 -3-3
3.3.4
Mean (n=5) nurnber of viable and non-viable AMF spores
recovered from soil at 12 different field sites cropped to (A) lentil
or (B) pea Nurnbers in parentheses adjacent to each bar
represents the SE of the mean for the total number of AMF spores.
41
Mean (n=5) &SEpercenuje of AMF-colonized root of (A) lentil
and (B) pea grown at 12 different field sites.
43
The !MF morphotypes recovered From (A) the soil trap cultures or
(J3) second cycle root trap cultures established using fieId soi1 or
pea roots obtained fiom the Man field site no. 8 in a wheat-pea
rotztion.
47
The AMF morphotypes recovered from (A) the soil uap cultures or
(B) second cycle root trap cultures established using field soi1 or
pea roots obtained from the BelIevue field site no. 10 in a barleypea rotation.
48
3 -3.5 The AMF morphotypes recovered from (A) the soil trap cultures or
(B) second cycle root trap cultures established using field soil or
lend mots obtained from the Moose Jaw field site no. 2 in a
wheat-lentil rotation3 -3.6
The adjusted response ratio of the FAME 16:l o5c to 12:O in
sorghum-sudangrass (soil and first cycle root trap) or maize
(second cycle root trap) roots recovered from (A) soil trap
cultures, (Fi) f i s t cycle and (C) second cycle root trap cultures.
4.3.1
Pea (cv. Trapper) plants inoculated with ca. 108 c h ml-1 of the
commercial inoculants RGP2 or PB 101 (2449) and grown for 6
weeks in a Leonard jar. Control refers to the uninoculated pea
plants.
52
68
4-32
4.3 -3
5.3.1
5.3 -2
5.3.3
5.3.4
6.3.1
Pea (W. Trapper) plants inoculased with ca. 108 cfu ml-1 of the
field isolates LX1, LX43 and LX48 and grown for 6 weeks in a
Leonard jar. Control refers to the uninoculated pea plants.
69
Lentil (cv. Laird) plants inocuiated with ca. 108 cfu ml-l of (A) the
commercial inoculants RGL 14 or fB 101 (2449) and (B) the
rhizobia isolates LX72, LX77 or LX84 and grown for 6 weeks in
a Inonard jar. Control refers to the uninoculated lentil plants.
74
Mean (n-4) grain N content of pea plants inoculated with the AiMF
species Glomus clarurn NT4 or G. mosseae NT6 andor 10
Rhizobium leguminosarum bv. viceae stnins and grown for 90 d
in soil containing indigenous AMF and rhizobia.
93
Mean ( n 4 ) grain P contcnt of pea plants inoculated with the AMF
species Glomus clarurn NT4 or G. mosseae NT6 andor 10
Rhizobium leguminosarum bv. viceae stszins and grown for 90 d
in soi1 containing indigenous AMF and rhizobia.
94
Mean (n=4) grain N content of lentil plants inoculated with the
AMF species G l o m s ciarum NT4 or G. mosseae NT6 and/or 9
Rhizobium leguminosarum bv. viceae shilins and grown for 110 d
in soi1 containhg indigenou AMF a d rhizobia.
1O5
Mean (n=4) grain P content of lentil plants inoculated with the
AMF species Glomus clarum NT4 or G. mosseae NT6 ancUor 9
Rhizobium leguminosarum bv, viceae strains and grown for 110 d
in soil containing indigenous AMF and rhizobia.
107
Mean (n=5) shoot N and P content of pea inoculated with the AMF
species Glomus clarum NT4 and/or the Rhizobium leguminosarum
bv. viceae strains 17~5P4and LX43 and g r o m for 95 d in soi1
amended with 0, 10 or 20 mg kg-1 of P and containhg indigenous
AMF.
127
6.3 2
6.3.3.
6.3 -4
Mean (n=S) grain N and P content of pea inoculated with the AMF
species Glornus clanm NT4 andior the Rhizobirim legrrmirzosarrrm
bv. viceae strains 175P4 and LX43 and grown for 95 d in soil
amended with 0.10 or 20 mg k g 1 of P and containing indigenous
PLW.
129
Mean (n=5) shoot N and P content of lentil inoculated \.ththe
AMF species Glornus clanrm NT4 andor the Rhizobium
legurninosamm bv. viceae strains PB 101 and LX77 and grown for
110 d in soil amended with 0. 10 or 20 mg kg1 of P and
containing indigenous AMF.
141
Mean (n=5) grain N and P content of lentil inoculated with the
AMF species Glomus c[anun NT4 andlor the Rhizobium
legurninosammbv. viceae strâins PB 101 and LX77 and grown for
110 d in soil amended with 0.10 or 20 mg kg-1 of P and
containhg indigenous AMF.
143
7 -2.1
Tryptic soy agar ( 1.5%)plates contahing the direct, diffusible and
volatile bioassays. Plates were streaked with the SWB isolates
and incubated for 24 h before the different biuassays were
assembled.
7.3.1
Mean (n=5)percentage contamination of G. c l a m NT4 spores
treated with chlorarnine-T at 30°C for 0,30,45 and 60 min. as
detected on various media- Spore contamination was rneasured
after (A) 24 h and (B) 72 h incubation on water agar (WA), 0.02%
yeast extract agar (YEA), 0.3% =tic soy agar (TSA), LuriaBertani agar (LBA) and 0.8% nutrient agar (NA). Vertical bars
represent the least significant difference (LSD) at pcO.05.
163
7-32
Effect of difhsibIe chernicals excreted by the S\VB isolates (A) B.
pubuli LA3 and @) B. chitinospo~usLA6a on the germination of
Glomzu clumm NT4 spores. Spores were observed for
germination or hyphal elongation 20 d after incubation on 1.5%
TSA plates.
7 -3-3
Effect of the FiMF Glornits clur-umNT4, the rhizobia isolate LX43
and the spore wall isolates B. chitinosponrs LA6a or B. pabuli
LA3 on the growth of pea at 6 weeks after planting Plants were
grown in an autoclaved 1:2 soil:sand mix.
1.0
INTRODUCTION
The biotic community of the hyphosphere and rhizosphere of al1 plants generally
comprises bacteria. fungi. actinomycetes. and various macroorganisms such as protozoa.
nematodes, earthworms, etc. (Edwards et al. 1988). These organisms may directly or
indirecdy influence the growth of plants. However. the interaction of rnost
microorgmisms with plants is resnicted to the rhizosphere or the rhizoplane. A few
groups of microorganisms CO-inhabitthe endorhizosphere. and therefore. may have a
direct effect on die growth and productivity of the associated plant speties (Brundett.
1991; Baldani et al. 1997; Nehl et al. 1996). However. relationships between plants and
these stricùy endophytic rnicroorgauisms are often determined by the interaction effects
on the individuals involved in the association (Burkholder, 1952: Bronstein. 1994). Two
groups of rnicroorganisms which are very intimarely associated with. and influence the
growth and nutrition of legume plants are rhizobia and arbuscular rnycorrhizal tùngi
Members of the Rhizobiaceae are among a special group of soil bacteria which
have the unique ability to convert atmospheric dinitrogen into ammonia through the
biological nitrogen fixation (BNF) process (Michiels and Vanderleyden. 1994). Rhizobia
are mostly associated with legume roots (Geurts and Fr=ssen. 1996: Freiberg et al.
1997). but occasiorially found within the endorhizosphere of non-Ieguminous hosts
(Yanni et al. 1997). It is estirnated that the rhizobia-mediated BNF process contributes
approximately 35 x 1012 tonnes Le., ca. 478 of the total N fixed annually to the global
nitrogen budget (Ekan. 1992). On an area basis, the Rhizobium-legume syrnbiosis
contributes 24-584 kg N per hectare per year (Elkan. 1992). The low cost of Rhizobium
inoculants snd high retum from the BNF process are some of the reasons for the world-
wide use of rhizobid inoculants for various l e p m e crops (Shantharam and Mattoo.
1997).
In conaast to rhizobia. AMF are Glornalean soil fun$ which associate with more
than 8 5 8 of land plants (Trappe, 1987: Bnindett. 1991). However. rnembers of the
plant families Cruciferae. Chenopodiaceae and Caryophyhceae do not benefit from a
mycorrhizae association (Harley and Harlcy. 1987: DeMars and Boerner. 1996). This is
because these plant species either inhibit the germination of LWIF spores by releasing anti-
AMF cornpounds (Schreiner and Koide, 1993a. Z993b). or limit M F root colonization
to the penetration of the hyphae and formation of vesicles. but not arbuscule formation
(DeMars and Boemer, 1996). Until recently. it was believed that because AMF have a
wide host range, they colonized ail plant roots equally and did not exhibit host specificity
(Bowen. 1987). However. there is a growing body of evidence to suggest that AMF do
discriminate among plant species (Giovanetti et al. 1994: Gianinazzi-Pearson. 1996).
This discriminatory colonization of roots by AMF is reflected in the benefits derived by
the plants from tlie AMF species. Earlier reports indicated that the mycorrhizal
dependency of a host plant was dependent upon the density and thickness of the root hair.
its nutrient absorbing organ (Baylis. 1972). and biotic or abiotic factors such as
interactions between AMF and other organisms in the rhizosphere. soil temperature. pH.
l
nutrient content. and water potential (Fitter. 1986; Mayo et al. 1986). However. while d
of the above factors can influence the outcome of a functional arbuscular mycorrhizal
(AM) symbiosis, there are reports indicating that metabolites in plant root exudates
directly affect spore germination and the establishment of a successful syrnbiosis resulting
in enhanced plant yield u s a i and Phillips. 1991: Becard et al. 1992). In addition. recent
reports indicate that mycorrhizal responsiveness may be a genetically conaolled m i t
(Hetrick et al. 7995; Giminazzi-Pearson. 1996). This indicates that the response of a
host to AMF rnay prirnarily depend on die specifc interxtions and compatibility between
the partners involved.
There are two important considerations in studying the AM symbiosis. First.
AMF are obligate biotmphs and meet ail of the carbon requirernent for their growth and
reproduction through hos t plant photosynthates (Smith and Smith. 1996). Therefore.
depending upon the nature of the AMF-host plant association. the AMF rnay increase.
decrease or have no effect on plant productivity (Bethlenfalvay et al. 1982: Sylvia et al.
1993: Trent et al. 1993). Second. rnycorrhizal plants have a greater productivitycompared to non-mycorrhizal plants in nuuient-deficient soils. The mechaisms through
which AMF enhance plant growth include improved P uptake (Ravnskov and Jakobsen.
1995), improved micronutrient uptake (Persad-Chinnery and Chinnery. 1996). enhanced
N uptake and assimilation (Johansen et aL 1996) or interplant N transfer (Frey and
Schuepp, 1993),altered phytohormonal activity (Frankenberger and Arshad. 1995).
improved drought resistance (Ruiz-Lozano et al. 1995). enhanced salinity tolerance
(Hirrel and Gerdemann, 1980) and increased resistance to herbivores (Gange and West.
1994) and plant pathogens (Caron. 1989: St-Arnaud et al. 1995). In addition. AMF rnay
also play an important role in the determination of ïnterspecific competitivz abilities of
plants (Cmsh. 1995) and revegetation strategies for the rcclarnation and rehabilitation of
desertified ecosystems (Michelsen and Rosendahl, 1990; Herrera et al. 1993). Other
unusual advantages confened to mycorrhizal plants include enhanced iron uptake
1995), limited
possibly by siderophore production (Cress et al. 1986: Ha~elwandter~
plant uptake of toxic heavy metds such as cadmium frorn soil (Gildon and Tinker. 1983)improved soil conservation by enhancing soil atgregate formation (Miller and h t r o w .
1990; Schreiner and Bethlenfalvay, 19 9 3 , and minvralization/utilization of organic
phosphates (Jayachandran et al. 1992; Joner and Jakobsen. 1995).
One of the various atternpts made to increase the benefits derived from the AM
symbiosis is the simultaneous addition of select groups of microorganisms and Ah4F tci
plants (El-Raheem et al. 1989: Azcon et al. 1991: Carnprubi et al. 1995: Walley and
Germida. 1997: Gryndler and Vosatka. 1996). However. interictions between these
organisms and AMF may result in increased (El-Raheem, 1989: Azcon et d. 1991:
Carnprubi et al. 1995; Walley and Germida. 1997). or decrcased @miels-Hetrick et al.
1988; Walley and Germida 1997) plant yields. Although the interactions between AMF
and other soi1 oganisms influence plant yields. these may be the result of vanous tictors
such as the cornpetition between rhizosphere microorganisms and plants for nunients
(Baas, 1990). or the synthesis of growth stimulatins (Gryndler and Vosatka. 1996) or
inhibiting substances by the bacteria (Walley and Germida. 1997).
In contrast to other Npes of AMF-plant growth promoting rhizobactena (PGPR)
interactions. the tripartite association between legumes. AMF and rhizobia appears to be
controlled at the molecular Ievzl of the host plant (Gianinazzi-Peanon. 1996: Shirtliffe
and Vessey. 1996). It has been shown that nodulation-defective mutants of pea are
resistant to mycorrhizal colonization (Duc et al. 1989: Balaji et al. 1994: GianinazziPearson et al. 199l), indicating that one gene probably controk the expression of
nodulation and mycorrhization of the host. These findings aside. studies conducted usin2
various legume species have dernonsuated enhanced growth. yield and N and P nutrition
when plants were CO-inoculatedwith rhizobia and AMF (Pacovsky et al. 1986: Vejsadova
et al, 1992; Ruiz-Lozano and Azcon, 1993; Ahmad. 1995: Ibrahim et al- 1995: Ahiabor
and Hirata. 1995). Furthemore, these positive growth responses appear to be the result
of specific interactions between AMF and Rhizobium strains (Ames et al. 1991: Azcon et
al. 1991). However, most of the studies have not included more than one AMF and/or
Rhizobizinz strain combination (Redente and Reeves, 1981: Manjunath et al. 1984:
Pacovsky et al. 1986; Kucey and Bonetti, 1988: Ahiabor and Hirata. 1995: Ibrahim et al.
1995). It is not clear from the existing reports whether the positive et'ficts of an et'fective
AMF will compensate for an ineffective Rhizobium strain and vice versa. or whether the
addition of P fertilizer will elirninate the positive effects of AMF.
Pea (Pisrrrn sativum L.) and lentil (Lens esculenra L.) are important pulse crops
worldwide. In Saskatchewan. pea and Ientil occupy an area of 860.000 and 690.000
acres. respectively (Anonymous. 1997). These crops are routinely inoculated with
Rhizobirrrn inoculants to enhance crop yield and seed nitrogen content (Hynes et al.
1995). Previously. Kucey and Paul (1983) and Talukdar and Germida (1993a) reponed
that Saskatchewan sous contain active AMF spore communities that may influence crop
production. Some of these indigenous AMF isolates have enhanced the growth and yield
of lentil and wheat in s t e d e (Talukdar and Germida 1994) and non-sterile soils (Xavier
and Germida. 1997. 1998). My study was undertaken to study the factors that idluence
the specific interactions between A M F and Rhizobirrm legumirtosm-zinzbv. Mceoe. a n d to
i d e n m specific combinations of AMF and rhizobia that enhance the growth. yield and
N
and P content of pea and lentil.
The objectives were as follows:
(i) to isolate and identify effective AMF that readily colonize roots. and enhance
the growth and P content of pea and lentil,
(ii) to isolate and identify effective rhizobia that enhance the growth and nitrogen
content of pea and lentil,
(iii) to identie and develop specific combinations of AMF and Rhizobiuin
leguminosarrun bv. viceae for pea and lentil.
(iv) to study the etiect of soi1 phosphorus levels on the specific interactions
between AMF. rhizobia and pea and lentil. and
(v) to assess the influence of AMF spore-wall iissociated bacteria on the Pisuin-
Glomus-Rhizobium symbiosis.
2.0 LITERATURE RIEVXEMr
2.1 The Rhizosphere
The term 'Rhizosphere' refers to the zone containing soil that closeiy adheres to
the roots, and is an area of intense rnicrobial activity (Lynch. 1990). The rhizosphere
includes the ectorhizosphere which refers to the soil adhering ta the root. the rhizoplane
or root surface. and the endorhizosphere which is the root inarior. A prÎmary reason for
the enhanced microbial activity in the rhizosphere is the supply of nutrients from host
plant root exudates containkg various stimulatory chemical molecules. sloughed off root
hair cells and mucigel surrounding some plant roots. Although most soi1 rnicroorganisms
scavenge the rhizosphere for nutrients for their growth and reproduction. this activity
does not appear to be common for dl organisms.
There are a few groups of rnicroorganisms which respond to chernical signals
released by plant roots and actively colonize the rhizosphere and endorhizosphere. For
example. the AMF colonize the roots of most known land plants and influence plant
growth. Similady. members of the Rhizobiaceae colonize most leguminous h o s a and
typically enhance plant growth by fixing atmospheric nitrogen (Men and Allen. 1981:
Brockwell et al. 1995). In addition. there are other non-pathogenic and pathogenic
bacteria and fun& which. depending upon the nature and composition of the root
exudates. colonize the roots and influence plant growth (Darvill and Albersheirn. 1984:
Nehl e t al. 1996). Therefore, mutualistic and parasitic associations are often rnediated by
the presence of the appropriate root exudate mixtures.
2.2
Rhizobia
Members of the Rhizobiaceae form intimaie associations with lepminous plants and fi
atmospheric nitrogen w i ~ l the
n rootfstem nodules of their hosts (Brockwell et al. 1995:
Boivin et al. 1997). The process ofniuogen furation is preceded by the nodulation
process (Hirsch. 1992). This is because the rhizobia-mediated BNF process occurs only
within the c o ~ n e of
s a highly regulated low oxygen environment such as the nodule.
The most important step prior to host recognition of Rizizobiunz species is the binding of
the NodD protein with specific flavonoids released by legume roots. resulting in the
transcriptional activation of other nod genes (Heidstra and Bisseling. 1996). The
stimulation of other nad genes results in the synthesis of 'Nod' factors. The nod operon
contains genes that code for 11 difTerent proteins that are crucial for the proper
recognition and functioning of the nodulation process (Heidstra and Bisseling. 1996).
Al1 Rhizobium species synthesize 'Nod' factors but not a l l rhizobia are compatible
with ail hosts. This is because of the specificity of 'Nod' factors. Based on specifIcity.
the genus Rhizobium contains eight species including R. legurninosarum bv. phuseoli
which nodulates Phaseolus (Krieg and Holt, 1984). R. leguminosanrm bv. ti-ifolii which
nodulates Trifoliurn (Heidstra and Bisseling, 1996) and R. leguminosamrn bv. viceue
which nodulates Pisum. Vicia, Lathyrus and Lens (Heidstra and Bisseling. 1996): R. etli
which nodulates Phaseolus (Segovia et al. 1993): R. fredii which nodulates Phaseolrts
and Glycine (Scholla and Elkan, 1984); R. galegae which nodulates Galega orientalis
(Lindstrom, 1989); R. huakii which noduTates Astralagus (Chen et al. 1991): R. loti
which nodulates Lonrs (Jarvis et al. 1982); R. meliloti which nodulates Melilotrw.
Mediutgo and Ttïgonella (Krieg and Holt, 1984); and R. nopici which nodulates
Phasedus and Leucaena (Poupot et al. 1993).
The nitrogen benefits derived from interactions between rhizobia and their hosts
depends on several factors. Although abiotic factors such a s salinity and exnemes of
temperature and pH affect the survival and efficacy of rhizobia in soil (Elsheikh and
Wood. 1989: 1990), nuaient levels may have the most profound effect on functioning of
the syrnbiosis. For example. different levels of N. P and micronutrients such as Fe and
Mo can affect the BW process (Munns. 1977: Novak et al. 1993). Whereas high lzvels
of N negatively a e c t the BNF process. moderate to high levels of P are most benetTcid
(Atkins and Raïnbird, 1982). The amount of nitrogen fixed by the bacteroids living
within the root/stem nodule depends on the amount of P available for nitrogen fixation.
This is because nitrogen fixation is a high enerm (ATP) requiring process. For every
molecule of ûmmonia produced, 16 ATP molecules are required (Ackins and Rainbird.
1982). Among ail the rnicronutrients in soil that affect the efficacy of the BNF process.
Fe and Mo are the most important The leghemoglobin protein. which effechvely
replates the oxygen levels within the nodule cells to preserve the activity of the
nitrogenase enzyme is produced as a result of interaction between the appropriate host.
which synthesizes the protein component (globin) and the Rhizobium species. which
synthesizes the heme prosthetic groups (Broughton and Dilworth. 1971). In addition. Fe
and Mo are components of the nitrogenase enzyme complex (Fischer. 1994). However.
it may be important to maintain dl the nunients at suitable levels for the overdl growth
and health of the plant: a healthy plant also ensures a steady supply of nutrients to the
bacteroids.
2.3 Arbuscular mycorrhizal fungi
2.3.1 AMF-host specificity
Plants colonized by AMF are either highly mycotrophic. facultatively mycotrophic or
non-mycotrophic. Baylis (1972) reported that plants with fewer or thicker roots and a
shaIlow root system benefit from mycorrhizal coloniation, and therefore are referred to
as highly mycoaophic. In this case. the AMF extemal rnycelium acts as an extension of
the root to absorb P x ~ other
d
nutrients (N and micronutrients) fiom soil (Jakobsen et al.
1992). Exarnples of highly mycotrophic hosts include members of the plant h n i l y
Liliaceae comprising Allirrnz cepa. A. sutiimm and A. pomtrn which possess very thick
root hÿirs. In contrasr facultatively mycotrophic hosts have a relative-elyl q e r root
system andfor fine roots which are suited to nutrient absorption. Exmples of
facultatively mycotrophic hosts include members of the plant f d e s Gramineae and
Solanaceae. The third category of plant. is the non-myconhizal group which comprise
members of the plant families Chenopodiaceae. Caryophyllaceae and Cmciferae. Nonmycotrophic hosts are plants that neither depend on M F for the absorption of nutrients
from soil nor aid AMF growth and reproduction. These hosts allow the entry of AMF
within their roots, but do not allow the formation of arbuscules or vesicles (Hirrel et al.
1978: DeMars and Boemer. 1996). For example, Demars and Boemer (1996) found that
132 of 649 Brassicaceae hosts were colonized up to 19% by AMF: however. arbuscules
were not formed. Early reports suggested that the non-rnycotrophic nature of these hosts
was due to the exudation of anti-AMF compounds such as glucosinolates by the host
plants (Erre1 et al. 1978). Hirrel and CO-workers(1978) found that cruciferous hosrs
such as Brassica had been colonized by Glomus mosseae to a small extent but did not
form arbuscules within roots. They attnbuted this response to the presence of anti-AMF
cornpounds released by the hosts. Similarly, Ocampo (1980) found that the entry of
AMF hyphae into Brassica mots was limited. However. El-Atrach et al. (1989) reported
that the AMF non-host plant roots presented a physical barrier to Ah4F entry. which may
result in limited root colonization. More recently. Schreiner and Koide (1993a. 19% b)
found that root exudates of mustard plants contained cornpounds that were mti-fungal,
and limited the entry of AMF within the mots of non-AMF hosts. Whatever the cause of
limitation of AMF entry, cruciferous hosts such as cmola do not allow the formation of
arbuscules within the root cortex.
The recent interest in AMF-host specificicy is because of thz ability of AMF to
discriminate not only between host and non-host plants (Giovanetti et al. 1993a. b). but
also between difi-erent host plants (Schenck and Kinloch. 1980: Rosendahl et al. 1989:
Bever et al. 1996). For example. Rosendahl et aI. (7989) used roots from seven different
plant species collected from the same location te inoculate Ccrcrrmis sativrrs plants. and
found that the AMF which colonized bait plant roots varied aith the host plants and the
harvest time. Their report indicates that although different plants share the same location.
they do not necessarily share AMI? endophytes. Altem2tivelÿ. this may be interpreted as
the host discriminating behavior of AMF. This AMF-host specificity not only reflects on
the subsequent effects on plant gowth, but also on AMF sporulation under different host
plants (Bever et al. 1996). For example, Bever and CO-workers(1996) found that in
experimental microcosms established in the field with hosts such as Allicrrn.
Anthoxanthurn, Panicum and Plantago plants. AMF sporulation rates varied with the host
plant species. They concluded that the variation in AMF spore counts was mediated by
host-dependent differences in fungoal growth rates. This occurrence was reported as early
a s the 1980s by Schenck and Kinloch (1980) who found that Gigaspora margarzk. Gi
gregaria and Gi. gigantea spores were abundant around soybean roots, whereas G.
fusciculutus and G. clarum spores were abundant around bahia grass roo ts. They also
reported ùiat Cotton and peanut preferentiaily supported increased sporulation of
Acuz~losporaspp. The recent interest in AMF-host specificity also opens the possibility
of exploiting this phenornenon in agriculture for the selection of appropriate AMF species
for different plants.
2.4 Interactions between AMF and beneficial bacteria
2.4.1
Interactions between AMF and non-nodulating bacteria
Microorganisms in the rhizosphere live in close proximity because of the steady supply of
nutnents around the roots- Due to the high density of bacteria in the rhizosphere and the
ubiquitous nature of AMF.interactions may occur. Sorne of these interactions may be
potentially benef'icid to plant grorvth. Mmy workers have assessed the CO-inoculation
response of mycorrhizal hosts to AMF and important ancilor beneficial bacteria (Bagyaraj
and Mente, 1978: Raj et al. 1981; Barea et aI. 1983; Pacovslq-et al. 1985: Rao et al-
1985: Meyer and Linderman, 1986: Azcon. 1989: El-Raheem et al- 1989: Will and
Sylvia. 1990: Paula et aI. 1992). For example- Buea and CO-workers(1983) foiind that
l~mz
and a yellow-vacuolate AMF spore
dual inoculation of maize ~ i t h ~ o s p i ~ - i lbl-usilense
type most closely resernbling G. rnosseue produced plants of a similar size and N content
but a higher P content than those fertilized with N and P. Pacovshy et al. (1985) studied
the influence of edaphic factors on the interactions betwem Azospirillum species and G.
fusciculuncrn. They found that plants inoculated with AMF and &ospirillrtnt had higher
plant dry weight, shoot-root-ratios and N content than those inoculated with either AMF
or the bacterium doue. They dso found that inoculation with Azospirillum stimulated the
AMF colonization of roots. Althouth there are generd interactions between AMF and
AzospiriIIum in the rhizosphere, not all of these interactions result in a synergistic
response (Rao et al. 1985). For example, Rao and CO-workers(1985) studied the
response of barley grown in potted field soi1 to combinations of an Azospirilirtrn inoculant
andor five different M F species. They found that while some combinations of AMF
and bacteria increased seed and shoot yield of barley. others did not have an effect.
h o n g the diazotrophic bacteria that enhance plant growth. Acetobacter
diazon-ophicus is a recently described species that colonizes the aerial parts of sugar cane
and sweet potato plant (Gillis et al. 1989). However. unlike other AMF-associative
diazotroph associations. A. diarotrophicus is beneficial only when CO-inoculatedwith
AMF (Paula et al. 1991). In this context. Paula and CO-workers(1992) assessed the
synergistic effects of co-inoculabng sweet potato (Ipomoea butarus) with G. clarum and
A. diazomphicus in fumigated and non-fumigated soils. They found that tuber
production by sweet potato occurred only in the fumigated soil with plants CO-inoculated
wiùi A. diazotrophiccrs and G. clamm. Furthemore. A. diazo rrophicrrs infected aerïal
plant parts only when CO-inoculatedwith G. clai-rrmspores. In addition.
Am
intraradicd growth and sporulation was enhanced by the bactenum. This effect.
however. was not so pronounced in non-furnigated soils. Hence. these authors
suggested that this specific interaction between AMF and Acetobucrer may be indicative of
a CO-evolutiontowards mutual benefit.
Diazotrophic Azotobacter spp. are free living bacteria thaî enhance plant g r o h
through fixation of amosphenc nitrogen (Azcon et al. 1973) or the production of plant
growth p r o m o ~ substances
g
(Barea and Brown. 1974: Azcon and Barea. 1975). Azcon
(1989) and El-Raheem et al. (1989) examined the effects of interactions between
Azotobacter and AMF on the growth and nuaient content of tornato plants. For example.
Azcon (1989) exarnined the response of tomato ro CO-inoculationwith Azotobacter
vinelandii strain ATCC 12837 and G. rnosseae, G. fascicdarcmz or Glomus sp. type E3
in a sand-vermiculite medium. They found that the response of tomato plants to the
difTerent Azotobacter-AMF associations was not the same. A- vinelurzdii did not alter the
response of tornato plants to the G. mosseae or the E3 inoculant, but enhmced the growth
response of plants inoculated with G. fasciculatum. Although the Azotobacter suain
influenced the growth of mycorrhizal tomato plants. the AMF colonization of tomato
roots was not altered by the Azotobacter strain. Similarly, El-Raheem and CO-workers
(1989) studied the effects of Azorobacter chroococcum -G. fascicdatum interactions on
tomato growth and nutrition in a steam-sterilized sandy soil. They reported that duÿl
inoculation of tornato with A. chroococc~rmand G, fasciculaarrn resulted in enhanced
AMF root colonization. and improved shoot growth and N, Ca. Mg and K contents.
Therefore. it appears that the simultaneous application of diazotrophs and AMF Knproves
thz N and P nutrition of the host. rzsulting in enhanced plant gmwth compared to
uninoculated plants.
Interactions between AMF and other rhizosphere bacteria such as phosphate
solubilizïng bacteria (PSB) have also been studied (Raj et al. 1981: Azcon-Aguiiÿr et al.
1986~:Toro et al. 1998). The rationale for CO-inoculatingplants with AMF and PSB is to
exploit the ability of the PSB to release P from bound or organic P sources in soil. which
may be subsequently absorbed by the AMF hyphae and effëctively translocated to the
host plant. For example, Raj et al. (198 1) inoculated finger millet (Eleusine corucunu)
with G.fasciculahls and a PSB Bacillus ci.rculans and grew it in a 1:1 stenlized soil: sand
mixture amended with either radio-labeled superphosphate or tricalcium phosphate. They
found that plants inoculated with the AMF removed more P from soil. and plants coinoculated with G.farciculatus and B. circulunr yielded more dry rnatter and P.
Similarly. Azcon-Aguilar and CO-workers(1986~)exarnined the interactions between
soybean, taro unidentifïed PSB and two AMF species. G. mossec<eand a Glomris sp. Eg
in gamma-imdiated çoïi amended with three lzvels of tricalcium phosphate. They found
that only inoculation with Glomus sp. E3 improved the abovegound dry matter
production and utilization of tricalciuni phosphate. However. they also found that the
proportion of P from the radio-labeled tricalcium phosphate in the shoot material was
lower in mycorrhizal plants. indicating that most of the available P in soil was probably
derived from other sources. Recently, Toro and CO-woskers(1998) studied the response
of Medicago sativa to CO-inoculationwith a PSB Enrembacter sp. and G. mosseur in soi1
containing indigenous populatims of PSB and amended with rock phosphate. They
found that mycorrhizal plants absorbed more P from soil rornpared to non-mycorrhizal
plants, and that the Enterobactei inoculant improved the use of rock phosphate in all
plants. This indicates that the P released by the PSB was effectively transported to the
host by the AMF. resultïng in enhanced host P numtion.
Arbuscular mycorrhizal tùngi have d s o been found to specifically intenct with
plant g o w t h promothg rhizobacteria (PGPR) specifically Psertdomorius spp. (Meyer and
Lindeman, 2986; GryndIer and Vosatka, 1996: Walley and Germida. 1997). For
example. Meyer and Linderman (1986) exarnined the response of submranean clover
grown in non-sterile soil to dual inoculation wirh an indigenous AMF mixed culture and
Pseudomonasputidu. The shoot dry weight and rnycorrhizai colonization of coinoculated plants was significantly greater than plants left uninoculated or inoculated with
the AMF mixture or PGPR alone. Furthermore. nodulation of these plants by h e
indigenous soi1 rhizobia was enhanced. and the concentrations of Fe. Cu. AI. Zn. Co and
Ni were greater in CO-inoculatedplants. Similady, Gryndler and Vosatka (2996) studied
the response of maize to G.fisnrloszmz and various culture fractions of P. putida. They
found that living ceus of P. putida and G.fistzclosurn Ïncreased the kaf area and shoot dry
weight of plants more than by G.fisndosurn inoculation alone. They also reported that
mycorrhizal colonization of rnaize roots was higher in plants CO-inoculatedwith Iiving
cells or a dialyzed cell extract of P. putida. They also found that mot infection by
extraneous filamentous fun$ was reduced when pIants were CO-inoculatedwith live P.
putida cetls, indicating the protective effect of the PGPR on plants. Walley and Germida
(1997) investigated the response of spring wheat to interactions benveen Pseudomonas
spp. and G. clurunz NT4 under gnotobiotic conditions and in a non-sterile soil: sand mi..
They found that there were specific interactions between G.clanrm NT4 and the PGPR
strains. but that the effect on plant growth was dependent on the PGPR strain.
Furthermore. they found that in the presence of some Pseudomoïzas spp. G. clarum NT4
spores did not germinate in virro. and suggested that a non-volatile substance may have
inhibited spore germination.
Interactions betvÿeen AMF and various other bacteria have also been examined by
workers (Azcon. 1989; Will and Sylvia, 1990). For example. Azcon (1989) inoculated
tornato seedlings with an indigenous Enterobacteriaceae isolate and G. mosseue. G.
fascicrdanrrn or GZomus sp. Eg isolate. They found that mycorrhizd tomato plants
showed enhanced growth in the presence of this bacteritm. They reported. however. that
the extent of AMF root colonization was not related to the effect of this bacterium on plant
growth. indicating that both orgmisrns did not act together. but exerted their individgai
effects on plant growth. In an attempt to establish sea oats on replenished beaches
quickly, Will and Sylvia (1990) inoculated sea oats (Uniolupanic~rluruL.) with a mixture
of Glom~tssp. S238 and G. deserticala and Klebsiella pneumorziae. Bacillus poiyrnyxu
and Alcaligenes denimficans. However, they faund no consistent evidence supporti!.nga
synergistic effect on plant growth.
Ln most cases. the interactions between AMF and bacteria result in enhanced host
*orowth and nutrition. Thus, AMF interact with bacteria which may influence host plant
growth and nutrition through a number of mechanisms: atmospheric nitrogen fixation. P
release from less readily available sources. phytohormone production. siderophore
production. production of antibiotics, and/or agressive colonization of the
endorhizosphere. Interactions between these beneficial bactena and AMF may directly
influence plant growth or indirectly alter rnicrobial activity in the rhizosphere.
2.4.2
2.4.2.1
Interactions between AMF and rhizobia
Nutritional benefits
Rhizobia interact very intimately with lepminous hosts and enhance plant growth
typically by improving host N numtion. Similady. arbuscular mycorrhizal fungi enhance
plant growth by absorbing P from soi1 and transporthg it to the roots (Jakobsen et al.
1992). Many workers have studied combinations of rhizobia and AW in an attempt to
improve l e p m e nutrition and productivity (Redente and Reeves. 1981: Manjunath et al.
1984: Pacovslq et al. 1986; Kucey and Bonem, 1988). Redente and Reeves (198 1) co-
inoculated sweenretch with G. fascic~iZanisand a Rhizobirrrn sp. and found that coalone produced more
inoculated plants and those incculated with G. fuscic~rlut~rs
aboveground dry matter than those left uninoculated or inoculated wïth rhizobia alone.
Similady. Manjunath et al. (1984) inoculated Lericuena leucocrphula with G.fasciczilatim
and a Rhizobizrm sp. and found that plants CO-inoculatedwith AMF and rhizobia had
increased root nodulation. mycorrhizal colonization. dry weight. and N and P content
compared to plants inoculated wiih AMF or rhizobia done. Pacovsky and CO-workzrs
(19 86) inoculated soybean with G. fuscic~ilatzrmand BI-udyrhi~obizi17~
japot? iczct~z- and
found that CO-inoculatedplants produced sipificantly higher shoot biomass than
uninoculated p l a t s or those inoculated with only Rhkobiwz. Kucey and Bonetti (1988)
reported that field beans inoculated vcritii a miunire of native AMF from several southern
Alberta soils enriched on a saawberry host had up to 54% higher dry matter production
than plants not receiving AME They also reported that plants CO-inoculatedwith AMF
and R. phuseoli had a higher N content compared to plants inoculated with only R.
phaseoli. A sirnilar positive response of nodulated plants to G. fuscicz~Zatuminoculation
for Leucaena ZeucocephaZu was noted by Purcino et al. (1986). The above studies
demonstrating positive responses with AMF and Rhizobium CO-inoculatedlegumes
typically involve combinations of one Rhizobium and AMF isolate.
In order to select for the best AMF endophyte for CO-inoculationpurposes. it is
important to understand interactions between dBerent AMF species in conjunction with
appropriate host-rhizobial combinations. Several workers have included various AMF
species into the dual inoculation schemes to evaluate this response (Ianson and
Linderman, 1993: Ibijbijen et ai. 1996; Saxena et al. 1997). For exarnple. Ianson and
Linderman (1993) studied the response of pigeonpea (Cajanus cajan) to an effective
Rhizobium strain and seven diffèrent AMF isolates of G.ehlnicaturn. G. uggreganlm
(microcarpum).G. deserticola. G. mosseae. Gigaspora rnargarita and G. innaradix.
They found that thz AMF species exerted very different effects on nodulation and
nitmgen fixation. They also suggsted that these differences were probably mediated by
a specific inter-endophyte interiction between the Rhizobium strain and the diffèrent AMF
species. which became evident under N-lirniting conditions. Similarly. Ibijbijen et 21.
(1996) inoculated three bean varieties with an effective Rhizobiclm leguminosarum bv.
phaseoli suain and four AMF species. G. clanrin. G. enmicutrrrn. G. ~nanihotisand Gi
murgarira. They found that although AMF inoculation increased plant dry matter
production on an average by 843%. anci plant P concentration by 160-3358. there were
significant differences between AMF in their effect on nutrient uptake and plant growth.
They also reported diat the shoot P concentration and N accumulation was increased by
AMF inoculation. Saxena et al. (1997) found that inoculating Vigna radiata with a
effective Bradyrhizobium sp. strain and eight different AMF species (G. mosseue, G.
fasciculatum. G. versiforme, G. macrocarpum. Gi. gilmorei. Gi.marganta.
Sc~ltellosporacalospora and Enaogone duseii) produced significantly different results.
Inoculation with Scritellospora calospora increased the nitrogenase activity and dry
biomass of the Brudyrhizobium-inoculatedplants more than other AMF species.
Moreover, the nodulation competitiveness of the Bradyrhizobium strain was significantly
increased by up to 9-12% in the presence of the AMF species. G. mosseae. G.
fasciculamm and S. cdospora, relative to when the Brudyrhizobi~tmmain was inoculated
alone. There was a corresponding increase in the percentage of AMF colonization of
roors containing nodules occupied by the Bradyrhizobium strain.
Some workers have examined the response of mycorrhizal leguminous hosts to
different stmins of rbizobia. in order to understand the contributions by the Rhizobizirn
species to the tripartite symbiosis (Vejsadova et al. 1992; Thiagarajan and Ahmad. 1993).
For example, Vejsadova and CO-workers(1992) studied the effect of two Bi-adyrhizobium
strains arid an AMF species on soybean plants grown in a steam-sterilizèd soil-perlite
mix. They found that the nitrogenase activity. growth and yield of plants increüsed in the
presence of the AMF. but did not v q between the Bradyrhizobinnz stf'ains. Similarly.
Thiagarajan and Ahmad (1993) compared the influence of three B~nfil-hizubiirmsp.
strains on the G. pallidum-cowpea syrnbiosis in nonsteiile SOLThey observed thüt in the
presence of G. pallid~rm.the nodule competitiveness of d l the three Br-adyrhizobitïm
strains increased sitnificantly. when compared to native rhizobia. However. no
information was provided on the effect of the various combinations on plant growth or
dry matter production.
The selection of appropriate M F species for pulse crop production is as
;mportant as the selection of Rhizobium for inoculation purposes. This is because
nodulated plants benefit from the P uptake by AMF extraradical mycelium. which is
required for the process of nitrogen fixation. In addition. it has been shown that some
legumes do not respond to inoculation with diazotrophs in the absence of AMF.
indicating the importance of AMF in the tripartite symbiosis (Azcon-Aguilar et al. 1982:
Paula et al. 1992). Therefore. it is very important to CO-selectcompatible rhizobia and
AMF which will result in the desired plant effects.
Various workers have atternpted to develop supenor combinations of AMF and
rhizobia for different legume species (Azcon et ai. 1991: Ruiz-Lozano and Azcon. 1994;
Ahmad. 1995: Redzcker et al. 1997). For example. Azcon and CO-workers(199 1)
studied the cornbined and separate effects of inoculating Medicago suriva L. with G.
mosseae, G. fasciculatum. and G. caledoniun2 and six strains of R. meliloti. They found
that after 70 days, plant growth, N and P benefits to the host were dependent on the
particular combination of AMF and rhizobia. However, it appeared that in most cases G.
farciculamm produced the best results on Medicago sarim. whatever the associated
Rhizobium strain. Similady, Ruiz-Lozano and Azcon (1993) exmined the development
and ûctivîq of the tripartite symbioses between G. nzosseue or G. fu~*ciculatunzand
B~-u&rhizobirrn~
strüins on Cicei-ci?-ierizrcin.They found diit the symbioac efiiciency of
the association as rneasured by plant growth and N and P nutrition was dependent on the
particular combination of AMF and Brrrdyrhizobirnn s ~ a i n .In order to seIect For the best
combination of A M F and rhizobia for red kidney bean cultivars. Ahmad ( 1995) coinoculated plants grown in sterilized and nonsterilized soils with the AMF G. pullidrrm.
G. UggregUhfmor Sclerocystis microcarpu. and four scrains of Rhizoobitu~rphuseoli He
too found t h ~the
t response of a red kidney bean cultivar depended on the particular
combination of the AMF spzcies. Rhizobiron phuseoii main and cultivar of red kidney
bean. Redecker et al. (1997) studied the effect of various Rhizobizlrn spp. on the
response of Phaseolus vrilgaris inoculated with G. manihotis. Gluinus sp. S329 or
Enrophospora colornbiana in nonsterilized soil using 'SN isotope dilution technique.
Their results indicated a specific involvement by the AMF species in the accumulation of
N by plants through the BNF process and from soiI. These authors sugested that an
enhanced rnycorrhizal symbiosis may aid in die N fixation by leguminous plants. Thus.
it appears that plants benefit from a functional symbiosis that c m be obtained from the
selection of appropriate combinations of AMF and Rhizobim species.
2.4.2.2
Non-nutritional benefi ts
Leguminous plants which fonn tripartite symbioses with AMF and rhizobia
benefit from enhanced âtmospheric N fixation. P nutrition and sometimes. micronunition
(Section 2.4.2.1). However. there arc no reports of non-nutritional benefits (e.g.,
tolerance to adverse environmental conditions. disease resistance. etc.) from this
symbiosis, other than salinity tolerance (Dixon et al. 1993; Azcon and El-Atrach. 1997).
For example, Azcon and El-Atrach (1997) reported that alfalfa plants CO-inoculatedwith
G. mosseae and R. nzeliloti and grown in potted soi1 receiving a mixture of NaCl. CaC12.
and MgCl2 yielded significantly higher levels of shoot biomass, and shoot N and P
content than plants inoculated with the Rlzizobirnn strain only with or without addcd P. A
similar observation for Letrccrerra lecicocephaIu and P i - o q i s CO-inoculatedwith a Clonzrrs
sp. isolate and a Rhizobi~rmsp. scnin wÿs noted by Dixon et al. (1993). These reports
indicate that AMF and rhizobia protect Ieguminoris hosts from salinity stress. Attempts to
investirate the effects cf co-inoculation of legurnes with AMF and rhizobia on disease
resistance rnay prove to be worthwhile. given the individual effkcts of rhizobia and AMï
on plant pathogens (St-Amaud et al. 1995: Robleto et ai. 1998).
2.4.2.3 Effects of inter-endophyte compatibiii ty on legume gro+wth
The response of legurnes to various AMF and rhizobia depends on the particular
combination of A M . and rhizobia (Azcon et al. 1991:Ruiz-Lozano and Azcon. 1994:
Ahmad. 1995: Redecker et al. 1997). This may indicate inter-endophyte compatibility or
incompatibility. An extrerne case of this compatibility was demonstrated using pea
mutants with the phenotypes Nod- and Myc-. Le.. plants that do not form nodules and
are resistant to mycorrhizal coionization D u c et al. 1989: Gianinazzi-Pearson et aI. 1991:
Balaji et al. 1994: Sagan et al. 1995). In Myc- pea mutants, the host-AMF recognition
occurs but further entry and f u n p l differentiation are restricted. whereas in the wild type
pea normal entry and f u n p l differentiation are observed. These Myc- pea were obtained
as a result of mutation at a single locus that pertains to three complementation groups.
Le.. p. c. and a that conrspond to the sym8, syrnl9 and sym30 nodulation genes
(Gianinazzi-Pearson, 1996). Since the Myc- trait does not separate from the Nod- trait in
the Fî progeny, it is possible that the same gene controls both traits. Interestingly.
whatever the growth conditions. the Nod- Myc- pea mutants are not colonized b y field
AMF populations or laboratory isolates (Sagan et al. 1993; Gidnazzi-Pearson. 1996).
suggesting that the products of the genes that define the Nod- and Myc- mutations must
play a central role in the establishment of the mycorrhizal and rhizobid symbioses.
Results from Nod- Myc- pea and soybean mutant studies contradict the effect of
the Nod- phenotype on Myc- plants and vice versa. For exmple. Duc et al. (1989).
Balaji et al. (1994) and Sagan et al. (1995) report that Nod- pea mutants are also Myc-.
In contrist. Wyss et al. (1990) found that Nod- soybean mutants and the wild type plants
are colonized by AMF equally well. They also found that the translational products of
polyadenylated RNA obtaùied from nodule-free and wild type plants cross reacted with
antiserum against soluble nodulins and membrane-bound nodulins. Sirnilarly. Xie et al.
(1995) observed that G.mosseae colonized non-nodulating mutants inoculated with an
effective Bindy-hizobfirmjuponic~rmstrain and wild type soybean plants inoculated with
ineffective rhizobia strains equally. indicating that a functional rhizobial symbiosis wiis
not r e d y necessary for mycorrhizae formation. They further noted that a Rhizobium sp.
mutant (NGEUnodABC) did not have any effect on mycorrhization of soybean. and that
the addition of 10-7 to 10-9 M concentrations of highly purified nodulation (Nod) factors
(acetylated NodNGR-V and sulfated NodNGR-V) obtained from strain NGR234 had
different effects on the colonization of soybean roots by G. rnosseae. It is unclear from
these studies what aspects of the nodulation process or what nod genes are involved in
the stimulation of the mycorrhiza symbiosis of the sarne legume host. Furthemore. it is
not very clear frorn the elàsting reports whether both symbionts in a tripartite association
nced to be complementary (in ternis of effectiveness) to each other.
Some of the questions raised by these studies are: (9 If the Nod-Myc- phenotype
is conaolled by the sarne gene. will one infenor endosymbiont reduce the effects of the
other on host performance or irnprove the overall bznefits despite the performance of the
inferior endobiont ?
(il]
C m the concept of 'one gene- two symbioses' explain the
functionality (Le.. growth, yield and nutrition of the host plant) of the tripartite
symbiosis? (iii) 1s the functionality of the tripartite symbiosis regulated only by the
nutrient supply and the dernand made by the Pamiers involved in the tripartite symbiosis?
It is clear ti-orn the existing literature that combining an effective rhizobid s r i n with
AMF dways produces superior ?lants. and the functioning of the tripartite symbiosis is
diI'ferent for the same AMF when combined with difirent Rhizobi~iinstrains. However.
it is not clear whether thc performance of an inferior bacterid symbiont c m be rectified by
a supenor fungal symbiont. and vice versa. Unfortunately. there are no reports
e x e g the effects of combinations of inferior or superior M F andlor rhizobia.
Reasons for the differences in the response of legume hosts to diEerent AMF-rhizo bia
combinations are not clear. but ùidicate the need to screen not only for superior AMF but
d s o rhizobia for specific plant species or genotypes.
Factors infhencing interactions between AMF and rhizobia
2.4.2.4
2.4.2.4.1
Host species
It is cornmon knowledge that other than soi1 nutrient leveIs (especidy P), the
mycotrophicity of a host plant is dependent on itç susceptibility to mycorrhization (Baylis.
1972: Talukdar and Germida, 1994). For example. Talukdar and Germida (1994) found
that lentil (coarse roots) benefited more from the colonization of three Glornus isolates
than wheat (fine roots). Therefore, it may not be surpnsing if leturnes with coarse roots
benzfit more from a functional AMF symbiosis than legurnes with fine roots. However,
given the tight replation of the tripartite symbiosis by the host. it is possible that other
factors will have to be considered.
Very few studies have assessed the effects of ;\MF-rhizobia combinations on the
growth and productivity of different host species or genotypes of the same host (Ahiabor
and Hirata, 1995; Ahmad. 1995; Ibrahim et al. 1995; Ibijbijen et d.1996: Thakur and
Panwar. 1997). For example, Ahiabor and Hirata (1995) studied the effects of coinoculating cowpea (Vignn unguiculatu L.), pigeonpea (Cajanus cajmt L.) and groundnut
(Arachis hypogaea L.) with compatible Rhizobium strains and G. efunicarum or Glomus
sp. Eg. They found that G. er~micaru?n
inoculation stimulated the growth of d l three
plant species. but E3 generally had no effect. The mycorrhizal dependency (as
detemined by the harvest index and P content of the host) of cowpea and pigeonpea were
hi_&er than that of grooundnut plants. indicabng differences in the hosts' response to
AMF-rhizobia combinations.
The interactions between plants. AMF and rhizobia that result in enhanced plant
growth and nutrition varies with different genotypes of the same host (Ahmad. 1995:
Ibrahim et al. 1995; Ibijbijen et al. 1996: Thakur and Panwar. 1997). For example.
Ahmad (1995) studied the effects of CO-inoculatingthree local cultivars of red kidney
bean (PhaseolusvulgariF L. cv. Miss Kelly. Portland Red and Round Red) with diree
species of AMF (G. pallidum. G. aggreg-emm and Scl- microcarpa) and four Rhizobium
phmeoli suains. He found that the effectiveness of the symbionts on host plant growth
and irnproved N and P nutrition depended on the combination of the three parmers
involved. and therefore. suggested that suitable partners need to be carefuIly selected for
obtaining the best results. Similady, Ibrahim and CO-workers(1995) found that growth
and yield response of groundnut to AMF and Bradyrhizobium sp. depended on the
groundnut genotype. and recornmended that AMF effects on plant yield and nutrition be
integated into plant breeding and agronomie approaches for stability in lepme
productivity. Similar genotype-mediated ciifkences in the response of plants to AMF
and rhizobia were noted for common beans (Ibijbijen et al. 1996) and greengam varieties
(Thakur and Panwar. 1997). These studies emphasize the signifïcance of the individual
parmers involved in the tripartite symbiosis.
2.4.2.4.2
Efficacy of the microsymbionts
It is generally accepted that for the effective functioning of the iripartite syrnbiosis, all
partners have to be effective andlor compatible. It is relatively easier to assess the
effectiveness of a Rhizobirrrn strain compared to an AMF species. f i s is because of the
uniform conditions under which aRhizobiurn SV'&
hnctions. For example. a R.
I~gumiiiosa~-um
bv- viceae strain has a lirnited host range and is u s u d y repressed b y high
levels of soil N (Novak et al. 1993). Furthemore. in the absence of other cornpetitors
and adverse soil conditions such as suboptimal pH and salinity. rhizobia cm fix nitrogen
and enhance its host growth. However. it is unclear whether a functiond rhizobial
syrnbiosis is critical for mycorrhizae formation. This issue has not been addressed in
detail so far (Xie et ai. 1995). Workers attempting to enhance kgurne productiviv
through the use of microbial inocrrIants have selected rhizobia because of their
effectiveness on the host or because of strain avaïlability (Azcon et al. 1991: Ahmad.
1995). However, in attempting to identie the importance of 'Nod' factors on the
elicitation of AMF, Xie et al. (1995) inoculated soybean plants with ineffective stxains
and found that mycorrbizal colonization was not diminished in the soybean plants. and
suggested that a functional rhizobial symbiosis was not critical for mycorrhization of the
host. It is dificult to malce inferences on host growth or productivity from this study. as
the authors did not include any information on host growth parameters. On the other
hand, the effectiveness of an AMF partner is not very well defined with regard to soi1 and
plant species. Therefore, depending on the soil nutrient levels (P. N. and micronutrients)
and host species. the AMF-host association may be mutualistic or parasitic. However.
although the response of a host to different AMF may v q . it is important to select for
AMF with appr~priatehosts and their rhizobia to maximize the benefits from the tripartite
symbiosis.
2.4.2.4.3
Nutrient levels
Most agiculmal soils do not have a sufficient amount of essential nutrients for
maintaining plant growth. Therefore. in most cases. large amounts of nutrients are
supplied to maintain adequate levels critical for plant growth. However, the application
of large amounts of nuaients. especially N and P. may be deaimental to some organisms
in soil. For example. hi& levels of N repress the potentiai N benefits from rhizobia to
plants. Therefore, most studies involving legurnes and rhizobia are uivÿnably conducted
in minera1 or marginal soils. or are not ferrilized with N fertilizers.
The requirement for P by the BNF process and the rnycorrhizal syrnbiosis places
phosphorus at a cenual place in the tripartite symbiosis. The reduction of one molecule of
nitmgen to ammonia through the rhizobia-mediateci BNF process requires 10 ATP
moIecuIes (Atkins and Rainbird. 1982). Therefore. an increase in the availability of P
may directly increase the N-fixation capacity of the rhizobial strain. In contrasr to
rhizobia, the effect of P on the AMF species depends on the tolerance of the A!'.!'species
to P and the P requirement of the host. For example, Schubert and Hayman (1986)
studied the response of onion (Allium cepa L.) plants to G. mosseae. G. epigaeum. G.
macrocarpum. G. caledonirrm, Glomris sp. E3, G. clurum and Gi.inargarita in sterilized
soit amended with different levels of P. They found that plants responded positively to
G. rnosseae and G. epigaeum at low to moderate P levels. However. G. clarum was
ineffective at low P, whereas G. mocrocarpum and Gi.rîrgarita enhanced piant growth
at the medium P level. In contrast. G. caledonium and Glumus sp. E3 were effective at
low, medium and high P levels. High levels of P are generally detrimentd to the
oermination of spores andfor the gowth and differentiation of intrarddicd and extraradical
C
hyphae (Miranda and Harris. 1994).
The requirement of P for the tripartite syrnbiosis is an aspect that warrants
attention. The participation and performance of rhizobia in the mpartite syrnbiosis is only
ensured when critical factors such as nutrient levels are rnaintained. On the other hand.
high levels of P inhibit most AMF species. Therefore. the selection of an appropriate
concentration of P is crucial for the proper functioning of the tripartite syrnbiosis.
Various workers have studied the eEect of P on the aipartite symbiosis (Barea et al.
1980: Azcon-Aguilar et al. 1986c: Morton et al. 1990: Rahman and Parsons. 1997:
Starnford et ai. 1997). For example. Barea et al. (1980) examined the effects of different
P sources such as K*PO4
and rock phosphate on the nodulation and nutrition of
Medicugo surivu L. inoculated with G. mosseue and R rnefiloti in phosphate-fixing
stznlized and non-stenlized soiIs. They found that inocuIation with G. mossecre not only
increased the nodulation by Rhizobium compared to the corresponding uninocuhted
controls. but also produced plant shoots that were similar in weight to the KH-04supplied plants in non-sterile soil. They concluded that myconhizae can substitute for
soluble P fertilizers. However, these effects were only observed in non-sterile soils and
not in sterilized soils. i n d i c a ~ the
g contribution of native endophytes. Sirnilarly. Azcon-
Aguilar and CO-workers( 1 9 8 6 ~observed
)
the effect of a mixture of two unidentified
phosphate-solubilizirig bacteria (PSB) on the tripartite symbiosis between soybean.
Rhizobi~imjaponicum and two Glomus spp. in a gamma-irradiated nzutral-calcareous soi1
amended with O, 1 and 5 g per kg of 32~-tricalciumphosphate (TCP). They found b a t
the pod yield of plants inoculated with G. mosseae and Glomus sp. Ej were significantly
enhanced compared to the control at O and 1 g of TCP. The application of phosphate and
the PSI3 did not affect pod yield for E3. However, increasing the level of TCP to 5 g and
inoculating with G. mosseae significantly increased the yield of plants by 21% over PSBinoculated plants supplied with 1 g of TCP. This suggests thar compared to E3. the
symbiotic efficiency of G. mosseae was greater at higher P levels. Mycorrhizal plants
inoculated with PSB had a higher N content at low P levels when compared to plants not
treated with PSB. indicahng die positive effect of AMF inoculation on die B h F p r o c e s
The increase in pod yield noted in E3-inoculated plants was accompanied by an increase
in the myconhizal colonization of roots.
Morton et al. (1990) assessed the soil solution P necessary for the nodulation and
nitrogen fixation of mycorrhizd clover inoculated with R. ineMoti in a stearn-sterilized
soil amended with up to 75 pg per ml of P as KHzPO4. They found that inoculation with
G. diaphanum signifcantly increased the shoot dry weight of clover in the unamended
soil and in soil amended with 40 and 60 pg P per ml cornpared to the control. However.
supplying more than 40 pg P per ml to mycorrhizal clover did not enhance the
nodulation. N and P uptake or shoot dry weight of plants. Rahman and Parsons (1997)
inoculated Sesbunia rostrata with Atorhizobium cacrlirzodans and GZomzrs mosseue in
sterilized soil amended with and without rock phosphate at an equivalent of 150 kg P
ha-1. There were no significant differences in the shoot dry weight and mycorrhizal
colonization of plants between any treatment Kowever. plants supplied with rock
phosphate and inoculated with AMF and rhizobia had siificantly higher shoot N and P
concenmtions than unuioculated plants establishing the positive effect of P on N2
fixation. Stamford and CO-workers(1997) examined the response of Mimosa
caesalpirziaefolia to inoculation with two effective Bradyrhùobium strains and a mixture
of G i margarita and G. ehmicahun in sieved, unsteriLized soil amended with tnple
superphosphate at an equivalent of 40 kg P ha-1. They found that mycorrhizal
colonization of roors was not affected by either of the rhizobia or the AMF treatment:
although, plants receivlng fertilizers had significantly higher levels of mycorrhizd
coZonization than untèrtilized plants. They also found that the lower symbiotic efficiency
of one Bradyrhizobium strain was increased when P f e d i z e r was applied, and both
strains had the same effect on plant growth. indicating that the availability of P enhanced
the syrnbiotic efficiency of rhizobia. Azcon and El-Amch (1997) studied the influence of
G. mosseae, Rhizobium meliloti and four (0.50, 100 and 150 mg P per kg soil) levels of
KH$?O4 on the growth and nodulation of Medicago sativa at four salinity levels in a
steam-sterilized soil. The growth and nodulation response of plants appeared to decrease
with increasing levels of soi1 salinity. However. plants inoculated with G. mosseae in the
non-saline soi1 produced a similx amount of biomass. more nodules and total N and P
than that of uninoculated plants supplied with up to 150 mg P per kg soil. These studies
not only ernphasize the importance of adequate P nutrition for the effective functioning of
the tripartite syrnbiosis. but also the efYect of AMF as a potential substitue for or
supplement to P fertilizers.
Although some nutrients assume greater importance than others because of the
special roles they play. it is generally accepted that dl nuuients essential for plant growth
must be present at optimal levels for the maximum growth of plants. In addition to the P
requirement, trace elements such as Fe and Mo are essentid for the proper functioning of
the nitrogenase enzyme complex consisting of the dinitrogenase and dinitrogenase
reductase (Munns. 1977; Fischer. 1994). The lack of these important elements has not
been studied on the tripartite symbiosis. but it is safe to assume diat the lack of these
elements may have a siCenificanteffect on nitrogen fixation and therefore. on the tripartite
sym biosis.
3.5
Conclusions
The partnership between legumes. AMF and rhizobia occurs naturally. and can
benefit al1 the partners involved. It appears that this association is tightly regulated by the
plant which initiates the chernical signal exchange between the microbial partners
(Stafford. 1997). However? the best partnership is not always ensured in nature due to
many cultural and other edaphic factors. Therefore. in order to ensure the best possibIe
effects on host growth. yield and nutrition. it is important to select for specific
combinations of AMI? and rhizobia which complement each other and the plant. In
addition. it is also important to assess the effects of combining an infenor AMFlrhizobia
with an effective microsymbiont on plant productivity in order to understand the relative
contributions of the different microsymbionts. Such studies will not onry help strengthen
the understanding and applications of appropriate AMF and rhizobia for lepmes. but also
help fine-tune the understanding of the tripartite synbiosis in t e m s of the evolun'onary
processes which have broughr together both the A M F and rhizobia synbioses into one
genetic locus.
3. O
OCCURRENCE AND ACTIVXTY OF ARBUSCULAR
MYCORRHIZAL FUNGI IN THE RHIZOSPHERE AND
ENDORHIZBSPLHERE OF LENTIL AND PEA IN
SASKATCHEWAN SOLLS
3.1
Introduction
Arbuscular mycorrhizal fun@ (AMF) are ubiquitous. and f o m an obligate symbiosis with
>85% of land plants. However, the occurrence and activity of AMF propagules is
affected by various host-soil-environment factors. The activity of AMF is intluenced by
soi1 characteristics such as texture (Fitter. 1989: Land et al. 1989: Lippman et al. 1989).
For example, Lippman et al. (1989) found that AMF colonization of winter wheat
growing in Ioamy and chernozemic soiI was greeater than for winter wheat growing in
sandy or clay soils. Other soi1 factors such as nutrient levels especially N and P and
micronutrients such as Mn, influence AMF activity and subsequent AMF spomlation
(Vivekanandan and Fixen, 1991: McGonigle and Miller, 1996). For example. high soil
N or P levels can inhibit AMF colonization of roots of some plants, and high levels of P
may inhibit AMF spore germination (Graham et al. 1981: Douds and Chaney. 1986:
Miranda and Harris, 1994).
The abundance and activity of AMI? in soil is affected by cropping sequences
(Black and Tinker, 1979: Harinikumar and Bagyaraj, 1988: Johnson et al. 1992). For
example, continuous monocultures reduce the activity of beneficial .W.which can lead
to plant stunting and yield reductions (Hendrix et al. 1992: Johnson et al. 1992).
However, growing a non-host crop or a fallow season reduces the nurnber of AMF
spores and affects the activity of AMF more profoundly. For example. Black and Tinker
(1979) derzrmined the percentqe AMF colonization of biirley and AMF spore abundance
in barley rhizosphere following a previous rotation of barley. kale or fdlow. They found
that after 70 d growth. barley roots tiom the barley-barley rotation had 100% more
colonization than barIey roots from the barley-kale or bariey-fallow rotation. These
authors also reported that the nurnber of AMF spores was ca. 21 cm-3 soil in the barleybarley rotation. but only ca. 10 and 8 cm-3 soi1 in the barley-kale and barIey-fallow
rotations, respectively. It is clear that the abundance and activity of AMF in any soil may
be influenced not only by these sofi and plant factors, but a combination of these factors.
Although AMF are considered to be generalists. there is growing body of
evidence to suggest that the AîîF symbiosis is in fact regdüted by the host (Boyetchko
and Tewari. 1990; Simpson and Daft. 1990; Hetnck et al. 1995: Gianinazzi-Pearson,
1996) or that A M F exhibit host preference (Hetrick and BIoom. 1986: Rosendahl et ai.
1989; h3cGonigle and Fiaer, 1990: Hendrix et al. 1995: Bever et al. 1996). For example.
Boyetchko and Tewari (1990) found that the level of root colonization by Glomus
dimorphicum of red clover and maize plants was si,@ïcantly higher than beans. alfalfa
and onion plants. and that barley was the least susceptible of ail the hosts tested.
Similarly, Simpson and Daft (1990) reported that sporulation by G. clarum was greatest
with a maize or sorghum host than a chickpea host. Bever et al. (1996) found that co-
occuming plant species such as Allium. Anrhoxanthum, Paninrm and Planrago supported
different AMF sporulation rates as a result of different AMF growth rates. Therefore. the
AMF community associated with the four different plant species was different The
authors suggested that these differences were not influenced by the time of plant harvest
or host-dependent timing of spomlation. but by host-dependent AMF growth rates. and
conchded that this relative fungd growth rate as controlled by the hosts probably
influences AMF species abundance. activity and diversity.
Similady. host plants are not equaliy efièctive at eliciting a response from dl the
members of the AMF comrnunity [Schenck and Kinloch. 1980: Rosendahl et al. 1989:
Bever et ai. 1996). For example. Schenck and Kinloch (1980) reported that sorghum
rhizosphere contained a more diverse AMF comrnunity compared to other hosrs such as
soybean. bahiagrass. cotton or peanut. These and other reports suggest that even thoueh
the rhizosphere soil of dBerent plant species may contain various AMF species.
colonization of the endorhizosphere or root intenor rnay be a function of host preference
or other host-soil-environment conditions. The preferential colonization of host ro Ots b y
M m a y be reflectiveof the level of AMF-host specificity. which rnay be important in
the selection of suitable AMF species as inoculants. This study evaluated the occurrence.
activity and apparent diversity of AMF in the rhizosphere and endorhizosphere of lentil
and pea grown in Saskatchewan soiIs.
3.2
Materials and methods
3.2.1 Sample collection and soi1 nutrient analyses
Field sites from various typically lentil and pea growing areas ôt Moose Jaw. Rosetown.
Drinkwater, Allan and Bellevue. Saskatchewan were selected for study. Site selection
was according to the suggestions of Dr. A. E. Slinkard (Crop Development Centre.
Sask.). Smples were collected during 1995 when the legume crop was at flowering to
pod formation stage. AI1 the field sites had been previousIy fedized with recommended
levels of fertilizers. except for both fields at the Drinkwater site where no fertilizers or
pesticides were applied. Previous (Le.. 1994) rotation and other details for the lentil and
pea crops are presented in Table 3.2.1.
At each field site. five sample locations separated by at l e s t 10 m were selected at
random. At each sample location. six replicate bulk soil cores (0-50 cm depth) were
collected between crop r o m and placed in individual plastic bags. From the same
Table 3.2.1- Field site location. number and select characteristics of the Saskatchewan
field sites cropped to lentil and pea.
Field site
1994-1995 crop
Soi1 zone
Inoculatedd
Moose Jaw
Canola-lentil
Dark Brown Chernozem
Yes
Moose Jwdw
Wheat-lentil
Dark Brown Chemozem
Yes
Rosetown
B arley-lentil
Dark Brown Chernozern
Yes
Rosetown
Lentil-lentil
Dark Brown Chernozem
Yes
Dridavater
F d o w-lentil
Dark Brown Chernozem
Yes
Drinkwater
Wheat-lentil
Dark Brown Chernozem
Ys
Allm
Wheat-Ientil
Dark Brown Chemozem
Yes
Man
Wheat-pea
Dark Brown Chernozem
Yes
Bellevue
Wheat-pea
Black Chernozem
No
Bellevue
Bariey-pea
Black Chernozem
No
Bellevue
FI=-pea
Black Chemozem
hTo
Field no.
Canola-pea
Bellevue
Black Chemozem
ahoculated with a commercial Rhizobium inoculant-
No
location. six intact plants (Le.. with shoots and roots) with adhering rhizosphere soil (O-
50 cm) urerecolIected as an intact core and placed in individual bags. Soi1 and plant
samples were placed in a cooler irnmediately. Upon retumiog to the laboratory. the six
replicate buk soil sarnples from each of the five sampling locations in a field site were
bulked together and stored at 4OC (five sarnples per field site). Plant shoots were
p
Root
rernoved and discarded. and the roots were thoroughly washed in t ~ water.
sarnples (n=6) from each of the five sampling locations were bulked together and stored
at -20°C. A composite soi1 sarnple from each field site was analyzed for nuûient content
and selected soil characteristics such as texture, pH. conductivis. and organic matter at the
Enviro-Test Laboratones, Saskatoon, Saskatchewan (Table 3 - 2 2 ) . The field sites varicd
in their N and P levels and organic rnatter content although soil pH was between 7.7 and
8.7.
3 . 2 - 2 Trap culture generation using field soil or plant mots
The AMF in the soil fiom the different field sites and associated lentil and pea roots were
trapped according to the procedure of Morton et al. (1993). Trap cultures were
estabfished using sorghum-sudangrass hybrïd (var: 877F). Sorghurn-sudangrass was
used as the host plant because of its broad host range for AMF (Morton et al. 1993). The
N'VIF were trapped from soils collected at each sampling location (n=5) within each field
site. Equal volumes of the field soi1 (1000 ml) and autoclaved (60 min. 121O C . 15 psi)
coarse sand (Silsilica, Grade 7) were thoroughly mixed and potted in 15-cm dia. pots.
Pots were seeded with 40-50 sorghum-sudangrass seeds and grown in a growth chamber
for 120 d with the following gowth conditions: 23 OC day/l8 OC night temperature for the
first 10 days and 25/20° C thereafter: a photoperiod of 14 hlday during the first week. 15
h/day during the second week and 16 Wday thereafter; the relative humidity was en. 60%.
and irradiame levels were 375-400 j
M m-2 se&
One hundred m W t e r s of a rnodified
Hoagland solution (Le., low P) (Talukdar and Germida, 1993b) were added when plants
showed symptoms of nutrient deficiency. Water was added to maintain the soil-sand mix
at -60% rnoisture holding capacity. The AMF trapped using this procedure will be
referred to as "soil trap" (ST) cultures hereat'ter.
Sorghum-sudangrass hybrid (var: 877F) \vas used to trap AMF from the roots of
field-grown lentil and pea. according to the procedure of Morton et al. (1993). The sandy
loam soil used for this procedure was collected at Outlook. Saskatchewan. This soi1 was
mixed with equal amounts (1:1) (w/w) of sand, and two ldograms potted in 15-cm dia.
pots. The soi1:smd mix contained 3.5 and 2.5 pg of N and P g-l. respectivdy. Pots
were sterilized at 121°C (15 psi) for 60 min- for two days with a 24-h interval and
allowed to equilibrate for seven days. A representative subsample of lentil and pea roots
collected from the different field sites and stored at -20°Cwere cut into 1-cm pieces.
surface-sterilized with 70% ethano1 for one niin- and washed thoroughly in sterilized
distilled water. Twenty surface-sterilized root pieces from each field location were placed
at 5-cm depth from the soil surface over which 40-50 sorghum-sudangrass seeds were
pIanted. Plants were grown for 120 days with the same growth chamber conditions as
descnbed previously for the ST cultures, except that the irradiance level was 400-450
m-2 sec-1. One hundred milliliters of a modified Hoagland solution were added when
plants showed syrnptoms of nutrient deficiency. The AMF trapped using this procedure
will be refemd to as "first cycle root trap" (1"RT) cultures hereafter.
A second successive root trap culture was established in the soi1 from 1°RT
c u h r e s using maize (Zeum q s L. cv. Early Golden Bantam) as host to enhance spore
production and AMF species richness. without the production of large amounts of root
matcriai a was the case with sorghum-sudanLms. One kilogran of soil from the 1ORT
cultures containhg spores. colonized mots and hyphae was mixed with equal amounts
(1:l) ( d w ) of autoclaved sand and two kilograms potted in 15-cm dia. pots. Two
surface-sterilized and pre-germinated maize seedlings were planted at 5-cm depth from the
soil surface and grown for 90 d. The growth conditions used were similar to that for the
2' RT cultures (as described eadier). The AMF mpped using this procedure will be
referred to as "second cycle root trap" (2ORT) cultures hereafter.
After 90 (for 2 O RT) or 120 d (for ST and 1' RT) p w t h . plants were allowed to
desiccate for 10 d in the growth chmber. Plant shoots were removeci at the soil surface
and discarded. The roots were cut into one centimeter pieces. added back to the soil. and
the contents of each pot were m k e d thoroughly. The trap cultures were maintained at 4OC
for at l e s t 30 days before processing.
3 . 2 . 3 Parameters
3.2.3-1
Spore enurneration
AMI? spores were retrieved from soil usiog the wet sieving and decanting method
of Gerdemann and Nicholson (1963). Two 10-g soil samples from each field sample
location were weighed separately and mixed with 100 mL of water. This soil suspension
was stined for 30 sec and allowed to settle for 10 sec. The supernatant was decanted
over nested sieves with porc sizes rmging from 500 to 38 p.The sievings were
collected. pooled and subjected to 20:60% (w/v) sucrose gradient centrifiqation for five
minutes at 2800 rpm (Menz et al. 1979). The AMF spore mixture at the interface was
rzmoved using a Pasteur pipette, washed thorou-ghly with water and vacuum-filtered ont0
a 47-mm dia 0.8 p pore size filter m i p o r e Corporation, Bedford. MA). The filters
containing AMF spores were placed in stenle Petri dishes (60 x 15 mm) and stored ~t
4 O C . Spores were enumerated with a stereo microscope (X 47). The rnoisture content of
each soil sample was deterrnined. and the total number of AMF spores was calculated on
an oven dry weight basis. An identical procedure was followed for spore enumeration in
the ST and 1' and 2 O RT cultures. In order to determine whether the pea and lentil hosts
dtered AMI? diversity at the different field sites- the number of different AMF
rnorphorypes (presumably M F species) in each of the ST and the correspondhg 1 and
2'RT cultures was counted. This was accomplished by separating morphologically
different AMF spore types based on spore size. color and other characteristics such as
texture of the outer spore wall (Le.. laminated or smooth. etc.). and grouping them
together. Photographic records of the different AMF spore morphotypes in the ST and
RT cultures were established for cornparison purposes.
The procedure of Walley and Germida (1995) was used to determine AMF spore
viability. The Millipore filters ccntiiining AMF spores were saturated with 1.5 mg rnL-l
iodonitrotetrazoliurn (INT) and incubated at 24OC for up to 48 h in the duk. Spores
which stained a bright red (mandarin to satum red) were counted as viable and recorded.
The percentage viability of AMF spores from each field location was then calculated as
foUows: % viability = Viable spores g-l/ Total number of spores pl x 100
3.2-3.2
Mycorrhizal colonization
The level of AMF colonization within plant roots was determined using
microscopy and fatty acid methyl ester (FAME) analysis. L e n a and pea roots collected
from the field and stored at -20°C were processed according to the method of Koske and
Gemma (1989) for the determination of AMF root colonization. Roots were cleared in a
5% KOH solution, acidified with 1% HC1. stained with acidic glycerol containing 0.05%
trypan blue and destained with acidic glycerol. The AMF colonization Ievels were
determined using a modified grid-line intersect method (Giovanem and Mosse. 1980)
with a Eght microscope (~100).The mycorrhizal colonization of sorghum sudangrass
(ST and I "RT culture) and maize (2"RT) roots was detemined using an identical
procedure.
The presence of AMF within host plant roots t'rom the different m p cultures was
also assessed using a fatty acid biornarker. FAME profiles of the roots tiom the ST. 1 Oand 2"RT cultures were analyzed for the presence of 16: 1 01%. an AMF FAiME biomarker
(Olsson et al. 1995: 1997: Bentivenga and Morton. 1996). This fatty acid is unusual in
fungi that do not forrn arbuscular mycorrhizas. indicating that it may be a biornarker for
AMF. Furthemore, 16: 1 w5c has been found in relatively high arnounts in roots
colonized by AMF when compared with uncolonized roots (Pacovslq. 1989).
Therefore. the presence or absence of this FAME biornarker was compared with the
sesults from the AMF colonization data to v e m the presence of AMF within roots-
Root samples were reaieved from the sorghum-sudangrass (ST and 1°RT) and
maize (2ORT) trap cultures. washcd thoroughly with tap water and dried iightly with paper
toweis. A representative root subsample of 3.0 g was placed in a MIDI extraction tube
(25 x 150 mm). and stored at -20°Cuntil processed for fatty acid extraction. Samples
were aiways handled with latex-glowd hands to avoid transfer of contaminant fatty acids
(i.e., from skin) to root samples.
Extraction of fany acids from the root tissues was carried out according to the
procedure of Caviglli et al. (1995). To 3.0 g of fresh root samples in 25 x 150 mm
extraction tubes. 5.0 ml of a saponification reagent (NaOH. 45 g: methanol. 150 ml and
deionized distilled water. 150 ml) were added. The tube was vortexed for 5-10 sec and
placed in a 100°C water bath for five minutes. The samples were vortexed again. and
returned to the 100°C water bath for an additional 25 min, removed and cooled to room
temperature. Ten milliliters of a methylation reagent (6.0 N HCl. 325 ml: methanol. 275
ml) were added to the saponified mixture and vortexed for 5-10 sec. The tubes were
placed in a 80rrl°C water bath for lm1 min. and cooled rapidly to room temperature. TO
the methylated samples, 1.5 ml of the extraction reagent (hexane. 200 ml: methyl-tert
bu@ ether. 200 ml) were added and the tube shaken (at 5-10 rev min-1) for 10 min. The
organic phase containing proteins and other material was removed using a Pasteur pipette
and discarded. To the rernaining aqueous phase, 4.0 ml of a base wüsh solution (NaOH.
10.8 g: deionîzed distilled water. 900 ml) were added and the tube shaken (at 5-10 rev
min-') for five minutes. Two-thirds of the top phase was then removed and transferred to
a viai which was capped and sealed ushg a crimper. The vials were placed in an
autosampler a a y for chromatographie analysis using a gas chromatopph (HP 5890
senes II). I:AME characterization and narning of FAME peÿks were done u s i y the
Microbial Identification System software (MD1 Corporation, Newark. DE). The FAMEs
were standardized by calculating the adjusted response area for each sample. This was
accomplished by multiplying the percentage composition of the appropnate FAMEs by
the total named area for that chromatogrm. The level of the FAiME 16: 1 w5c in the roots
of sorghum-sudangrass (ST) or maize (2ORT) plants from the different trap cultures is
presented as the ratio of the adjusted response area of the FAME 16: 1 w5c to that of the
FAME 120.
3.2.4 S tatistics
Percentage values for AMF spore viabiliq and mycorrhizal colonization were arcsinetransfomed before data analysis. Correlation analyses were carried out usïng SAS
(SAS. 1997). Al3 the values are expressed as mean t,stimdard error (SE).
3.3
ResuIts
3 . 3 . 1 Field
The total number of AMF spores at field sites cropped to lentil ranged from 13 to 23 gl
soil, whereas the peafieid sites contained 7 to 24 AMF spores g1soi1 (Fig. 3.3.1).
Fewer AMF spores were observed in Black Chernozem (i-e.. d l the Bellevue field sites)
than in Dark Brown Chemozem (i-e., all other field sites). There was no apparent
Moose Jaw#l -Canola-lentil
mm]
(8)
Moose Jaw#2-Wheat-lentil
Rosetom#3-B arley-lentil
Rosetown#4-Lentil-lentil
Drinkwater#5-FaUo w-lentil
- Viable
O
5
10
25
25
20
AMF sporedg soil
Viable
AMF morede soil
Fig. 3 -3.1.
Mean ( n 4 number of viable and non-viable M F spores recovered from
soil at 12 different field sites cropped to (A) lentil or (B) pea. Numbers In
parentheses adjacent to each bar represents the SE of the mean for the total
number of AMF spores. Additional details are given in Tables 3.2.1 and
3.2.2.
relationship between important soil characteristics such as N or P levels and îhs t o d
nurnber of AMF spores. However. it appeared that the relatively high level of P (Le.. 30
ppm) at the Bellevue field site no. 11 in a tlax-pea rotation rnay have reduced AhlF
sporulation. The previous canolü (-Moose Jaw field site no. 1). continuous lentil
(Rosetown field site no. 4). or fallow (Drinkwater field site no- 5 ) rotations had no eftèct
on the total nurnber of AMF spores. but reduced the percentage viability of spores (Fig
3.3.1). For exarnple. thz number of viable AMF spores in lentil-lentil rotation was lower
than the barley-lentil rotation at Rosetown. In some cases. the number of viable spores at
one field site was higher than the adjacent field site. despite similar soil nutrient levels at
both sites. For exarnple. the number of viable AMF spores at the Nlan field site no. 7 in
a wheat-lentil rotation was CU. 10 g-* soil, whereas the number of viable AMF spores at
the adjacent wheat-pea site (Allan field site no. 8) was only 4 g-1 soil. However. this
diKerence may have been mediated by the different crops (Le.. lentil vs pea). The four
Bellewe field sites no. 9, 10. 11 and 12 generally contained fewer viable AMF spores.
compared to other sites.
At all the field sites. lentil and pea roots were readily colonized by A M F (Fig.
3-32). AMF colonization of pea and lenàl roots was characterized by the presence of
intraradical hyphae. arbirscules andor vesicles, suggesting AMF involvement in nutrient
uptake. The percentage of AMF-colonized root of lentil was generaliy higher than pea.
The percentage of AM F-colonized root was not affectcd by a previous canola crop
(Moose Jaw field site no. 1 and Bellevue field site no. 1 3 , fallow (Drinkwater field site
no. 5 ) or continuous lentil crop (Rosetom field site no. 4). The low percentage of AMF
colonization at the Bellevue field site no. 11 in a flax-pea rotation rnay be due to the
relatively high-P levels at that site (Table 3.2.2: Fig. 3.3.2). There was no apparent
relationship between AMF colonization of leuaï and pea roots and any soil characteristics
except N03-N; a negative correlation (r-û.84: p<0.00 1) was noted beiween the AMF-
AMF-colonized root iength (%)
AMF-colonized root length (%)
Fig. 3.3-2.
Mean (n=5) ISE percenwe of AMF-colonized root of (A) lentil and (B)
pea grown at 12 different field sites. Additional details are a v e n in Tables
3.2.1 and 3.2-2.
colonized root and the N03-N levels at the diffsrent field sites.
3.3.2 Trap cultures
3-3.2-1
Soi1 trap cultures
The ST cultures should have uapped most. if not a l l of the MF species in the
rhizosphere of the lentil and pea plants. The number of AMF spores recovered fron soil
in the ST cultures varied h m 83 to 1693 per 100 g of soil. which was lower than the
number of viable AMF spores recovered from 8 of 12 field soil sampks (Table 3.3.1).
The highest number of AMF spores was found in the ST cultures produced using soil
frorn the Allm field sites no. 7 and 8. However. the ST cultures produced using soil
from the Bellewe sites except for the field site no. 10 in a barley-pea rotation contained
fewer AMF spores compared to aLl other sites (Table 3.3.1). The AMF in the ST cultures
readily colonized sorghum-sudangras roots. The AMF colonization of sorghumsudangrass roots in the ST cultures ranged from 29 to 71% (Table 3.3.1). There was no
particular trend in the AMF-colonization of roots. although ST cultures established usine
soil from both the Man field sites were generally observed to have high AVFcolonization levels. The AMF at the four Bellevue sites were agressive in colonizing
s o r g h u m - s u d a n p s roots. in spite of their low (spore) numbers.
The number of AMF spore morphotypes from the different field sites ranged fiom
four to nine (Table 3.3.2). For example, ST cultures produced using soil collected at the
Moose Jaw field site no. 1 in a canola-lentil rotation, Rosetown field site no. 4 in a lentillentil rotation, Drinkwater field site no. 5 in a fallow-lentil rotation and Man field site no.
8 in a wheat-pea rotation contained four AMF morphotypes each (Fig. 3.3.3A).
Although the Bellevue field sites no. 9,10,11 and 12 contained fewer viable AMF
spores, the average number of AMF morphotypes was relatively higher than other sites.
For example, ST cultures produced using soit collected at the field site no. 10 in a barley-
Fig.
The AMF morphotypes recovered from (A) the soil trap cultures or
(B) second cycle root trap cultures established using field soir or pea roots
obtained from the Allan field site no. 8 in a wheat-pea rotation. Bar
rnarker=137 Pm.
3.3.4-
The M F morphotypes recovered froin (A) the soi1 t ~ cultures
p
or
(B) second cycle root uüp ç~ilturesesstbblished using field soi1 or pea roi
obtained t'rom the Bellevue tield site no. 10 in a bwley-pea rokation. Ba
rnueker=l 37 Pm.
pea rotation contained nine different AMF morphotypes (Table 3 - 3 2 : Fig. 3 - 3 - 4 9 The
number of AMF morphotypes varied between the two field sites at each field site location.
For exarnple. the Moose law field site no. 2 in a wheat-lentil rotation yielded more AMF
morphotypes than the Moose law field site no. 1 in a canola-lentil rotation (Table 3.3.2).
This also indicates that in addition to reducing the v i a b i l i ~of AMF spores (Fig. 3.3.1)
compared to the Moose Jaw field site no. 2 in a wheat-lentil rotation. the previous canola
crop also reduced the apparent AMF diversity. The AMF spores isolated from the Black
Chernozerrïic soîls at Bellevue field sites no. 9. 10. 11 and 12 (Le.. ) were smaller in size
(i-e.. 53- 150 p m dia) than AMF spores recovered from the Dark Brown Chernozemic
soils at the other field sites (Le.. 90-300 p m dia).
3.3.2.2
Root trap cuItures
The RT cultures should have contained AMF species that colonized the roots of lentil and
pea collected at the different field sites. The number of AMF spores in ail the 1ORT
cultures except that of Bellevue field site no. 10 was very low. and ranged from 6-28 per
100 g soil (Table 3.3.1). Furthemore. the AMF colonization of sorghum-sudangrass
roots in the 1°RT cultures was very low and rmged from five to six percent. In nuiz of
12 of the 1°RT cuItures. roots were apparently not colonized by AMF. However, AMF
root colonization in the RT cultures established using lenril or pea roots collected from
both iUlan field sites no- 7 and 8 and the Bellevue field site no. 10 was minimal.
The second successivz RT cultures produced usuig maize had no effect on AMF
sporulation. except for the 2ORT ccltures produced from roots collected at field no. 10
(Table 3.3.1). However. enrÏching the 1ORT culture using a maize host enhanced the
AMF root colonization of maize plants. For example, A M F colonization of maize roots
from the 2ORT cultures representing the M a n field sites no. 7 and 8 was 52% and 62%.
and the AMF-colonized roocs of rnaize plants representing the Bellewe field site no. IO
was 38%. However. the high level of AlMF colonization was not associated with
enhanced spomlation. suggesting that AMI?spomlation was restricted by edaphic factors
or altematively. reflect the chwactztisa~sof the colonizïng AMF species or interactions
between the AMF species and the maize host.
The number of AMF morphotypes in eight of twelve of the 2ORT cultures was
reduced from that observed in the ST cultures. suggesting seiectîon of AMF morphotypes
(Le.. AMF species) by the lenhl and pea hosts at the different tield sites (Table 3.32).
For exarnple. at the Bellevue field site no. 10 in a barley-pea rotation. only one-thirds of
the AMF morphotypes (3 of 9) were selected in the 2ORT culture (Fig. 3.3.4). Similady.
at the Moose Jaa field site no. 2 in a wheat-lenhl rotation. only four of seven of the MLF
morphotypes were selected from the ST cultures (Fig. 3.3.5). These results suggest that
lentil and pea preferentially selected AMF morphorypes from the rhizosphere AMF
cornmunity to colonize the endorhizosphere and enhanced their proliferation. Ln addition.
seven of 12 RT cultures conrained at least one AMF morphotype that was previously not
noted in ST cultures (Table 3.3.2; Fig. 3.3.3). This suggests that AMF morphotypes thar
were present in very Iow numbers in the ST cultures (i.e.. in soil) were selectively
enhanced in RT cultures-
3 - 3 . 3 Correlation of AMF' root colonization with the FAME 16:lo5c
within roots of trap hosts
The presence of the FAME biomarker 16: 1 03% in the various ST and RT cultures is
presented as the adjusted response ratio between the FAME 16: 1 w5c and 120 (Fig.
3.3.6). The presence of the FAME biomarker was noted in sorghum-sudanpass roots
from all the ST cultures and one of the loRT culture, and in maize roots from 8 of 12 the
2ORT cuitures. The FAME biomarker 16:1 o 5 c was not present in uninfected maize roots
(data not shown). There was no apparent relationship between the adjusted response
5.
The AMI? morphotypes recovered fiom (A) the soil trap cultures or
(B) second cycle root trap cultures established using field soil or lentil
roots obtained f o m the Moose Jaw field site no. 2 in a wheat-lentil
rotation. Bar markeril37 Pm.
-- .--._ .==--' &--%p"
z
5
5
z
=
-& - -&,?
IZ 9
8 g%ess&Zi"
--.-
c
c
-
e
.L
c
-
-
-
-
-
cj
eJ
G
- ï . + + + - -
C
a
m
3 s m2 ' 5 s p s g E ZU
ratio and M coIonization of sorghum-sudangrass roots in the ST or 1ORT cultures.
However. the adjusted response ratio of the F M E biomarker was positively correlated
(r= 0.79; p<O.Ol) with AMF colonization of maize roots in the 3"RT cultures.
Furthemore. the presence of the FAME biomarker in the roors of eight of twelve of the
2"RT cultures suggests that determinahon of the FAME biomarker within plant roots may
be a sensitive method for the assessrnent of the presence of AMF. This is because 2ORT
cultures produced from both iMoose Jaw field sites no. 1 and 2 and the Dnnkwater field
sites no. 5 and 6 contained trace levels of the biomarker (Fig- 3.3.6). even though AMF
colonization of roots was not apparent using microscopy (Table 3.3.1).
3.4
Discussion
The diversity of AMF in a particular soil is detemined by species richness i-e.. the
number of AMF species (Magunan, 1988). Several workers have studied AMF diversity
in various habitats such as agricultural soils (Bakerspigel. 1956: Jensen and Jako bsen,
1984; Hetrick and Bloom. 1983; Talukdar and Germida. 1993a), grasdand soils
(Rosendahl et al. 1989: Bever et al. 1996), desert soils (Rose, 1981: Stutz and Morton,
1996) and forest soils (Schenck and Kinloch- 1980; Moutogolis and Widden. 1996). Al1
of these reports describe AMF species diversity andfor species abundance in these
habitats as uitluenced by factors such as plant species, soil acidiq. nutrient levels. soil
type, cultivation and temporal variations. However. one of the key factors that influences
AMF diversity at any a v e n site is the host plant species. My study also demonstrates that
the host plant hüs a profound influence on the sporulation and diversity of AMF in the
rhizosphere and the endorhizosphere.
To date, there are no reports on AMF that form mycorrhizae only with a restricted
group of hosts. Although AMF generally lack specificity. many workers have
hypothesized that AMF do preferentially colonize some CO-occunïnghost plants more
than others. or some hosts allow the preferential colonization of some AMF species
(Rosendahl et al. 1989: McGonigle and Fitter. 1990). Rosendahl et al. (1989) found that
the endorhizosphere of seven plant species CO-occu~nng
in a Danish p s s l m d cornmunity
was colonized by different AMF communities. which were dso diffierent fiom ùiat
occurring in the rhizosphere. Their report suggests that (i) contrary to previous
hypotheses. AMF-host selectivity does exisr and (ii) the selection of M F endophytes
depends on the host plant. McGonigle and Fitter (1990) found that the g r a s species
Holnrs lulzatlrs was preferentially colonîzed by the "fine endophyte" G. renrre rather than
by other AMF species in the rhizosphere. Bever et al. (1996) indicated that each of the
four plant species Allium, Anthoxmthum. Panicum and Pluntugo supponed the growth of
different AMF species. and their growth rates depended on 'host-dependent differences in
fungal growth rates'. Thus. it appears that the host plant exerts a great deal of influence
on the selection of AMF endophytes. It has been hypothesized that host plant replation
of AMF enay and intraradicd proliferation may be to effectively utilize the presence of the
endophytes in the acquisition of nutrîents from soi1 during the maximum growth stage of
the host (Smith and Read, 1997). However, few workers have attempted to study this
phenomenon, despite its practical importance (Rosendahl et al. 1989: Bever et al. 1996).
My study examined the phenomenon of AMF-host selectivity using pea and lentil. I
found that lentil and pea selected AMF morphotypes (species) from the rhizosphere AMF
community that preferentidy colonized and proliferated within the endorhizospherz.
Arbuscular mycorrhizal fun,@ spores were abundant in dl the field sites sarnpled.
The number of AMF spores found in these soils was at least 250% higher than those
reported for a_gicultural soils by Abbott and Robson (1977). Hayman and Stovold
(1979)- Malibari et al. (1988), Land et al. (1989) and Talukdar and Germida (1993a).
Generally. AMF sporulation under lentil was ca. 58% higher than that observed for pea.
Similarly, the average percentage of AMF-colonized root was ca. 6% higher for lenal
than pea. Key factors that influence the density of AVIF propaples include plant species.
soil nutrient levels and other soi1 characteristics such ÿs texture (Lippman er al . 1989:
Brundetr 1991: Douds, 1994). Lipprnan et al. (1989) observed that AMF colonization
levrls of winter wheat grown in loam soils was higher than that of winter wheat g o w n in
clay soils. and suggested that AMF colonization of host roots may also be influenced by
soi1 texture. In my study. the texture of soil at thz IentiI field sites was clay or clay loam.
whereas soil texture at the pea field sites was generally loam. However. AMF
colonization levels were higher in the clay soiIs (Le.. lentil sites) tfian in the ioarny soils
(Le.. pea sites). suggesting that AMF sporulation and root colonization were influenced
by factors other than soi1 texture. One important difference between the lentil and pea
hosts is that Ienril is more dependent on mycorrhizae than pea (Baylis. 1972; Hetnck,
1984). Therefore, differences in AMF spore density and root co1onization betsveen the
lentil and pea field sites were probably mediated by interactions between members of the
AMF community and plant genotype.
A compa5son between the number of AhiIF spores in the field soil and the ST
cultures revealed a reduction in spore numbers in eight of twelve ST cultures. but an
increase in the spore numbers of four of twelve cultures. AhlF sponilation in soil is
generaily reduced by extreme nutrient levels (Douds and Schenck 1990), non-responsive
host species (Black and Tinker. 1979; Harinikumar and Bagyaraj. 1988). hyperparasitism
(Lee and Koske. 1994). and predation by soi1 fauna (Rabatin and SMner, 1988).
Changes in AMF sporulation c m also be regulated by the fun@ genotype with or
without the involvernent of the host (Gazey et al. 1992; Franke and Morton, 1994: Bever
et al. 1996). The changes in sporulation in my study could not have been caused by the
nutrient levels. hyperparasitism. predation or plant species. as the ST cultures were
established using the field soil. However. as suggested by Bever et al. (1 996). it is
possible that the difference in AMF sporulation was related to a change in the growth rate
of the AMF species in association with sorghum-sudmpss (host used for establishment
of ST cultures). compared to that of pea or lentil in the field.
Morton et al. (1995) proposed that the only reliable measure of AMF species
diversity and species abundance is the extraction. enurneration and identification of ALMF
spores. 1 assessed AMF diversity in the rhizosphere and endorhizosphere of lentil and
pea using rnorphological characteristics of the different AMF spore types found in the ST
and 2ORT cultures. The number of AMF spores and AMF morphotypes in the 2ORT
cultures was generally lower than that of the correspondhg ST cdtures. In eight of
twelve of the 2"RT cultures. the number of AMF spore morphotypes (Le.. species) was
lower than that observed in the ST cultures (Table 3.3.2). This suggests that AMF
preferen-iallycolonized the roots of pea and lentil. In some cases, new AMF spore
morphotypes not observed in the ST cultures were seen in the Z0RT cultures (Fig. 3.3.3:
Table 3.3.2). These results indicate that the hosts not only allowed preferential
colonization by some AMF species. but also enriched rnorphotypes present in low
niimbers in soil. which points to AMF-host selectivity.
The establishment and intraradical proliferation of an AMF species precedes
sporulation in soil. Therefore. as suggested by Gazey et al. (1992). a threshold level of
AMF colonization is required for spore production by an AMF species. In rny snidy.
soghum-sudangass roots in ail the ST cultures were readily colonized by AMF.
However. the sorghum-sudangrass roots in the loRT culnires either exhibited no AMF
colonization or very low levels, resulting in poor sporulation. In contrast to the I ORT.
some 2ORT cultures had high levels of AMF root colonization. i n d i c a ~ gthat successive
trap cultures benefited AMF growth (Morton et al. 1995: Stutz and Morton. 1996). S m e
and Morton (1996) found that the second and third successive trap cultures established
using field soil contauiing spores, colonized roots and extemal hyphae helped increase the
spore numbers of various AMF species in the rhizosphere of Prosupis i~elutim.
However. 1 found that the intramdical prolifëration of M F in the P U T cultures was not
associated witlï enhanced AMF sporulation. Stutz and Morton ( 2 996) hypothesized that
the lack of AMF spores in soil did noz: indicate the absence of AMF sporulation. Rose
(198 1) found that some desert plants of Baja. California were colonized by AMF.but no
AMF spores could be recovered from soil. Similarly. Pond et al. (1984) found that
altbough tomato plants in a saluiized soil were colonized by AMF, no AMF spores could
be recovered from soil. These fmdings suggest that there rnay fiot be a definite
relationship between AMF sporulation and root colonization. and that the AMF rnay be
non-sporulating for the @ven combination of host soil and environmental conditions.
The lack of AMF sporulation is seen as a result of changes in soil nument levels and host
species (Brundett, 1991, Douds. 1994; Mente et d. 1978b), and is dependent on the
interaction of the AMF species with plants and edaphic factors (Pearson and Schweiger,
1993). The low Ievel of AMF sporuiation. but high AMF colonization levels in my study
suggest that some members of lentil and pea endorhizosphere cornmunity rnay have
become non-sporulating fun@ in association with sorghum-sudangrass in the growth
chamber.
The presence of AMF within the trap host roots was also veritïed using 16: 1 ~ 5 c .
a FAME biomarker for AMF species (Grzham et al. 1995: Olsson et al. 1995: Bentivenga
and morto on. 1996; Olsson et al. 1997). In my study, the presence of 16: 1 o5c was
observed in the roots of al1 the ST cultures. Similar FAME data interpretations were used
by Olsson et aI- (1995) to estimate AMF biomass in SOLThe significant positive
correlation between the adjusted response ratio of 16:l o5c/12:0 and percentage of AMFcolonized root in the 2ORT culture samples suggested that this FAME biomarker may be
used as a supplement or substitute for the microscopical determination of AMF
colonization. Alternatively, this FAME biomarker may be used for a preliminary
determination of AMF presence in roon. Furthermore. the presence of AMF as
determined by the presence of the FAME biomarker in eight of twelve of the 2"RT
cultures, but only four of twelve of the 2"RT cultures as determined using microscopy
indicates that the FAME biornarker determination may be more sensitive than microscopy-
This study shows that although various hctors influence AA4.F diversity. a key
factor is the host plant species. The lentil and pea hosts preferentially selected M F to
coIonize their roots which resulted in different AMF communities in the rhizosphere and
endorhizosphere. Furthermore. this study pmvided evidence to suggest that depending
upon the combination of host. soi1 and environmental conditions some AMF may not
sporulate. but that the lack of sporulation did not imply the absence of AME This
condition leads to the extensive coIonization of roots but very little production of AMF
spores. The high correlation between AMF root colonization in the 2ORT cultures and
presence of the FAME biomarker in maize mots suggests rhat this FAME biomarker may
be used for the determination of AMF presence within plant roots.
4 0
ISOLATION AND IDENTIFICATION OF RHIZOBIA AND
EVALUATION OF THEIR EFFICACY ON PEA AND L E N T E
4 1
Introduction
Leguminous hosts form a symbiosis with Rhizobium species that rnay enhance plant
productivity and N nutrition. Kowever. the symbiotic effectiveness (i-e.. effects on
shoot dry weight and N content of plant hosts) of different Rhizobiuin strains c m v q
under the same growth conditions. Thus. depending on the zfficacy of the Rhizobium
strain, the host rnay or may not benefit Iiom the association. Furthemore, the
effectiveness of a Rhizobium strain can be modified or limited by host specificity
(Trinick. 1980). and external factors such as temperature (Gibson. 1967: Hely. 1964),
pH (Howieson et al. 1988; Richardson and Simpson. 1989). rnoisture (ttreisz et al. 2985:
Zahran and Sprent. 1986), salinity (Sprent. 1972: Serraj et al. 1994). weed competition
(Martin et al. 1992: Rerkasem et al. 19881, soil-N, -P or micronutrient levek (Munns.
1977: O'Hara et al. 1988), and strain competition (Thies et al. 1991; George and Robert.
1992; Robleto et al. 1998). In addition, strain effectiveness can be affected by the ability
of the straîns to produce siderophores (Barton et al. 1996; Berraho et al. 1997). or to
effectively recycle hydrogen during the nitrogen fixation process (El-Hassan et al. 1986:
Zargar and Kahlon, 1995: Maier and Tnplett. 1996).
Rhizobia are an important component of the tripartite Rhizobium-AMF-le,wme
symbiosîs. The N contribution by rhizobia can significantly influence the effectiveness of
the tripartite symbiosis (Hoflich et al. 1995; Ahmad. 1995). Therefore. it is important to
evaluate the efficacy of the Rhizobium strain on appropriate host plants pnor to coinoculation of the host with AMF and rhizobia. This study (i) isolated rliizobia from the
root nodules of lentil and pea grown at various Saskatchewan field sites. and (ii)
evaluated the efficacy of these Rhizobirrm strains relative to commercial inoculant and
reference culture collection strains on the growth and N content of pea and lentil under
=otobiotic conditions.
4.2
Materials and Methods
4 . 2 . 1 Isolation, purification and maintenance of Rhizobium cultures
Pea and lennl pl-
were collected frorn field sites at Moose Jaw. Rosetown.
Drinhwater. AUan and Bellevue. Saskatchewan. Details on plant sample collection and
processing are given in section 3-2.1. The nutrient content and selected soil
characteristics of the field sites were dewrmined at the Enviro-Test Laboratories,
Saskatoon, Saskatchewan (Table 3-22).
Isolation of nodule bacteria from pea and lentil roots was according to the
procedure of Vincent (1970). Pea and lentil roots were washed thoroughly with distilled
water and dried with paper towek. Ten nodules h m plants collected at each field site
were selected at random, separated from the roots and placed in a stenlized ghss Petri
dish. Nodules were surface-sterilized by immersion in 95% ethano1 for 10 sec, then
soaked in a O. 1% mercuric chloride solution for four minutes and washed thoroughly in
severd changes of s t e d e distilled water. The surface-sterilized nodules were transferred
to a clean. sterilized g l a s Petri dish and crushed in three mifiliters of sterile distilled tap
water with a flame-sterilized glass rod. One rnilliliter of the solution was pipztted ont0
triplicate yeast extract mannitol (YEM) agar plates containing Congo Red and incubated at
27OC for up to 72 h. Ten isolated, large. white, opaque colonies were selected and
restreaked for purity. A total of 120 isolates ( L X 1 to LX120) were ob tained from the 12
I7eld sites (Table 4.2.1). However, the field isolates LX3. LX4 LX23, LX24, LX95
LX96, LX109 and LX1 10 did not grow after the initial isolation. The remaining 112
Table 4.2.1. Bacterial isolates obrained h m mot nodules of pea and lentil grown ar
various field sites in Saskatchewan during 1995.
IsoIate no.
Field site locations
Field n o 3
Crop
LX1 - LX10
Bellevue
9
Pea
LX11 - LX20
Bellevue
12
Pea
LX21 - LX30
Man
7
Lentil
LX3 1 - LX40
Alkan
8
Pea
LX41 - LX50
Bellevue
II
Pea
LX51 - LX60
Bellevue
10
Pea
LX61 - LX70
Drinlcwater
5
Lentil
LX71 - LX80
Drinkwater
6
Lentil
LXSZ- L X ~ O
Rosetom
4
Lentil
LX91 - LX100
Rosetown
3
Lenti!
LX101 - LX1 10
Moose Jaw
2
Lentil
LX1 10 - LX120
Moose Jaw
1
Lentil
aAdditional details on the previous (1994) crop and relevant soil chariicteristics of the
field sites are given in Tables 3.2.1 and 3.2.2.
isolates were maintained on YEM agar slants at 4OC and in a sterîle 5050% (v/v) YEM
broth:glycerol mixture at -80°C.
4 . 2 - 2 GommerciaI inoculant and reference Rhizobium strains
Strain PB IO1 (for pea and lentil) was obtained from Philom Bios. Inc. Saskatoon, and
strains RGP2 (for pea) and RGLI4 (for lentil) were obtained from MicroBio Rhizogen
Corporation. Saskatoon. Dr. L. M. Nelson (Plant Biotechnology Institute. Saskatoon)
provided the reference strains for pea and lentil. Strain Cl was origindly obtained from
Agrkultural Laboratories Inc. Columbus, Ohio. Strain Star B4 was isolated from the
noduIes of field-grown pea at Star City. Sask. Strains 1-ICAR-LE13 and I-ICAR-SYR-
LE20 (hereafter, referred to as LE13 and LE20, respectively) were originally obtained
fiom the International Centre for And Research and Development of Agriculture
(ICARDA). Syrîa. whereas the saain 1-ICAR-SYR-LE16 (hereafter. referred to as LE16)
was originally obtained from ICARDA, Morocco. AU the other reference strains (Le..
128C52, 128C54, 128C56, 128A12, 175F15, 175P4, 92AL92B1,99A2. 175PZ and
175M1) were obtained from Nitrakgin Co., Liphatech Corporation. Milwaukee,
Wisconsin.
4.2.3 Evaluation of rhizobia on pea and lentil
The symbiotic effectiveness of commercial Rhizobitcm inoculants. reference strains and
field isolates was evaluated on pea or lentil. Pre-washed and autoclaved vermiculite (CU.
75 g) was placed in the top portion of a Leonard jar c e o n a d . 1943). The nutrient
reservoir (bottom portion of the Leonard jar) was filled with 750 ml of N-free Fihraeus
solution (Fahraeus. 1957). The whole Leonard jar assembly was autoclaved at 121°C for
one hour. Pea (cv. Trapper) and lentil (cv. Laird) seeds were immersed in 70% ethanol
for three minutes. then soaked in a 1.5%sodium hypochlorite solution for four minutes
and rinsed thoroughly with sterile disciUed water. The surface-sterilized seeds were
placed on 0.3% w t i c soy agar (TSA) and held at 24'C for up to seven days in the diirk
to allow for germination. Three aniformly germinated pea or lentil seeds were placed at
3-cmdepth in the vermiculite and inoculated widi 3.0 ml (1 .O milseed) of a Rhkobium
culture containing 108 colony forming unitdm1 (duml-1). The rhizobia cultures were
prepared by inoculating a loop of cells fiom a stock culture into 100 ml of YEM broth and
growiog on a a r o t o r y shaker (150 rev min-1) at 28'C for 72 h. The Leonard jars were
placed in a growth chamber with the followins conditions: 25OC 16 h day and 20°C 8 h
night with ca. 60% relative humidity and 350-400 ph4 m - sec-' of iradiance. An
uninoculated control was also maintained. After three weeks of growth. 400 ml of 1/4
strengtb sterilized (12 1OC; 1 h) Fahraeus solution were added to each nutrient reservoir.
The Leonard jars were randomized in the growth chamber and repositioned weekIy.
4.2.4 Parameters
Plants were grown for six weeks, harvested, the shoots dried (65OC: 48 h) and
weighed. The roots were removed. washed with distilled water and dned using paper
towels. The number of nodules on the main and laterd roots was counted, and
nodulation was categorized as follows: low. 0-20: medium. 21-40; and high. >40
nodules. The N contelit of the shoot tissues was determined using a CNS-2000
Elernental Analyzer (LEC0 Corporation, Mississauga. ON).
4.2.5 Statistics
AU data except nodulation results were analyzed using the ANOVA procedure and the
means separated using the LSD test in SAS (SAS, 1997). Unless indicated otherwise. all
means were tested for significant differences at p<0.05.
4.3
ResuIts
The control plants and those inoculated with ineffective rhizobia appeared chlorotic.
whereas plants inoculated with effective rhizobia were healthy and green. Regardless of
the Rhizobium snain used. pea plants produced niore shoot biornass than lentil plants.
Ali of the commercial inoculants and rekrence Rhizobium strains nodulated pea and
lentil. Forty of 48 isolates from pea root nodules nodulated pea. and 4 4 of 64 isolates
from lentil root nodules nodulated lentil. Less developed nodules appeared smali arid
white, whereas mature nodules appeared pink to reddish brown.
4 . 3 . 1 Effect of rhizobia on pea
There were ~i~onificant
differences in the symbiotic effectiveness betwezn the pea rhizobia
(Table 4.3.1). Inoculation of pea with the Rhizobium strains RGP2 and PB 1O 1 resulted
in statistically similar levels of shoot dry matter, N content and nodulation: although
RGP2-inoculated plants yielded ca. 1 7 2 more shoot dry matter than PB IO 1-inocdated
plants (Fig. 4.3.1). The reference Rhizobium saains had difTerent effects on shoot
growth. For example, strains 128A12.128C.52, 128C54. 128C56 and C l were
significantly more effective at e n h a n c e the shoot dry matter of pea compared to stmins
175F15 and 175P4 (Table 4.3.1). This growth enhancement was positively associated
wiih an increase in shoot N content, but not root nodulation. For example. although
straùis IZ8C52. 128C54 and 128C56 bad statistically similar levels of shoot biomass and
N content, 128C54 induced more nodules than 128C52 or 128C56 (Table 4-3.1).
The field isolates varied in their ability to influence the shoot dry weight of pea.
For example. pea inoculated with the best Rhizobium isolates LX1, LX43, LX48 and
LX57 yielded at least 188% more shoot dry matter than that of the uninoculated control
plants (Fig. 4.3.2: Table 4.3.1). On the other hand, the shoot dry weight of plants
inoculated with the least effective Rhizobium isolates LX13.LX40 and LX50 was not
Table 4.3.1. Mean (n=3) shoot dry weight. percentage and total N content and
nodulation of pea (cv. Trapper) inoculated with commercial inoculants. reference
Rhizobîzrm strains. or field isolates (LX) and grown for six weeks in a Leonard jar.
LSD- Least Sicpifkant Difference
Rhizobium strain,
Shoot dry
Shoot N
Nodulation
isolate
weight (.@pot)
%
(rng/pot)
Control
0.9 8
1.3 1
12.7
None
RGP2
2.23
2.58
57 -2
High
PB 101
1.91
2.90
55.8
High
Star B4
2.3 3
2.93
67.9
High
128A12
2.70
2.94
79.1
Mediun
128C52
2.77
3.00
81.3
Medium
228C54
2.67
3.27
87.0
High
128C56
3.57
3 -28
84.5
Medium
175F15
1.92
2.14
41 -3
Medium
175P4
1.12
1.38
15.4
Medium
Ci
2.53
2.55
64.6
High
LX1
2.8 9
2.88
83.0
Medium
LX5
2.5 1
3 .O4
76.2
High
LX6
2.52
3 .O7
77.4
High
LX7
2.33
2.84
64.6
Medium
LX8
2.39
3 -37
80.8
Medium
LX9
2.32
2.84
66.0
High
LX1 I
2.60
2.85
73.9
Medium
LX13
1.O0
1.38
13.8
Medium
LX15
2.5 1
2.86
7 1.7
High
LX16
2.03
3.30
65.3
Medium
Table 4.3.1 (continued)
Rhizobium strainf
Shoot dry
Shoot N
isolat.
weight @/pot)
G/C
LX17
2.28
2.76
63 -2
High
LX18
2 -45
3 -24
78 -9
High
LX19
3.08
2.96
61.3
High
LX20
3-52
2.89
72.8
High
LX3 1
2 -50
2.85
70.5
Medium
LX32
2 -29
2.80
64.3
High
LX35
2 -44
2.74
66-4
High
LX36
2 -25
2.66
60- 1
High
LX37
2 -40
2.85
68.1
High
LX38
2.5 1
3.59
65.0
Kgh
LX39
2.67
2.76
73 -7
Medium
LX40
O -94
1.23
11.6
Low
LX41
3.3 8
2.65
62.0
Low
LX42
2.75
2.73
73 -9
High
LX43
3.85
2.96
83.6
High
LX45
2.43
3.13
75.9
Medium
LX46
2.7 1
2-85
76.7
Medium
LX47
2.23
2.94
65.4
Medium
LX48
3.O3
3.O2
89 -7
Medium
LX49
2.49
2.87
71-1
High
LX50
1-10
1.40
15.2
Medium
LX5 l
1.75
3.13
55 -4
Low
LX53
2.30
3.10
7 1.2
High
Nodulation
(rngpot)
Table 4.3-1 (continued)
Rhkobiccm straid
Shoot dry
Shoot N
isolate
weight(g/pot)
%
LX54
2.16
2.86
61 -3
Medium
LX55
2-29
2.87
65 -4
Medium
LX56
2.52
3-02
742
High
LX57
2-82
2.95
82.9
High
LX58
2.42
2.89
69 -8
High
LX59
2-35
2.93
68 -3
High
LX60
2.68
2.89
77 .O
Meàium
LSD (0.05)
O -60
0.46
15.9
-
Nodulaaon
(mt/pot>
Fig . 4.3.1.
Pea (cv. Trapper) plants inoculated with ca. 108 cf%ml-1 of the
commercial inoculants RGP2 or PB 101 (2449) and grown for six weeks
in a Leonard jar. Conaol refers to the uninoculated pea plants.
Fig. 4.
Pea ('cv.Trapper) plants inoculated with Ca. 108 cfu ml-1 of the
field isolates LXI, LX43 and LX48 and grown for six weeks in a
Leoniard jar. Control refers to the uninoculated pea plants.
s i ~ c a n t l different
y
from that of the control plants. The symbiotic effectiveness of the
most and least effective isolates was noi related to their ability to nodulate host roots. For
example, the nodulatïon of pea by the Rhizobium isolate LX13 was similar to that of the
isolate LX1 and LX48. However. plants inoculated with LX1 and LX48 yielded at l e a s
15 9 8 more shoot biomass and ca. 500% more shoot N than LX13 (Table 4.3.1). Of d l
the pea rhizobia evduated in this study. the straïns 128C52. 128C54. 128A12, LXL
U 4 3 , LX48 and LX57 were superior to all other rhizobia in terms of their ability to
enhance pea growth and N nutrition. whereas strains PB 101. Z75P4. 175F15. LX13.
LX40, LX50. LX5 1 were the least effective.
4.3.2 Effect of rhizobia on lentil
The commercial inoculants RGL14 and PB 101 significantly increased the shoot dry
matter of lentil by ca. 157% and N content by at l e m 298% over the uninoculated control
(Table 4.3.2; Fig. 4.3.3). The effect of both commercial inoculants on lentil shoot dry
matter production and N content was statisticdy sirnilar. However. the number of
nodules induced by these inoculants was considerably different. The efficacy of the
reference mains on the lentil host varied siLigd5cantly. For example. plants inoculated
with strains 175M1 and 99A2 yielded at least 379% more shoot dry rnatter than the
uninoculated control and 3 19%more than the least effective strain LEZ3 (Table 4.3.2).
These increases in dry matter were associatcd with an increase in the shoot N content but
not nodulation.
The field isolates also varied in their ability to influence shoot dry weight and N
content. For example, lentil plants inoculated with the Rhizobirirn isolates LX72. LX77.
LX8 1 and LX84 yielded at least 42 1% more shoot dry matter than the control plants (Fig.
4.3.2; Table 4.3.2). and 306% more than the least effective field isolate LX61. On the
other hand. the growth and N content of lentiI ininoculared widi some ineffective isolates
Table 43.2. Mean (n=3) shoot dry weight. percentage and total N content. and
nodulation of lentil (cv. Laird) inoculated with commercial inoculants. rekrence
Rhizobium strains. or field isolates (LX) and grown for six weeks in a Leonard jar.
LSD- Least Simificant Difference
Rhizobium straid
Shoot dry
Shoot N
isolate
weight (@pot)
5%
Nodulabon
(mgfpot)
None
Low
Mediun
Low
Low
High
Medium
High
Mediurn
Medium
Mvdiurn
Medium
Medium
Medium
High
Medium
High
Medium
High
High
Medium
Table 4-32 (continued)
Rhaobnm strain/
Shoot dry
Shoot N
isolate
weight (@pot)
t/c
Noduiation
(mgipot)
Medium
High
Medium
Hi&
Medium
Medium
High
Medium
Medium
Medium
Medium
Medium
Medium
Medium
High
Medium
Medium
Mediurn
Medium
Low
Medium
Medium
Table 4.3.2 (continued)
Rhizobiumstrainf
Shootdry
Shoot N
isoiate
weight (g/pot)
Cï%
(mgpot)
LX92
0.29
2.55
7.40
LX94
0.5 1
2.57
13.05
LX98
0.3 1
2.47
7 -59
Hig h
LX101
0.3 8
2.62
9 -96
Medium
LX1 13
0.6 1
2.55
15.40
Medium
LX1 14
0 -5 6
2.72
15.14
Medium
LX1 15
0.58
2-58
14.98
High
LX1 16
0.69
2.46
16.99
Medium
LX1 18
0.59
2.24
13.39
High
LX1 19
0.57
2.46
14-10
figh
Nodulation
High
Medium
6
Hi h
LX120
0.68
LSD (0.05)
0.09
0.43
3.01
-
Fig.
Lentil (cv. Laird) plants inoculated widi Ca. 108 c h ml-1 of (A) the
commercial inoculants RGL14 or PB101 (2449) and (B) the rhizobia
isolates LX72, LX77 or LX84 and grown for six weeks in a Leonard jar,
Control refers to the uninoculated lenhl plants.
was not different from that of the unuioculated control. For example. the shoot dry
weight and
N content of controi plants and lentil inoculated with the straïns LE 13.91A1.
92B 1. LX6 1. LX62 and LX66 were not significanrly diftérent (Table 4-32). The
effkctiveness of these strains did not appear to be associated with root nodulation. For
exarnple. die nodulation of lentil by the Rhizobium isolate LX66 w u similar to or higher
than that of the isolates LX81 and LX84- However. plants inoculated with LX8 1 and
LX84 yielded at least 320% more shoot biomass and CU. 423% more shoot N than LX66-
In terms of their ability to enhance the shoot dry weight and shoot N content, the rhizobial
l other lentil
strains 175M1199A2. LX72, LX77.LX8 1 and LX84 were supenor to d
rhizobia. whereas straùis 92Al. 92B 1, LE13. LX61 and LX66 were the least efiective
(Table 4.32). The percentage increase in the shoot dry weight a n d o r shoot N content
between the different lentil Rhizobium field isolates was much greater than that of pea
Rhizobirirn field isolates.
4.4
Discussion
Inoculation of pea and lenti! with the commercial inoculants and rnany of the reference
strains siLonificantlyincreased the shoot dry weight and N nutrition of plants compared to
the control. The symbiotic effcctiveness of the field isolates on pea and lentil varied
significantly. Many of the rhizobial field isolates were effective on pea and lentil and
enhanced growth and N nutrition. However. the growth and N nutrition of plants
inoculated with some ineffective rhizobial field isolates were similar to those of the
control plants. As hypothesized by Quigley et al. (1997). the differences in the
effectiveness of the various strains used in the present snidy rnay be due to genetic
differences in the rhizobia. These genetic differences may be a consequence of
interactions between the rhizobia and factors such as soi1 nutrient levels and srrain
cornpetition at the site from which the rhizobia were originally isolated.
Nodulation (Le.. number of nodules. nodule dry weight) is an important
panmeter considered by workers in the evaluation of a Rhizobium strain on host plants.
The increases in N content and plant growth following inoculation of a legume with a
R h i d i u m strain c m be positive!^ correlated to (Franco et al. 1973: Tang. 1979) or not
related to (Zaroug and Munns- 1980: Anand and Dogra. 1997) the nurnber of nodules
formed. In my study. the stimulation of plant growth or increase in the shoot N content
of pea and lentil plants by the most effective rhizobia was apparently not related to
increases in root nodulation. These results agree with the observations made by de
Almeida et al. (1973) for beans, Zaroug and Munns (1980) for Cliroria tematea and Vigna
trilobuta and Anand and D o s a (1997) for Cujanus cajan plants.
A cornparison of the effectiveness of the Rhizobium field isolates to commercial
inoculants used in this study on pea nutrition and growth reveded that some of the field
isolates were superior to the commercial inoculants. For example. isolates LXl. LX43.
LX48 and LX57 were superior to the commercial inoculants RGP2 or PB 101. These
Rhizobium isolates (Le,, LXI, LX43. LX48 and LX57) were obtained from different
field sites at Bellevue (Table 4.2.Q which were not inoculated with rhizobia. In
e v a l u a ~ gthe effectiveness of indigenous Rhizobium isolates to isolates from other
geographical locales (i.e.. other Afncan counaies) on TrifDlium spp.. Friedericks et al.
(1990) and Lupwayi et al. (1997) found that Ethiopian Rhizobium isolates outperfomed
rhizobia isolated from other African countries. These observations reflect the cornpetitive
ability and effectiveness of the indigenous rhizobia on plant productivity.
The Rhizobium field isolates obtained from the same field site varied significantly
in their effectiveness on the host For example. the nine Rhizobium isolates, LX7 1 to
LX80 obtained from the mot nodules of lent3 at the Drinkwater site (field no. 6 in Table
4.2.1) varied significantly in their effectiveness on lentil plants grown i n h o n a r d jars.
Weaver and Wnght (1987) assessed the variability in the effectiveness of bradyrhizobia
n o d u h ~ siratro
g
(Macroprilium an-opiopro-eirin)in cone-tainers containing sterilized
vemiculite. They found that isolates from different nodules of siratro forrned by the
same Brudyrhizobium straïn varied in their effectivenzss on the host plant. Batzli et al.
(1992) reported that the variabifity in the symbiotic effectiveness amon2 bliick locust
rhizobia was high even among Rhizobium isolates obtained from one tree. Similar results
were reported for Leucaena by Swelim et al. (1997). These differences may be explained
by genetic variations in hydrogen recychg (Maier and Triple t t 1996). suMval strategies
such as antibiotic production (Robleto et al. 1998). or variations in generation time of the
isolates (Turco et al. 1986; Weaver and Wnght. 1987).
The effect of the rhizobial microsymbiont on the tripartite AMF-Rhizobium-
legume symbiosis is mostly N-mediated (Azcon-Aguilar and Barea. 1992). Niuogen
fixed by the rhizobia is utilized by the legume host towards maintainuig the ove& health
of the plant which supports both microsymbionrs. The aim of this study was to evaluate
the response of pea and lentil to commercial Rhizobium inoculants. reference strains and
field isolates under gnotobiotic conditions, and select for effective and ineffective rhizobia
strains for CO-inoculationwith AMI? species. Based on their effectiveness on pea or lentil
N nutrition and plant growth. the rhizobia were ranked, and effective snains (e-g..
RGP2. PB I O 1, LX1, LX43,LX48 and LX57 for pea and RGL14. PB I O 1. LX72.
LX77, LX8 1 and LX84 for lentil) and ineffective strains (e.g.. LX13, LX36 and LX5 1
for pea and 92A1. LE13 and LX66 for lenhl) were selected for CO-inoculationstudies
(sections 5.0, 6.0 and 7.0). It remains to be seen whether these Rhizobium strains will
be cornpetitive against native rhizobia in non-sterile soils.
EFFECTS OF INTERACTIONS BETWEEN TWO GLOMb'S
SPECkES ANP) RHIZOBIUM LEGUMINOSAR U M BV. VICEAE
ON THE GROWTH, YIELD AND NUTRITION OF PEA AND
LENTIL IN NON-STERKLE SOIL CONTAl[riUTXNGINDIGENOUS
A m AND RPEIZOBIA
Introduction
Among the various rhizosphere microorganisms which influence l e p m e growth ar.d
productivity, AMI? and Rhizobium spp. are particularly important because of their
inrimate associations with legurnes. The tripartite association between AMF. rhizobia and
legurnes is also significant because of the drain on host carbon by the AMF and rhizobia
for all or paxt of their carbon requirements. Up to 47% of the carbon f ï e d by dual
symbiotic (i.e., rnycorrhizal and nodulated) plants c m be transferred to the roots for
supporthg the growth of microsymbionts (Pang and Paul, 1980: Kucey and Psul, 1982;
Harris et al. 1985). Although the amount of carbon transferred to dual symbiotic host
roots may Vary depending on the plant, Rhizobium and AMF species. these results
underscore the importance of host carbon utilization by the AiW and rhizobia in dual
symbiotic systems.
Various workers have assessed the influence of many AMF species on the g o w t h
of nodulated l e e u e s (Ianson and Lindeman, 1993; Ibijbijen et al. 1996; Saxena et al.
1997). For example. Ianson and Linderman (1993) suggested that a specfic interaction
occurs between AMF and the Rhizobium strain which influences nodulation and AMF
colonization of roots, but not host P nutrition. Saxena et al. (1997) reported that the
nodulation and growth of Vigna radiata inoculated with a Bradyrhizobium sp. varied
~ i ~ c a n tdepending
ly
upon the CO-inoculatedAMF species. Several workers have
examined the interactions between different AMF species and Rhizobium strains (Arnes et
al. 1991; Azcon et al. 1991; Ruiz-Lozano and Azcon. 1993, 1994: Ahmad, 1995:
Redecker et al. 1997). The b a i s for selecting these Rhkobiunz strains was strain
availability (Azcon et al. 199 1). effectiveness on host (Ames et al. 1991). or not specified
(Ruiz-Lozano and Azcon. 1993. 1994: Ahmad, 1995: Redecker et al. 1997). In a l l cases.
growth and productivîty of the lepmes were dependent on the specific combination of
AMF and rhizobia, indicating that synergistic interactions between compatible
microsyrn bionts resulted in g o wth and yieId increases.
Field-grown l e p m e s are invariably inoculated with effective Rhizobium strains to
enhance nutrition and productivity. However, it is clear from previous reports that the
response of a nodulated host can be modified by the AlMF species involved in the
tripartite association. It is not clear whether an effective Rhizobium straui in a d u d
symbiotic system is rendered less effective on the host because of an incompatible AMI?
species. Similarly, it is not clear whether an ineffective Rhizobium strain can be made
effective, because of the activiry of a compatible AMF species. In addition, the
importance of the rhizobia in the AMF-legume symbiosis has not been addressed in
detail. The aim of this study was to evaluate the effects of interactions between the AMF
species Glomus clarurn or Glomus mosseae and Rhizobium legtcminosarunz bv. viceae
strains varying in efficacy on pea and lentil, on the growth. yield and nutrient content of
pea and lentil grown in soi1 containhg indigenous AMF and rhizobia
5.2
Materials and rnethods
5 . 2 . 1 Effect of AMF-rhizobia interactions on pea
5.2.1.1
Treatments set-up for pea
The following treamients were set-up for pea: uninoculated control. the AMF species
Glarnus clarum NT4 and G. nzosseae NT6. Rhizobium legrrminosalzrrn bv. viceue
strains RGP2, PB 101 and 17SP4,Rhizobilrm isolates LX 1, LX13. LX36. LX43.
LX48. LX51 and W 7 , and combinations of the AMF species and the rhizobia. A total
of 33 factorid treatment combinations (3 AMF x 11 rhizobiaj each replicated four tirnes.
were assessed on pea.
5.2.1.2
AMF inocula
The AMF species G. clamm NT4 (INVAM no. SAlO1) and G. mosseae NT6 (INVAM
no. SA103) are two dominant AMF species found in Saskatchewan field soils (Talukdar
and Germida, 19933, and enhanced the g o w t h and yield of wheat and lentil in a sterile
soi1:sand mix (Tdukdar and Germida. 1994). Monospecific C
U ~ Wof
~ both
S
AMF
species were produced in a (1: 1) soil:sand mut using rnaize (Zeam y s L. cv. Early
Golden Bantam) as host for 90 d. The AMF inocula consisting of spores. externd
mycelium and AMF-colonized roots were stored at 4OC. The G. clanrm NT4 inoculurn
contained 330 propagules per 50 g, whereas the G. mosseae NT6 inoculum contained
390 propagules per 50 g, as determined using the "Most probable number" assay of
Porter (1979).
5.2.1.3
Rhizobium leguminosarum bv. viceae strains
The Rhizobium strains and isolates used in diis study are listed in Table 5.2.1. The
commercial inoculants RGP2 and PB 101 are widely used in Saskatchewan, and were
therefore included. The Rhizobium isolates ml, LX43, LX48 and LX57 enhanced the
growth and N content of six week-old pea under potobiotic conditions, whereas isolates
Table 5.2.1.
Sources of the R. le.grmzinosa>-~~m
bv. viceae suains and isolates used.
and their ef'fectiveness on pea-
Straidisolate
EfYectivea
RGPî (Commercial inoculant)
Yes
Source
MicroBio RhizoGen Corporation,
Saskatoor,, SK
PB 101 (Commercial inoculant)
Yes
Philom Bios Inc. Saskatoon, SK
NG
Nitrak& Inoculants, Liphatech
175P4
Corporation. Milwaukee. WI
AU LX isolates were obtained from
LX1, LX43, LX48. LX57
Yes
LX13, U 3 6 , LX51
hTo
the nodules of pea plants collected
from uninoculated fields at
Bellevue, Saskatchewan.
aEffectiveness was determined based on the ability of the Rhizobium strains to enhance
pea (cv. Trapper) growth and N content after 6 weeks of growth. Germinated pea seeds
were inoculated with CO. 108 colony forming units of rhizobiafml and grown in Leonard
jars containing a N-free nutrient solution. For additional details see section 4.0.
LX13, LX36. LX5 1 and strain 175P4 were ineffective on p e a The Rhizobi~mzcultures
were prepared by inoculaMg a loopful of ceils from a stock culture into 100 ml of YEM
broth and growing on a gyrotory shaker (150 rev min-1) at 2S°C for 72 h. The
Rhizobium cultures contained ca. 108 colony forming unit. per ml (cfü ml-').
5.2.1.4
Soi1
The loamy sand soil used in this study was collected from Bradwell. Saskatchewan. The
soil was air-dried, passed through a 4-mm sieve, and the nutrient content determined at
the Envïro-Test Laboratories, Saskatoon. Saskatchewan. The nutrient content of the soil
was as follows (pg gg-l): N, 10; P, 13: K. 300: S, 9.4: B. 0.72: Cu, 0.56; Fe. 11.2: Mn.
9.6; Zn. 3.18. The pH of the soi1 was 8.1. the electrical conductivity. 0.2 rnS/crn.
organic carbon, 1.1 %, and the organic matter, 1.9%. The soil was nUxed 1:1 ( w h )with
silica sand and two kilograms potted in 15-cm dia. pots. Two hundred milliliters of
distiUed water were added to each pot and the soil:sand mixture was thoroughly mixed.
The soil:sand mix was allowed to equilibrate for 10 d before planting. The number of
indigenous rhizobia and AMF propagules in the soi1:sand mix were 5.5 per g and 170 per
50 g respectively. as determined using the n o s t probable number technique of
Somasegaran and Hoben (1994) for rhizobia and Porter (1979) for AMF.
5.2-1.5
Inoculation and plant growth
For aU the eeatments, pea seeds (cv. Trapper) were planted at 5-cm depth from the soil
surface, For the Rhizobium treatments, 2.0 ml of the Rhizobium culture were added over
four pea seeds. For the AMF treatments. 10 g of the appropriate AMF inoculum (Le.,
NT4 or NT6) was placed at 5-cm depth from the soil surface over which four pea seeds
were placed. For the MdF+Rhizobium treahnents, 10 g of AMF inoculum were placed
at 5-cm d q t h from the soi1 surface over which four pea seeds were planted, and 2.0 ml
of the appropriate Rhizobium c u l ~ r were
e
added. M e r seedling emergence, plants were
h e d to two per pot. Autoclaved polypropylene beads were applied on the soi1 surface
to prevent cross contamination and excessive moisture loss. Plants were grown in a
growth charnber with the following conditions: X 0 C . 16 h day and 20aC. 8 h night: 375-
400 pm mm-2
sec-1 of irradiance and ca. 60% relative humidity. Soil was rnaintained at
-606 moisture holding capacity throughout the study by periodically adding water. Pots
were randomized in the growth chamber and repositioned once a week.
5.2.1.6
Parameters
Plants were grown for 90 days and harvested. The above-ground plant material was
separated. dried (6S°C; 48 h) and weighed. Grain was separated from the dried shoot and
weighed. The harvest index of plants (grain yield/total above-ground dry matter x 100)
was calculated according to Ahiabor and Hirata (1995), and is indicative of the
partitionhg of dry weight into seeds. The roots were arashed thoroughly in running tap
water and reserved for assessing nodulation and AMF colonization. The number of
nodules on the main and lateral roots was counted, and nodulation was categonzed as
follows: low, 0-20; medium. 21-40; and high, A 0 nodules. The whole root system was
dried (65°C; 48 h) and weighed. Oven-dried roots were rehydrated and cut into 1-cm
pieces for the determination of AMF colonization. A representative root sarnple (-50 mg)
was cleared in KOH, acidified with HCl, stained with trypan blue. destained in acidic
glycerol (Koske and Gernrna. 1989) and observed with a cornpound microscope (100 x).
The percentage of root length colonized by AMF (%AMFroot colonization) was
determined using the gridLine intersect method (Giovanetti and Mosse. 1980). Shoot and
grain were digested using a mixture of H2S04-H202and the N and P concentrations
determined (Thomas et al. 1967). The N and P content of the shoot and grain were
calculated by rnultiplying the shoot JX grain weight by the N or P concentration. The P
use efficiency was determined according to Raju et al. (1990) and expressed as gram
shoot or grain dry weight per gram P absorbed.
5.2.2 Effect of Am-rhizobia interactions on Ientil
The AMF inocula. rhizobia culture preparation. soil. inoculation procedures. plant growth
conditions. harvest and plant parmeters were identical to that of pea except for the
foUowing modifications: (1) the rhizobia used far i n o c u l a ~ glentil were different (Table
5.2.2) and (2) plants were harvested at 1 10 d after planting.
5.2.3 Statistics
Percentage values for AMF colonization were arcsine-transforrned before statistical
analysis. AU data except nodulation results were analyzed using the ANOVA procedure
and means separated using the least sipificant difference (LSD) test in SAS (SAS.
1997). The relative ranking (i.r.. low, medium and high) assigned to nodulation results
was converted into numerical data (i.e.. 1= low. 2= medium and 3= hi$)
for correlation
analysis (R. J. Baker, personal communication). CorreIation coefficients between
growth, yield, nutrition parameters, AbIF colonization and nodulation were calculated
using PC-SAS. Unless indicated otherwise, al1 ueatment means were tested for
significant differences at pc0.05.
5.3
Results
For al1 the parameters, statistical significance for the main effects (Le,, effect of the AMF
species irrespective of the rhizobia strains. and the effect of the rhizobia strains
irrespective of the AMF species) and interaction effects (i-e.. efTect of interactions
between AMF species and rhizobia strains), and mean values for the main effects are
presented in Appendix Tables A. 1- A.4 for pea and Appendix Tables AS- A.8 for lentil.
Table 5.2.2.
Sources of the R. leguminosarum bv. viceae s h n s and isolates used.
and their effectivensss on lentil.
Straidisolate
aEffective
Source
(yesho)
RGL 14 (Commercial inocuIant)
Yes
MicroBio RhizoGen Corporation.
Saskatoon. SK
Philom Bios Inc. Saskatoon. SK
Nitmgin Inoculants, Liphatech
Corporation, Milwaukee, WI
LE13
No
ICARDA, Syria
AU LX isolates were o b tained from
LX72, LX77, LX8 1. LX84
Yes
LX66
No
the nodules of lentil plants
collected fiom inoculated fields at
Moose Jaw. Rosetown, Drinkwater
or Allan. Saskatchewan.
aEffectiveness was determined based on the ability of the Rhizobium strains to enhance
lentil (cv. Laird) growth and N content after 6 weeks of growth. Germinated lentil
seeds wzre inoculated with ca. 108 colony forming units of rhizobidml and grown in
Leonard jars containing a N-free nutrient solution. For additional details see section
4.0.
PB 101 (Commercial inoculant)
92AI
Yes
No
5 . 3 . 1 Effect of AMF-rhizobia interactions on pea
5.3.1.1
Dry matter production
The shoot dry weight of plants was not afYected by G.clarum NT4 compared to the
control, and was significandy lower than that of G. mosseae NT6-inoculated plants.
irrespective of the rhizobial strain (Appendix Tables A S and A2). The effect of the
rhizobial snains on thz shoot growth of plants varied depending upon the saain.
irrespective of inoculation with AMF (Appendix Tables A. 1 and A-2). For exarnple. the
rhizobial strains 175P4 and LX48 were significantly less effective at stimulatùig shoot
growth compared to the best inoculants LX43 and LX57.
The efficacy of some Rhizobium saains under potobiotic conditions (Table
5.2.1) was not similar to that in soil. For example, the effective strain LX48 was not
effective on pea in soil, whereas the ineffective strain LX51 enhanced the shoot biomass
of plants relative to the control. Co-inoculation of pea with NT3 or NT6 resulted in very
different effects on the shoot growth of plants inoculated with the same Rhizobium strain
for five of 10 rhizobial strains (Table 5.3.1). For example. the shoot growth of pea
inoculated with the NT3+PBlOl combination was significantly greater than the
NT6+PB 101 combination. Similarly, the NT6+LX57 combination sitonificantly (by
30%) increased the shoot growth of pea relative to the NT4+LX57 combination.
Irrespective of the rhizobial strain used, the total root dry weight of plants
inoculated with NT4 or h i 6 was not significantly different from each other (Appendix
Tables AS and A.2). Most of the rhizobial inoculants except 175P4 significantly
increased the total dry weight of plants relative to the control, irrespective of the AMF
species (Appendix Tables A.l and A.2). Co-inoculation of pea with AMF and rhizobia
either significantly increased (NT4+LX51, NT6+LX5 1). decreaszd (e.g ., NT6+LX43,
NT6+LXl). or had no effect on the total root dry weight of pea inoculated with only the
Rhizobium strain (Table 5.3.1).
Plants inoculated with NT6 only yielded more grain than the controI or hT4inoculated plants. irrespective of the rhizobial saain (Appendix Tables A. 1 and A.2). The
effect of the NT4 inoculant on the grain yield of pea was inferior to the native AMF.
Inoculation of pea with the rhizobia sipificantly altered the yield of pea plants; and
regardless of the AMF species, most inoculants (7 of 10 rhizobia) increased grain yield
relative to the control (Appendix Tables A.1 and A.2). The AMF species had very
different effects on the yieield response of pea to inoculation with the same Rhizobium
strain (Table 5-32). For example, the NT4+ LX43 combination resulted in higher yields
than the NT6+LX43 combination (Table 5.3.2). This yield increase was Ca. 116%
higher than the control plana, ca. 34% higher than plants inoculated with LX43, and ca.
48% higher than the NT6tLX43 combination. Similarly. the NT6+l75P4 combination
produced a higher grain yield than the NT4+175P4 combination. However, the yield of
plants inoculated with NT4+LX43 was Ca. 1 12% higher than the NT6+175P4
combination, indicating that although AMI? inoculation incrcased the yield of an iderior
Rhizobium, the yield was higher for a superior Rhizobium.
There were no siguficant differences between the harvest index of uninoculated
plants and plants inoculated with NT6 or NT4 irrespective of the rhizobia (Appendix
Tables A. 1 and A.2). However, there were significant differences between the rhizobial
saains. irrespective of the AMF species used (Appendix Tables A. 1 and A.?). For
example. the harvest index of pea plants inoculated with L X 1 or WC43 was significantly
higher than that of plants inoculated with LX13, LX48 or 175P4. Co-inoculation with
the AMF species NT4 or NT6 caused sipificant differences in the harvest indices for
plants inoculated with the same Rhizobium strain (e-g., 175P4.LX43 and LX51) (Table
5.3.2). It appeared that the effectiveness of the Rhizobium strain had no impact on the
harvest index of the CO-inocuiatedplants. It was notewoïthy that the hT4+LX43
combination signi.fkanùy increased the harvest index by ca. 28% compared to the
NT6+LX43 combination and ca. 17% compared to LX43 only. The proportion of dry
matter allocated to &grainwas dependent on the specific combination of AMF and rhizobia
and not on the efficacy of either microsyrnbiont. as noted for other parameters.
5.3.1.2
Nutrient parameters
The shoot N content of plants inoculated with NT6 was not sizuficantly different from
that of the control or plants inoculated with NT4 (Appendix Tables A S and k3).On the
other hand. the rhizobial strains had a signifcant impact on the shoot N content of pea.
irrespective of the AMF species (Appendix Tables A. 1 and A.3). For exarnple. strains
LX1, LX43 and LX57 were more effective than strains 175P4 and LX48. The
effectiveness of the Rhizobium isolates on pea N nutrition under gnotobiotic conditions
was not similar to their effect on the shoot N content of pea in soi1. However. for the
same Rhizobium strain, inoculation with NT4 or NT6 significandy increased. decreased
or in most cases, had no effect on the shoot N content (Table 5.3.3). For example, the
NT6+LX57 cornbination significantly increased the shoot N content of pea by CU. 8 0 9
compared to the NT4+LX57 cornbination. A sieiificant positive correlation was noted
between the shoot dry weight and the shoot N content of pea (rd.93; p<O.OS). The P
content of pea shoots was not afTected by the AMF treatments or the AMF x rhizobia
interactions (Appendix Tables A.1 and A.3). However, there were significant differences
in the effect of the rhizobia straùls on the shoot P content of pea. when averaged over the
AMF species (Appendix Tables A. 1 and A.3). For example. plants inoculated with the
typically ineffective rhizobia strains 175P4 and LX13 had significantly higher levels of
shoot P than the other stsains.
Table 5.3.3. Mean (n=4) shoot N iind P content of pea plants inoçulated witti the AMF species Glori~iisclwrii~tNT4 or G. r~ios~secic
NT6
andlor 10 Rliizobiiini 1rgrci~iino.suriii11
bv. viceac strüins and growii for 90 d in soi1 cont;iiiiing indigenous AMI; and rliizobiii. Al1
treatment rneans witliin a parameter wese separated using the least significant diffesence (LSD) test at pc0.05.
Sliaot N content (mglpot)
Slioot P content (mglpot)
Rhizobiirnt
straiidisolate
AMF speçies
None
NT4
NT6
None
NT4
NT6
Control
32.74
29.73
42.1 O
4.29
3.92
4.72
RGP2
46.76
42.67
35.24
4.48
4.63
3.77
PB101
42.79
48.30
29.08
4.1 O
5.1 O
2.62
175P4
4 1.45
34.86
36,74
6.22
5,70
5.94
LX 1
46N
48.05
47.6 1
3.9 1
3.62
S.7O
LX13
46.0 1
4 1 ,O2
37.42
5.77
5.97
4-68
LX36
52,17
36.59
46.45
4.5 1
3.94
4.60
LX43
49.06
58.42
60.6 1
4,05
4.13
4,6S
LX48
31S 4
27 -95
4 1.50
4.08
4.33
5.37
LX5 1
50.59
42.85
39.93
4.13
3.63
3,63
LX57
48.28
37.62
67.57
4.69
3.90
5.89
LSD (0.05)
13.27
NS
There were no significant differences between the grain N content of control
plants or plants inoculnted with NT4 or NT6. irrespective of the rhizobial strain
(Appendix Tables A.1 and A.3). However. the rhizobial suauls varied in theïr ability to
enhance g a i n N content irrespective of the AMF species (Appendix Tables A. I and
A.3). For example. strains LX1 and LX43 siL&canùy
increased the grain N content of
pIants compared to the control plants or those inocdated with snains LX48 and 17354.
It appeared that the relative effectiveness of some Rhizobium st&s in soi1 was sirnilar to
.
their effect under gnotobiotic conditions ( e g . LX1 LX43)- There were no siemcant
differences in the grain N content of plants CO-inoculatedwith NT4 or NT6 and the same
Rhizobium strain, in seven of 10 cases (Fig. 5.3.1). However, CO-inoculationof pea
with NT4 and LX1 or LX43 significantly increased the grain N content by up to 4: %
over the respective rhizobia+NT6 combination (Fit. 5.3.1).
The grain P content of plants inoculated with NT4 wcts significantly lower than
those inoculated with NT6. irrespective of the rhizobial strain (Appendix Tables A. 1 and
A.3). The effect of the rhizobial strains on the g a i n P content of pea varied sigdicantly
irrespective of the AMF species (Appendix Tables A. 1 and A3). For example, pea
inoculated with the rhizobial strains LX1,LX43 and LX5 1 had ~ i ~ p i f i c m thigher
ly
levek
of grain P content than that of plants inoculated with LX48 or 175P4. As noted for g r a h
N content. there were no significant differences in the grain P content of plants co-
inoculated with NT4 or NT6 and the same Rhizobium s ~ & .in seven of 10 rhizobia
(Fig. 5.3.2). For example. there was no sign5cant difference between the grain P
content of pea inoculated with LX1 and NT4 or LX1 and N T 6 In contrast. coinoculation of pea with NT4+IX33 s i ~ c a n t l increased
y
the grain P content by 35%
over the NT6+LX43 combination. In addition, a significant positive correlation (r= 0.99:
pc0.05) was noted between the gain yield and grain P content.
The AMF inoculants h l 4 and NT6 did not affect the shoot PUE of plants relative
to the conuol. irrespective of the rhizobid treamients (Appendk Tables A. 1 and A.4).
Sunilarly, interactions between M and rhizobia had no effect on the shoot PUE of peü
(Table 5.3.4).
On the other hand. the rhizobial strains significantly influenced the shoot
PUE of pea. irrespective of the AMF species (Appendix Tables A. 1 and A.4). For
example. the shoot PUE of plants inoculated with an ineffective rhizobial strain such as
175P4 or LX13 was very low compared to other strains such a s LX43 and LX5 1. A
sirnilar non-significant trend with the main AMF effects and AMF x rhizo bia interaction
effects was observed for grain PUE of pea (Appendix Tables A. 1 and A.4; Table 5.3.4).
However. the rhizobial strains varied in their effects on the grain PUE of pea (Appendix
Tables A.l and A4). For example, plants inoculated with the srnains LXI. LX43 or
LX51 had significantly higher levels of grain PUE than those inoculated with strains
LX13 or LX48 .
5.3.1.3
AMI? coionization and noddation
Irrespective of the rhizobial strâin, inoculation of pea with NT4 significantly increased the
AMF colonization levels compared to NT6 (Appendix Tables A. 1 and A.4). Inoculation
of pea with some rhizobial strains enhanced (e.g., LX43) or restricted (e-g., RGPZ.
LX48) AMF colonization of roots relative to plants not inoculated with rhizobia.
irrespective of the AMF species (Appendix Tables A. 1 and A.4). The AMF inoculants
NT4 and NT6 had different effects on the rnycomhizal colonization of plants inoculated
with the same Rhizobium strain (Table 5.3.5). For example. NT4 increased the
mycorrhizal colonization of pea inoculated with RGP;! and LX43.but reduced that of
175P4. No measured plant parameter was correlated with the AMF colonization of pea
roots.
Table 5.3.4. Meün (n=4) slioot and gisin P use efficiençy of pea plants inoculated with the AMF species Gloi~iitsclurici~iNT4 o r G.
niosserre NT6 and/oi*10 Rltizobirrr~i.lcgirri~iriosctr*ii~~~.
bv. viceoe strltins and giawn for 90 d in soi1 coiitaining indigenoiis AMF and
rhizobia. Al1 treatment means witliin a paraineter were sepanted using the least signifiçmt difference (LSD) test at p<0.05.
P use efficiency (g d ~ yinatter g-l P absorbed)
Rh izubium
Shoot
straidisolate
None
NT4
NT6
Noiie
NT4
NT6
910
87 1
1173
325
316
329
Control
LSD (0.05)
NS
--
- --
Grairi
--
-
---
--
NS
W the plants were nodulated either by the uidigcnous rhizobia and/or the
introduced Rhizobirim strain. Apparently, NT6 stimulated noduhion of pea by
indigenou rhizobia more than NT4 (Table 5.3.5). In rnost cases. nodulation by the same
Rhkobizim strain was not altered by NT4 or NT6. However. in some cases. inoculation
of pea with NT4 or NT6 enhanced (e.g.. LX57 vs. NT4/NT6+LX57) or reduced (RGP2
vs. NT4jNT6tRGP2) nodulation by rhizobia. Nodulation was positively correlated with
the shoot (r=0.37: p<0.05) and root (r=0.47: pcO.05) dry weights. grain yield (-0.43:
p<0.05). and grain P content (r=0.44:pc0.05) of pea plants.
5 . 3 . 2 Effect of AMF-rhizobia interactions on lentil
5.3.2.1
Dry matter production
Statistical analyses indicated that irrespective of the rhizobial saain. there were no
si,pificant differences between NT4 or NT6 in their ability to enhance the shoot growth
of plants. but both AMF species were superior to the native AMF colonizing the control
plants (Appendix Tables A S and A.6). Plants inoculated with the different rhizobial
strains exhibited si,onificcant differences in shoot growth (Appendix Tables A S and A6).
For example, strains PB 101 and LX72 were more effective on the shoot growth of lennl
than strains LX66 and LE13. Furthemore, the A-MF NT4 and NT6 altered the shoot
gowth of plants inoculated with the same Rhizobium main (Table 5.3.6). For example.
the NT6+RGL14 combination si-dcantly increased the shoot dry weight of plants by
CU.
1598 over the NT4+RGL14 combination. Conversely. plants CO-inoculatedwith
NT4 and LX77 produced
Ca. 4 3 8
more shoot biomass than the NT6+LX77
combination.
Irrespective of the rhizobial sûain, the total root dry weight of plants inoculated
with NT4 was significantly higher than NT6 and control plants (Appendix Tables A S
and A6). Similarly, irrespective of the AMF species. the total mot dry weight varied
si,"dcantly between plants inoculated with the different rhizobia (Appendix Tables A S
and A.6). For example. lentil inoculated with the rhizobial s d n s LX72 LX77. LX8 1
or LX84 had a higher root biornass than that of control plants or plants inoculated with
strâins LE13 or 92A1. In six of nine rhizobia, CO-inoculationwith one AMF species
si@cantly
increased the total root dry weight of plants inoculated rvidi the same
Rhizobium strain, over the other AMF species. Co-inoculation of Ientil with AMF and
rhizobia inc~eased(eg.. NT4+LX72, NT6+LX77) or decreased (e-,o..Nï4+PB 101) the
total root dry weight of lentil cornparcd to that inoculated with only the Rhizobium strain
(Table 5.3.6).
Lentil inoculatcd with NT5 yielded s i ~ c a n t l less
y grain than the control and
NT4-inoculated plants. irrespective of the rhizobial strain (Appendix Tables A S and
A.6). Regardless of the AMF species, the yield response of plants ta the rhizobial strains
was significantly different (Appendix Tables A.5 and A.6). For example, inoculation of
lentil with RGLl4, LX77,LX72 or LX81 produced siEonificantlyhigher yields thm
92A1, LX66 or LX84 (Appendix Tables A 3 and A.6). The yield response of lentil to
NT4 or NT6 inoculation within the same Rhizobium treatment varied in six of nine
rhizobia (Table 5.3.7). For example, the grain yield of plants inoculated with the
NT4+LX77 combination was Ca. 156%higher than the NT6+LX77 cornbination, but
was not s i , ~ c a o t l ydifferent from that of plants inoculated with only LX77. The grain
yield of lentil inoculated with the NT6+LX81 combination was ca. 112% higher than the
NT4+LX8 1 combination. The number of CO-inoculationtreütments wherein NT4
increased grain yield was more than that for hT6.
The harvest index of control plants was higher than that of NT4-. or NT6inoculated plants. regardless of the rhizobial strain (Appendix Tables A.5 and A.6).
Irrespective of the AMF species, inoculation of lentil with some rhizobia strains increased
In
-
\S
C
Y
CI
-
G
?
V
Tt
Tl-
".
h
d
N
V
h
Tf
Oc
C1
N
the harvest index compared to others (Appendix Tables A S and A.6). For exarnple. the
harvest index of plants inoculated with rhizobia strains LX77. LX84 or RGL14 was
higher than that of control plants or those inoculated with strains PB 102 or WA 1. in
some cases, the M species NT4 or hT6 ~ i g ~ c a n taltered
ly
the harvest index of plants
inoculated with the different rhizobia (Table 5.3.7). For the same Rhizobium strain. COinoculation with NT4 or NT6 significantly increased the harvest index of lentil. For
exampk, NT4+LX77 increased the harvest index of lentil by ca. 58% over the
NT6+LX77 combination. In five of nine Rhizobium treatments. CO-inoculationwith NT4
sipLificantly increased the harvest index of lentil compared to NT6.
5.3.2.2
Nutrient parameters
Control plants contained the Least N in their shoots. indicating that the indigenous rhizobia
were not effective N2 fixers. There were no significant differences in the shoot N content
of plants inoculated with NT4 or NT6. but the shoot N content of these plants was
simcantly
higher than that of uninciculated plants (Appendix Tables A S and A.7).
DifYerences in the effectiveness of the rhizobial m a i n s were manifested as differences in
the shoot N content of lentil (Appendix Tables A 5 and A.7). For example. strallns
LX72, LX81 and PB 101 si,@ficantly increased the shoot N content of lentil plants
compared to strains LX66 or LE13, or the control. The NT4 or NT6 inoculant
signifcantly altered the response of lentil to five of nine rhizobia (Table 5.3.8). For
exarnple. inoculation of lentil with NT6 and RGL14 sipificanùy uicreased the shoot N
content over the NT4+RGL14combination and those inoculated with only RGL14 by up
to CU. 226%. In general. CO-inoculationwith NT4 appeared to be more beneficial than
with NT6.
Lentil plants did not benefit from the indigenous AMF as the shoot P uptake of
control plants was significantly lower than NT4 or NT6, irrespective of the rhizobial
Table 5.3.8. Meün (n=4) shoot N and P content of lentil plants inoculated with the AMF species Gloi?iiï.sclu~.itr?iNT4 or G. niosseue
NT6 andhi*nine Rhizoliirr~niegir~?ii~iost~rrn~
stiains and grown foi 1 10 d in soi1 containing indigenous AMF and rhizobia. Al1 tseeatnient
means withiri a paismeter were separated using the least significant difference (LSD) test at p8.05.
Shoot P conteii t (mg/pot)
Shoot N content (mg/pot)
C-r
W
Rhizobiw~
AMF s~ecies
Control
12.32
36.32
33,66
3.36
6.68
6.63
RGL 14
24.48
29.19
79.75
4.83
5.18
13.33
PB 101
24,69
69.63
41.68
5.12
1 1,CiCi
9,22
92A1
31 -24
35.60
27.6 1
6.73
8.20
5 91
LE 13
23.23
2395
30.29
5.85
5.88
7.53
LX 66
2 1 SI)
22.44
33.49
33 7
5.0 1
6.55
LX 72
28.9 1
71,15
49.45
498
11.81
79 3
LX 77
32.87
5 1.64
36.5 1
5.73
7.74
6.22
LX 81
39,O1
57.79
44.55
6.07
0.69
6.83
LX 84
26.6 1
34S9
32.53
4.30
5.35
4.89
LSD (0.05)
1 1.86
1 .Y3
treatment (Appendix Tables A.5 and A.7). Althouph significantly higher than the control.
the influence of one AMF species on the shoot P content was not significÿntly diftérent
from the other (Appendk Tables A.5 and A.7). Irrespective of the AMF species. the
rhizobial strains varïed in their ability to enhance the shoot P content of lentil (Appendix
Tables A.5 and A.7). For exarnple. s&s
RGL14. PB101 and LX72 were more
effective than strains LX66, LX84 and the control, The influence of NT4 or NT6 on the
shoot P content of lenal inoculated with the sarne Rhizobiunz strain varied in five of nine
rhizobia tested (Table 5.3.8). For example. lenhl CO-inoculatedwiùi NT6 and RGL14
had CU. 157%more shoot P than the NT4+RGL14 combination or the control. Coinoculation with NT4 increased the shoot P content more than NT6 in many
AMF+rhizobia treatmenn. A siCIonificantpositive correlation was found between the
shoot dry weight and the shoot N (rd.97: ~ ~ 0 . 0and
5 ) P contents (r= 0.94: p<0.05) of
lentil,
Inespective of the rhizobia strain, the NT4 inoculant significantly increased the
grain N content of plants compared to that with NT6 (Appendix Tables A 5 and A.7).
However. the grain N content of plants inoculated with 1V4 was not significantly
different from that of the control. There were sipifkant differences between the
rhizobial strains. irrespective of the AMF species uscd (Appendix Tables A.5 and A.7).
For example, plants insculated with LX77 had the highest grain N content. whereas those
inoculated with 92A1 or not inoculated with rhizobia contained the least grain N
(Appendix Tables A.5 and A.7). Co-inoculation with AMF altered the response of lentil
to rhizobia which resulted in sipificant increases (e-g., i\TT4+LX77) or decreases (eg..
NT6tLX77) in the grain N content It appeared that the grain N content of plants coinoculated with NT4 and rhizobia was considerably higher than NT6. in rnany coinocuIation treatments pig. 5.3.3).
Inoculation with W 4 si=onificantlyincreased the grain P content of lentil
compared to NT6-inoculated plants. irrespective of the rhizobial strain used (Appendix
Tables A S and A.7). However. lentil plants not inoculated with any AMF species
contained significantly higher levels of grain P thm plants inoculated with the AMF
species. The influence of the rhizobid snains on the grain P contcnt varied sitnifcantly.
irrespective of the AMF species (Appendix Tables A S and A.7). For example. plants
inoculated with strains LX72, LX77, LX8 1 and RGL14 contained higher leveis of grain
P than IentiI plants inoculated with LX66.92Al or plants not inoculated with rhizobia In
most cases. CO-inoculationwith one AMF species ~i~anificantly
altered the grain P content
compared to the other AMF species, indicating specific interactions between the AMF and
the rhizobia and with the host. As noted for other parameters. CO-inoculationwith NT4
was more often beneficial to increasing the grain P content of lentü than NT6 (Fig.
5.3.4). A s i p f ï c a n t positive correlation (r=0.99; pc0.05)was noted between the grain
yield and the grain P content of plants.
Irrespective of the rhizobid strain. the AMF NT4 si,pScantly increased the shoot
PUE of lentil compared to NT6 (Appendk Tables A S and A.8). However, the shoot
PUE of lentil planrs not inoculated with AMF was higher than that inoculated with either
AMF speàes. Lentil inocuiated with the different r k o b i a exhibited sipificant
differences in their shoot PUE levels, irrespective of the AMF species (Appendix Tables
A S and A.8). For example, plants inoculated with the rhizobiz strains LX77. LX84 and
RGL14 had higher levels of shoot PUE than that of lentil inoculated with strains PB101.
92A1. or not inoculated with rhizobia. For the same Rhizobium treatrnent, CO-inoculation
with one AMF species had a sitdcantly different effect on the shoot PUE froni the
other. in four of nine rhizobia (Table 5.3.9). In three cases, the NT4 inoculant was
sigmficantly better at improving the shoot PUE of lentil than 1V6.for the same
Rhizobi~rrnstrain. For exmple. the shoot PUE of lentil CO-inoculatedwith NT4+LX77
was ca. 107% higher than the NT6+LX77 combination, but not signficmtly difYerent
from the LX77 treatrnent. The main AMF and rhizobial effects for the grain PUE of lentil
were not significant (Appendix Tables A S and A&. However. the AMF species NT4 or
NT6 altered the grain PUE of plants inoculated with the same Rhizobium strain (TabIe
5-3.9).
5.3.2.3
AMF colonization and nodulation
Control plant roots were readily colonized by indigenous AMF. Plants inoculated with
NT4 had higher levels of AMF colonization than plants not inoculated with AMF or those
inoculated with NT6, irrespective of the rhizobid strain (Appendix Tables A S and A8).
The AMF colonization of lentil roots was sipificantly increased or decreased by the
Rhizobium straùis, irrespective of the AMF species (Appendix Tables A 5 and kg). For
example. the AMF colonization levels of lentil inoculated with the rhizobia strJins LX72.
LX77 or LX84 or colonized by the natil.~erhizobia were signifrcantly higher than plants
inoculated with strains LX8 1 or LE13. In seven of nine cases. CO-inoculationresulted in
sir:onificant differences in the AMF colonization levels between the AMF species NT4 and
NT6 for the s m e Rhizobium treatrnent (Table 5.3.10). For example, plants COinoculated with the NT6+RGL14 combination had sigifïcantly higher levels of AMF
colonization than the NT4+RGL14 combination. Similarly. plants CO-inoculatedwith the
hT4+LX77 combination had a ca. 2.5 fold increase in A M F colonization over that of the
NT6+LX77 combination. However, mycorrhizal colonization was not related to any
plant parameter measured.
Plants were mdulated by the indigenous rhizobia (control) and/or the uitroduced
Rhizobium strain. However, nodulation of lentil by the same Rhizobium strain was
affected by the AMF species (Table 5.3.10). For example, NT4 increased nodulation of
lentil roo ts by LE13 compared to NT6. Similady, nodulation of lentil roots by PB 101
was enhanced in the presence of NT6 rather tban hT4. A sipificiuit positive correlation
was observed between root nodulation and the grain yield (r=0.73: pi0.05).grain P
content ( ~ 0 . 7 3pc0.05)
:
and shoot PUE (rS.59: p<0.05) of lennl.
5.4
Discussion
Sprent (1989) listed steps which are imperative for the formation of functional le,=urne
nodules. These include the various chemical stimulants and growth regdators essential
for the nodulation and nitroten fixation processes. However. seldom is a legume host
root system not CO-inhabitedby rhizobia and AMF. Recent studies addressing the
sirnilarities between the 'nod' and 'rnyc' symbioses show that a high level of similarity
exists between both symbioses (Duc et al. 1989: Balaji et al. 1994; Gianinazzi-Pearson.
1996; Overholt et al. 1996: Stafford, 1997). and suggest that in fact one genetic locus
may control both symbioses. Several workers have reported that CO-inoculationof
legumes with AMF and rhizobia si@cantly
increases plant productivity and nutrition
relative to uninoculated plants or those inoculated with AMF or rhizobia. The present
study demonstrated that there were synergistic interactions between compatible ALMF
species and rhizobia which were rnanifested as growth or yield increases. However.
these growth and yield increases were not restrïcted to the effective AMF species or
rhiobia Based on the percentage yield increasr of inoculated over the control plants. it
appeared that lentil benefited more (in tems of grain yield. N and P content) from AMF
and/or rhizobial inoculation than pea. In evaluathg tbe response of nodulated Vigna
unguiculata, Cajanus cajaiz and Arachis bpogaea to two Glumus species. Ahiabor and
Hirata (1995) found that Vigiza ~rrzguiculataand Cajanus cajan benefited more from co-
inoculation with AMF and rhizobia than Arachis hypogaeu. The dramatic response of
lentil compared to pea in my study was probably because lentil is more dependent on
mycorrhizae than pea for nuaient uptake.
Many workers have s h o w that the growth and yield of 1egumes were iniluenced
by interactions between WfF species and rhizobia (Ames et al. 1991:Azcon et al. 1991 :
Vejsadova et al. 1992; Ianson and Linderman. 1993: Ruiz-Lozano and Azcon. 1994;
Ahmad. 1995; Saxena et al. 1997: Redecker et al. 1997). Azcon et al. (1991) found that
alfalfa growth was enhanced by the specific combination of Glomirs and Rhizobirm
meliloti strains. Similarly. in cornparhg the effects of interactions between G. pallidurn,
G. aggregaturn and Sclerocystis rnicrocalpa and four Rhizobium phusedi strains on the
growth and yield of three kidney bean cultivars, Ahmad (1995) found that the symbiotic
efficiency was "dependent on the particular combination" of the AMF species. Rhizobiim
strain and the host cultivar. The growth, yield and nutrition of both pea and lentil in my
study were dependent on the specific combination of AMF species and Rhizobiimz s ~ a i n .
This agrees with similar observations made by Azcon et al. (199 1) and Ahmad (1995) for
alfdfa and kidney bean. However, in contrast to o t k r studies, wherein only effective
rhizobia were included, ineffective Rhizobir~mstrains were also included in my study. It
was noted that a superior Rhizobium isolate such as LX43 was rendered less effective on
the host (e.g., in tenns of grain yield) when CO-inoculatedwith an incompatible AMF
species (e.g,, NT6). On the other hand, the yield of pea or lentil inoculated with a Iess
effective Rhizobirrm strain was enhanced when combined with a compatible AMF
species. However, this enhanced productivity was s a si,onificantly lower than that of an
effective Rhizobium or AMF-Rhizobium combination-
The role of AMF in the tripartite symbiosis may be important in soils with a low
a\ ailable P content (such as the soi1 used in this study) as nitrogen fixation is resüicted by
am inadequate P supply. The growth and yield increase of legumes inoculated with AMF
and rhizobia is generally due to enhanced N andor P uptake (Manjunath et al. 1984:
Pacovsky et al. 1986; Kucey and Bonetti, 1988; Azcon et al. 1991: Ruiz-Lozano and
Azcon, 1993). Correlation analyses revealed that in my study the grain yield increase
observed for both pea and lentil was associated with increased grain P content. but not N
content This indicates that che yield increase noted in compatible AMhrhizobia
ueatments was probably due to enhanced P uptake.
The grain yield and grain P content increases noted in some CO-inoculation
treamients were associatzd with increases in AMF colonÎzation of pea ( e - g .1LT4+LX43)
and lentil (e-g., Nï'4-1-LX77)plants. although correlation analyses revealed no signifknt
relationships between the Mi@ colonization levels and yield and/or I? content for both
hosts. In comparing the AMF root colonization response of 70 d-old alf;tlf-2plants to
combinations of G-fasciculaturn, G. niosseae or G. caledorziunz and six R. rneliloti
strains, Azcon and CO-workers(1991) found that plant growth was not dways related to
AMF colonization. Similar results were observed by Ruiz-Lozano and Azcon (1993) for
chickpea co-inoculated with combinations of G. mosseae or G.fascic~datumand two
Bradyrhizobium strains. It is possible that the enhanced P uptake by pea and lentil plants
inoculated with some AMF+rhizobial treatment. was mediated by the activity of the
extemal mycelium. the P absorbing organ of the AMF (Jakobsen et al. 1992). and not by
AMF colonization of roots.
CorreIation analyses revealed that the shoot dry weight, grain yield and grain P
content were positively correlated with nodulation of pea plants. Sirnilarly. the grain
yield, grain P content and shoot PUE were positively correlated with nodulation of lentil
plants. Sirnilar results indicating a positive relationship between nodulation and yield and
nutrition parameters were observed for fababeans by Kucey and Paul (1982), Leucaena
lerrcocephala by Manjunath et al. (1984) and soybeans by Pacovsky et al. (1956). It has
been proposed that in an effective tripartite symbiosis, the AMF supplies the high P levels
required for nitrogen fixation, and the Rhizobium supports the intemal and extemal
growth of AMF by enhancing the overall health of the plants (Azcon-ANilar and Barea.
1992). In keeping with this theory. it is possible that the enhanced P uptake by AMF
helped increase nodulation and nitrogen fixation and ultimately grain yield.
This study demonstrated that by caretiilly selecting for AMF and rhizobiü which
are compatible with each other and with the host plant, the growth. yield and nutrition of
pea and lentil can be sipificantly enhanced even in non-sterile soil containing indigenous
AMF and rhizobia. Furthemore. this study shows that in high yielding CO-inoculation
treatments. the increased grain yield may be due to enhanced N and P nutrition. In
addition. CO-inoculationwith one AMF species signifcantly increased plant p r o d u c t i v i ~
over the other for the sarne Rhizobium strain or isolate, irrespective of its effectiveness on
the host It appeared that in most cases, the LX isolates which were effective or
ineffective on the respective hosts in Leonard jars. remained the same in non-stede soil.
probably reflecting their aggressiveness/ competitiveness.
6.0
THE INFLUENCE OF PHOSPHORUS ON T m TRIPARTITE
SYMBIOSIS BETWEEN AMF, RHPZOBIA AND LEGUMES
6.1
Lntroduction
The activity of AMF in soils is influenced by various abiotic factors such as exnemes of
temperature (Tommerup, 1983), moisture @aniels and Trappe. 1980; Soedarjo and
Habte, 1995). pH (Green et al. 1976) and nutrient levels (Siqueira et al. 1982). Of all the
nutrients which influence AMF acti\lty and survival in soil, phosphorus may be
considered the most important High soil-P levels can inhibit AMF spore germination
(Miranda and Harris, 1994) and root colonization (Lu et al. 1994), and impair or resuict
the activîty of the AMF extemal mycelium (Miller et al. 1995)- In contrast. at very Iow
soil-P levels. the host, AMF and other soil microorganisms compete for P. resulting in
reduced AMF activity (Barber and Loughman, 1967: Same et al. 1983; Medina et al.
1988). These observations indicate that an optimum soil-P level is necessary for AMF
activity. This is further complicated by the fact that the level of P for optimum AMF
activity varies with AMF species and the host (Schubert and Hayman, 1986). On the
odier hand. the iiitrogenase-mediated reduction of aünosphenc nitrogen by rhizobia
requires high levels of P for the synthesis of high-energy reducing power (Phillips et al.
1980). In CO-inocdatedplants, a delicate P balance is achieved to accommodate AMF
and rhizobia activity in such a way that the activïq of both organisrns is not impaired, but
enhanced.
Many workers have examined the effect of P on the tripartite association between
AMF, rhizobia and legumes (Barea et al. 1980; Bethlenfalvay et al. 1982; Azcon-Aguilar
et al. 1 9 8 6 ~Morton
;
et al, 1990; Azcon and Barea, 1992; Azcon and El-Atrach, 2997:
Rahman and Parsons, 1997: Stamford et al. 1997). and found that mycorrhizae
substituted for the effects of soluble P fertdizers. In some cases, the addition of moderate
amounts of P fertilizers ~ i ~ c a n tincreased
ly
the growth of dual symbiotic plants more
than non-symbiotic plants or dud symbiotic plants not supplied with P (Bethlenfalvay et
al. 1982; Azcon-Aguilar et al. 1986c; Morton et al. 1990). Some workers noted that
available soil-P corrected the uieffectiveness of the rhizobia involved in the tripartite
association (Stamford et al. 1997).
In the previous study (Section 5 O ) , I found that the growth and yield response of
pea and lentil to the s m e AMF species but different Rhizobium strains varied
significatly. For example, the combination of Glomus clurum NT4+ Rhizobium
leguminosarurn bv. viceue LX43 sipificantly increased the grain yield and grain N and P
content of pea, whereas the combination of G. clarum NT4+R. leguminosarurn bv.
viceae 175P4~ i ~ c a n treduced
ly
the grain yield, compared to the uninoculated control.
Similarly, the combination of G. clanrrn NT4+R. legunrinosaium bv. viceae LX77
sigdicantly increased the grain yield and grain N and P content of lentil, whereas
inoculation of lentil with the combination of G. clarum NT4+R. legslminosarum bv.
viceae PB 101 did not benefit lentil yield or nutrition, compared to the control. It was not
clear whether these yield depressions or neutrd responses to dual inoculation noted in pea
and lentfi were mediated by incompatibility between the microsymbionts or between the
host and the AMF-rhizobia combination, or alternatively, due to other factors such as
nuaient levels. Since P is an important element which affects both microsyrnbionts and
host growth. this study was designed to examine the effect of different P levels on the
growth, veld and nutrition of pea and lenril CO-inoculatedwith the above effective (i.e..
NT4+LX43 for pea and NT4tLX77 for lentil) and ineffective (Le., NT4+175P4 for pea
and NT4+PB 101 for lentil) PMF+Rhkobiurn combinations.
6.2
Materials and methods
6.2.1 Experimental design for pea
Six treatrnerrts were assessed: (2) control. (2) G- clarurn NT4. (3) R. legwninosarrrm bv.
viceae isolate LX43, (4) R. leguminosarurn bv. viceae strain 175P4.(5) NT4 -t LX43
and (6) NT4 + 175P4. AU the treatments were assessed at three different added P levels
(i.e., 0, 10 and 20 mg kg-1 soil). resu1t.Q in eighteen factorial treatment combinations (6
treatments x 3 P levels), each replicclted five times.
6.2.1.1
AMF inoculum
The AMF G. clunun NT4 used in this study was oriEoinally isolated from Saskatchewan
soils (Talukdar and Germida. 1993a). A monospecific culture of G. clarurn NT4 was
produced on a maize (Zea mays) host grown in pots containing a 1:1 (w/w) mixture of
sterilized soil and sand (Talukdar and Gennida, 1993b). The NT4 inoculum consisted of
spores. colonized rooü and extemal hyphae. and was stored at 4OC for 12 months. This
inoculum contained ca. 490 propagules per 50 ,o. as detemiined using a 'Most Probable
Number' assay (Porter, 1979).
6.2.1.2
Rhizobium leguntinosarum bv. viceae strains
The Rhizobium leguminosarum bv. viceae strains used in this snidy were 175P4 and
LX43. The source of both rhizobia is listed in Table 5.2.1. The Rhizobium cultures
were produced by inoculaMg a loop of cells from a stock c u l ~ r into
e 100 ml of YEM
broth and growing on a gyrotory shaker (150 rev min-1) at 2 8 C for 72 h: this yielded ca.
108 cfu ml-1.
6.2.1.3
Soi1
The sandy loam soil used in this study was coilected at Aberdeen, Saskatchewan. The
soi1 was air-dried, passed through a 4-mm sieve, and the nument content detemined at
the Enviro-Test Laboratories, Saskatoon. Saskatchewan, The nutrient content of the soil
was as foüows (pg gl):N. 3.2: P. 11.4: K. 253: S. 4.5: Cu. 0.6: Mn. 4.1: Zn. 0.4: B.
0.9: Fe. 9.5: Cl. 1.8. The pH of the soi1 was 8.4. the electricd conduchvity. 0.2 mS/cm
and the organic matter content, 1.58. The soil was mixed 1:1 (wl'w) with silica sand and
two kilograms potted in 15-cm dia pots. The soil:smd mix contained
CU.
460 infccEive
AMI? propagules per 50 ,o. but did not contain any Uidigenous Rhizobium legurniizosanim
bv. viceae?as determined using the most probable number assay. Before seeding. each
pot was arnended with 0.043 g of ILI.03 and 0.097 g of K2SO4 in 100 ml of dislilled
water to provide 3 ppm of N. 9 ppm of S and 11 ppm of K. to maintain recommended
(Le., Enviro-Test Laboratories and the 1997 Crop Production Manual) levels of N. P. K
and S. Potassium dihydrogen phosphate (KH2P04) was dissolved in 50 n?l of water and
mixed thoroughly with the soi1 at the following rates: (Le.. 0. 10 and 20 mg kg1 soil).
The soil:sand mix was allowed to equilibrate at 24'C for seven days in a growth chamber
before plantinp.
6.2.1.4
Inoculation and plant growth
Pea (cv. Trapper) was used as the first test plant. For the control treatment, four pea
seeds were placed at 5-cm depth from the soil suiface. For the AMF treamienr seven
g r a m of the NT4 inoculurn (69 NT4 propagules) was placed at 5-cm depth from the soi1
surface over which four pea seeds were placed. For the Rhizobium treatment. two
milliliters of the Rhizobium culture was added on the seeds placed at 5-cm depth from the
soi1 saface. For AMFi-Rhisobiunr treatments. seven grams of the NT4 inoculurn was
placed at 5-cm depth over which four pea seeds were placed, and two milditers of the
Rhizobium culture was added on the seeds. After emergence, the plant count was
reduced to two per pot. Autoclaved polypropylene beads were spread on the soil surface
to prevent cross contamination and excessive moisture loss. Plants were grown in a
growth chamber witb the followhg growth conditions: W C , 16 h day and a 20°C. 8 h
nights: 375-400 pm m-2 sec-1 of irradiance. Soil was maintained at 6 0 9 moisnire
hoIding capacity throughout the study by the periodic addition of water- Pots were
randornized in the growth chamber and repositioned weekly.
6.2.1.5
Parameters
Plants were grown for 95 days and harvested. The above-ground plant material was
separated and dried (65OC:48 h). Grain was separated from the shoois and both
weighed. The harvest index of plants, hdicative of the partitioning of dry matter into
grain (gain yieldltotal above-ground dry matter x 100) was calculated zccording to
Ahiabor and Hirata (1995). The roots were washed thoroughly in rumint tap water and
reserved for assessing nodulation and Ah@ root colonization. The number of nodules on
the main and lateral roots was counted and categorized as follows: low. 0-20; medium.
21-40; and high, >40 nodules. The whole root system was dried (65OC: 48 h) and
weighed. Oven-dned roots were rehydrated and cut uito one centimeter pieces for the
determination of AMF colonization. About 50 mg of a representative root sample was
cleared in KOH, acidified with HCI, stained with aypm blue, destained in acidic glycerol
(Koske and Gemma, 1989) and observed with a compound microscope (100 x). The
percentage of root length colonized by AMF (%AMI?root colonization) was determined
using the =ridline intersect method (Giovanem and Mosse. 1980). Shoot and g r i n were
digested using a mixture of H2S04-Hz02 and the N and P concentrations determined
(Thomas et al. 1967). The N and P content of the shoot and grain were cdculated by
rnultiplying the shoot or grain weight by the N or P concentration.
6 . 2 . 2 Experimental design for lentil
L e n a (cv. Laird) was used as the second test plant. Six treatments were assessed: (1)
control, (2) G. clarum NT4, (3) R. Zegurninosanm bv. viceae LX77. (4)R.
legurninosarurn bv. viceae PB 101, (5) NT4 + WC77 and (6) NT4 + PB 101. AU the
treaments were assessed at three diftèrent added P levels (i.e.. 0. 10 and 20 mg k g 1
mil). resulting in eighteen factorial treatment combinations (6 treatments x 3 P levels).
each replicated five times.
The AMF inoculum, rhizobia culture preparation. soil, inoculation procecirires.
plant growth conditions. harvest and plant parameters for ientil were idenucal to that of
pea, except far the following modifications: (i) the Rhizobium leguminosarurn bv. viceae
strains used were PB101 and LX77 (Table 5-22)?and (ü) plants were hanrested at 110
days after planting.
6.2.3 Statistics
Values for percentage AMF colonization were subjected to ang~lartransformation before
statistical analysis. AU data except nodulation results were analyzed using the ANOVA
procedure and means separated using the LSD test in SAS (SAS, 1997). Unless
indicated otherwise, ail treatment means were considered significanùy different at
pe0.05.
6.3
Results
For ail parameters. statistical s i a d c a n c e for the main effects (i.e., effect of the various
inoculation treaments irrespective of the P fertilizer levels, and the effect of the P fertdizer
levels irrespective of the inoculation treaments) and interaction effects (Le., effect of
inoculation treatments and added P levels), and mean values for the main effects are
presented in Appendix Tables B. 1-B.4 for pea and Appendix Tables B -5- B.8 for lentil.
6 . 3 . 1 Effect of added P on the growth and yield of pea
There were significant ciifferences in the effect of the inoculation treatments on the shoot
dry weight of pea, irrespective of the P levels (Appendix Tables B. 1 and B.2). For
exampie. plants inoculated with NT4 or LX43 had higher levels of shoot biomass than
the uninoculated control plants or those inoculated with a combination of the AMF NT4
and the rhizobial strain 175P4. However. averaged over d l the inoculation treatments.
the shoot biomass of pea plants did not increase with application of P fertilizer (Appendk
Tables B. 1 and B .2).
Inoculation of pea with the AMF NT4 had no effect on shoot biomass of plants in
the non-fertilized soi1 (i.e.. soi1 with no added P). In contrast, the Rhkobiunz sbains
LX43 and 175P4 siZMcantIy increased shoot dry weight compared to the uninoculated
(control) plants r a b l e 6.3.1). However, CO-inoculationof plants with the combination
of NT4iLX43 or NT4+175P4 had no sienificant effect on the shoot dry weight of pea
compared to the uninoculated conaol plants in the non-fertilized soil. In fact. the
presence of NT4 reduced the benefit of the rhizobia inoculants.
The application of P fertilizer did not significantly alter the shoot dry weighr of the
uninoculated or NT4-inoculated plants (Table 6.3.1). Surprisingly, application of 20
ppm P f e f i z e r sipiiicantly reduced the shoot biomass of plants inoculated with only
rhizobia compared to plants in the non-fertilized soil. The shoot biomass of plants coinoculated with the NT4+LX43 or NTLk175P4 combination was generally not altered by
application of P f e d i z e r . Furthemore. the shoot dry weight of plants in the coinoculation treatments were not s i C ~ c a n t ldifYerent
y
at any added P level.
L~espectiveof the P levels. the total root dry weight of plants inoculated with the
rhizobia strain LX43 was significantly higher than all the otber inoculation treatments
(Appendix Tables B. 1 and B.2). The NT4+LX43 and hT4+175P4 combinations had the
same effect on the total root dry weight of p e s Regardless of the inoculation treatment,
pea planrs receiving 20 ppm P had higber Ievels of total root biomass than those receiving
no P (Appendix Tables B. 1 and B.2).
Inoculation of pea with only LX43 or NT4+LX43 significantly increased the total
root dry weight of plants compared to control plants in the non-fertilîzed soil. h contrast.
the 175P4 inoculant significantly reduced the total root dry weight of plants compared to
the control in the non-fertilized soil. The total root dry weight of uninoculated plants
increased with application of 20 ppm P fertilizer. However, the response of inoculated
and CO-inoculatedplants to P fertilizer varied, For example, applicatioc of P fertilizer to
LX43-inoculated plants had no sitonificant effect on root biomass, whereas it decreased
the total root dry weight of NT4+LX43-inoculated plants (Table 6.3.1). In contrast, the
application of only 10 ppm of P fermizer sigdicantly increased the total root dry weight
of 175P4-inoculated and CO-inoculatedplants (i.e.. NT4+175P4) cornpared to nonfertiiized plants.
Inoculation of pea with both rhizobia and AMFtrhizobia treatments enhanced the
grain yield compared to the control, irrespective of the added P level (Appendix Tables
B.1 and B.2). Furthemore, the gain yield of pea plants inoculated with the rhizobia
strain LX43 or a combination of hT4 and WC43 was significantly higher than the 175P4
or Ni4+175P4 treatments. The application of P fertilizer did not benefit grain yield of
pea, as plants receiving no P produced higher grain yields than those fertilized with 20
ppm of P f e d i z e r , regardless of the inoculation treatment (Appendix B. 1 and B.2).
The NT4 inoculant had no efrfect on the gain yield of pea in the non-fertilized
soil. However, inoculation of pea with rhizobia or rhizobia +AMF (LX43, 175P4.
NT4+LX43 or NT4+175P4) significantly increased the g a i n yicld of pea over the
uninoculated plants in the non-fertilized soil. In fact, the yield response of these
inoculated plants was 19-32% greater than uninoculated plants receiving 20 pprn of P
fertilizer (Table 6-3.2).
The application of P f e d ï z e r had no significant effect on the grain yield of the
control or NTPinoculated plants. Similarly, 10 pprn of P fertïiizer had no effect on grain
yield of plants inoculated with the rhizobia LX43 or 175P4. Surprisingly. application of
20 pprn of P ferrilizer significantly reduced the grain yield of pea inoculated with &se
rhizobia cornpared to plants in the non-ferùlized soi1 (Table 6.3.2). There was no
significant diference between grain yield of plants inoculated with NT4tLX43 or
NT4+175P4 in the non-fertilized soil, but the addition of 20 pprn P fertilizer siLnificantIy
ùicreased the grain yield of the NT4+LX43 treatment by ca. 6 0 8 over the NT4+175P4
treatment. In fact, application of P ferditer signifïcantly reduced the yield of
NT4+175P4 CO-inoculatedplants compared to the yield of those plants without added P.
Statistical analysis showed that irrespective of the P level. the NT4+LX43
treatment partitioned more dry matter into grain thac other treatments (Appendix Tabbs
B.1 and B.2). As noted for the grain yield, application of P fertilizer did not benefit the
harvest index of pea (Appendix Tables B. 1 and B.2). Plants receiving no P or 10 pprn of
P fertilizer had si,gicantly higher harvest index values than those receiving 20 pprn of P
fertilizer. regardless of the inoculation treatment (Appendix Tables B. 1 and B.2).
The NT4 inoculant had no effect on the harvest index of plants in the nonfertilized soil (Table 6.3.2). In contras4 the LX43 and 175P4 inoculants sipificantly
increased the harvest index of pea over the control plants, when inoculated alone or in
combination with NT4. Application of P fertilizcr had no effect on the harvest index of
the uninoculated or plants inoculated with LX43 or hT4+LX43 ,compared to the
respective treatments in the non-fertilized soil. However. application of 10 pprn of P
fertiLizer significanùy reduced the harvest index of plants inoculated with NTS+175P4.
whereas 20 pprn of P fertilizer was required to reduce the harvest index of NT4 or
17SP4-inoculated plants (Table 6.3.2j. At the highest added P level. plants inoculated
with NT4-LX43 partitioned CU. 24% more dry matter into grain than NT4+i75P4-
inoculated plants when fértilized with 20 ppm P (Table 6.3.2).
6.3.2 Effect of P on the nutrient content of pea
Irrespective of the P levels. the LX43 and W4 treatments, alone and in combination
significantly increased the shoot N content of pea compared to the control and
NT4+l75P4 treatments (Appendix Tables B. 1 and B.3). Neither application of P
fertilizei- nor treatment x P 1eveIs had a sigdicant effect on the shoot N content of pea
(Fig 6.3.1; Appendix Tables B.1 and B.3).
The inoculation treatments averaged over the P levels had a significant effect on
the shoot P content of pea (AppendUc Tables B. 1 and B.3). For example. inoculation of
pea with the AMF NT4 significantly increased the shoot P content over all the other
treatments. The rhizobia strain 175P4 was sipificantly better at improving the shoot P
nutrition of pea than the rhizobia strain LX43. Irrespective of the inocuIation treatment,
the application of 20 pprn of P f e d i z e r significantly enhanced the shoot P content of pea
over plants receiving O or 10 ppm of P fermizer (Appendix Tables B.1 and B.3).
In the non-fertilized soil. the shoot P content of pIants inoculated with NT4,
LX43 or i75P4 was not sipificandy dBerent from that of the control plants. The shoot
P content was even sienificantly lower when LX43 or 175P4 was CO-inoculatedwith
NT4 in the non-fertilized soil (Fig. 6.3.1). Application of P fertilizer had no effect on the
shoot P content of uninoculated plants. Sirnilar1y, P application had no effect on the
shoot P content of pea inoculated with LX43 or NT4+LX43. However. application of 30
Added P (ppm)
LSD (0.05)
NT4+LX43
NT4tL75P4
Added P (ppm)
Fig. 6 -3.1.
Mean (n=5) shoot N and P content of pea inoculated with the AMF
species Glomrcs clarum NT4 and/or the Rhizobium leguminosarum bv.
viceae strains 175P4 and LX43 and grown for 95 d in soil amended with
0, 10 or 20 mg kg-' of P and ccntaining indigenous AMF. AU treatment
means within a parameter were separated using the least signiticant
dzerence (LSD) test at p<0.05.
ppm of P fertilizer siConificantlyincreased the shoot P content of plants inocuIated with
NT4, 175P4 or NT4475P4 (Fig. 6.3.1).
The various microbial inoculants significantly influenced the grain N content.
irrespective of the application of P fertilizer (Appendix Tables B. 1 and B.3). For
example. the rhizobia straîn LX43 done and in combination with the AMF NT4
significantly increased the g a i n N content of pea compared to alI other treatrnents.
Averaged over the inoculation treatments. pea plants not receiving P fertiiizer or receiving
only 10 ppm of P fertilizer had higher levels of grain N than those fe~tilizedwith 20 ppm
of P f e f i z e r (Appendix Tables B. 1 and B.3).
AU of the different inoculation treatments except NT4 significantly increased the
grain N content of pea by 53-104% over that of control plants in the non-fertilized soil
(Fig. 6.32). Furthemore, the increase in grain N content of these treaments was greater
than the uninoculated plants fertilized with up to 20 ppm of P fedizer. In the nonfertilized soil, the NT4 inoculant had different effects on the grain N content of pea
inoculated with LX43 or 175P4. For example. in the non-fertilized soil. NT4 had no
effect on the grain N content of pea inoculated with 175P4, but significantly decreased
that of plants inoculated with LX43. The application of P fertilizer did not affect the grain
N content of the control, N T 4 LX43 or 175P4 treatments (Fit. 6.32). However. the
application of 20 ppm P fertilizer significantly iocreased the grain N content of pea
inoculated with the NT4+LX43 combination by ca. 31% over that of plants in the nonfenibed soil. In contrast, the application of P-fertilizer siConiflcantlyreduced the grain N
content of NT4+175P4-inocuIated plants.
Averaged over the P levels. the various inoculation neamients had different effects
on the gain P content of pea (Appendix Tables B.l and B.3). For example, the rhizobial
Added P (pprn)
l5
11
LSD (0.05)
Added P (ppm)
Fig. 6 - 3 2
Mean (n=5) grain N and P content of pea inoculated with the AMF species
Glomus clanlm NT4 andor the Rhizobium leguminosarum bv. viceae
strains 175P4 and LX43 and grown for 95 d in soi1 amended with 0, 10
or 20 mg k g 1 of P and containing indigenous AMF. AU treatment means
within a parameter were separated uing the l e a s simcant
(LSD) test at p d . 0 5 .
difference
strain LX43 alone and in combination with the AMF NT4 significantiy increased the grain
P content of pea compared to the uninoculated controI. However. the rhizobia strain
175P4 and the combination of NT4+175P4 had the sarne effect on the grain P content as
that of the uninoculated control. On the other hand. averaged over the inoculation
treatments, the application of P fertilizer had no effect on the grain P content (Append~x
Tables B. 1 and B.3).
None of the inocuiation treatments significantly increased the grain P content of
pea when no P fertilizer was added to soil. As noted for the grain N content the grain P
content of the control and the NT4-inoculated pIants were not significantly altered by P
application (Fe. 6-32). Furthemore, the application of P fercilizer had no effect on the
grain P content of plants inoculated with only LX43. However, application of 20 ppm P
fertilizer si@cantly
increased the grain P content of pea when LX43 was cornbined
with NT4. compared to these plants in the non-fertilized SOLApplication of P fertilizer
either had no effect (e-g., NT4+175P4) or reduced P uptake (e-g.. 175P4 at the 20 ppm
P level) by pea inoculated wlth 175P4 (Fig. 6.3.2).
Pea plants inoculated with the rhizobial strain LX13 or a combination of
NT4+LX43 siEnificantly increased the shoot PUE over that of uninoculared plants or
plants inoculated with 175P4 or NT4475P4, irrespective of the added P levels
(Appendix Tables B. 1 and B.4). Averaged over the inoculation treaments. plants
receiving no P f e f i z e r had higher levels of shoot PUE thao those receiving 10 or 20
pprn of P fertilizer (Appendix Tables B.1 and B.4).
Inoculation of pea with the rhizobial strains LX43 or 175P4 significantly
irnproved the P use eficiency of pea shoots over die uninoculated plants in the nonfertilized soil (Table 6.3.3). However, at the same P level (Le., O ppm), the AMF NT4
.c
ril
Z1
Ir,
h
inoculant had no signiticant elfect on PUE. The application of P fertilizer had no effect
on the shoot PUE of control plants or plants inoculated with LX43. but reduced that of
plants inoculated with only NT4 Similarly. application of 10-20 ppm of P fertilizer
reduced the shoot PUE of 175P4-inoculated plants and a 20 ppm P amendment reduced
the shoot PUE of pea CO-inoculatedwith NT4 and LX43. cornpared to these plants in the
non-fertiIized soil (Table 6.3.3).
The gain PUE of pea plants was significantly increased by the rhizobial strains
LX43 and 175P4 and the NT4-+LX43 treatment relative to the control or the NT4
treament, irrespective of the P level (Appendix Tables B. 1 and B.4). When averaged
over the inoculation treatments, as noted for the shoot PUE, pea not receiving P fertilizer
had sigificantiy higher levels of grain PT& than those receiving P (Appendix Tables B. 1
and B.4). However, treatment x P interactions were not significant (Table 6.3.3).
6 . 3 . 3 Effect of P on the AMF' colonization and nodunation of pea
Irrespective of the P fertilizer level, uninoculated plants and those inoculated with the
AMF NT4 or a cornbination of NT4 and 175P4had significantly higher levels of AMF
colonization than the other treatments (Appendix Tables B. 1 and BA). When averaged
over the inoculation treatments, application of up to 10 ppm of P fercilizer had no effect
on AMF root colonization relative to those not receiving P fertilizer, but a 20 ppm P
amendment sigificantly reduced AMF colonization of pea roots (Appendix Tables B. 1
and B-4).
The NT4 inoculant had no effect on AMF colonization compared to the
uninoculated plants in the non-fertilized soil. A cornparison between the rhizobial snains
revealed that the LX43 inoculant sipificantly enhanced root colonization by indigenous
AMF more than 175P4 in the non-fertilized soi1 (Table 6.3.4). Co-inoculation with NT4
'ci
C
and LX43 or 175P4 decreased AMF colonization of roots compared to uninoculated
plants in the non-fertilized soil. The activity of indigenous AMF colonizing the roots of
uninoculated plants was si,.nificantly enhanced by 10 ppm of P fertïlizer. but restricted by
the 20 ppm P amendment (Table 6.3.4). Furthemore. the application of only 10 ppm P
fertilizer significantly reduced the AMF colonization of plants inoculated with only N T 4
This suggests that NT4 was more competitive than the indigenous M F . and thus
responsible for the AMF colonization levels detected. It also indicates that this AMF
inoculant was sensitive to high soil-P levels. The AMF solonization of LX43-inoculated
pea roots was signScantly affected by application of P fertilizer. indicatint that the
indigenous A M F in this treatment were sensitive to increasing soil-P levels. In contrast,
no such cffect was apparent in the 175P4 treatrnent The application of P fertilizer altered
the AMT? colonization of CO-inoculatedplants. For example. the addition of up to 10 ppm
of P fertilizer had no effect on the AMF colonization of NT4+LX43-inoculated pea
compared to this treatment in the non-fertilized soil. but a 20 ppm P amendment resaicted
the A M F colonization. In contrast, the application of 10 ppm P significantly Uicreased the
AIVIF colonization of NT4+175P4-inoculated plants.
The indigenous rhizobia were not very active and nodulation of pea in the control
and NT4 treatments was low. even with the application of P fertilizer. Furthermore, the
application of P fertilizer had no effect on root nodulation by the rhizobial inoculants
(Table 6.3.4). However, application of P fertilizer increased nodulation of the
NT4+LX43 treatment, but reduced nodulation in the NT4t175P4 treatment.
6 . 3 . 4 Effect of B on the growth and yieId of Ientil
Irrespective of the P levels, inoculation of lentil with LX77 alone and in combination with
the AMF NT4 significantly increased the shoot dry weight when compared to the PB IO 1
alone and in combination with the AMF NT4 (Appendix Tables B.5 and B.6). However,
all the rnicrobial inoculants had a positive effect on shoot growth cornpared to the conaol.
Application of up to 10 ppm P fermizer signiticantly increased the shoot dry weight of
plants. but a 20 ppm P amendment decreased the shoot dry weight when averased over
the inoculation trearments (Appendix Tables B.5 zind B.6).
The AMF and rhizobial inoculants ~i~nificantly
increased the shoot dry weight of
lentil in the non-fertilized soil (Table 6.3.5). The NT4 inoculant increased the shoot dry
weight by ca. 97% whereas the rhizobia strains increased the shoot biomass by 201-
214%. These increases in shoot biomass were CU. 45432% greater than that of
uninoculated plants receiving up to 20 ppm P fertilizer. Co-inoculation of lentil with NT4
and LX77 did not significantly rno-
the response of plants to the LX77 inoculant in the
non-fertilized SOS.However, CO-inoculationof lentil with NT4 and PB 1O 1 sigfl~cantly
reduced the shoot dry weight of plants compared to the PB 201 treatment in the nonfertilized soil.
The addition of P fertilizer generally increased the shoot dry weight of control
plants, but decreased that of NT4-inoculated plants (Table 6.3.5). There was no clear
trend in the response of rhizobia-inoculated plants to the application of P fertilizer. For
example, 10 ppm of P feràlizer sipificantly increased the shoot dry weight of lentïi
inoculated with LX77,but sir,pifïcantly reduced that of PB 20 1-inoculated plants.
Inoculation of lentil wiib the various microbial inoculants had different effects on
thz total root dry weight of plants. when averaged over the P levels (Appendix Tables B.5
and B.6). For example, the rhizobia strain LX77 alone. and in combination with the
AMF NT4 enhanced the mot dry weight of pIants when compared to all other treatments.
Plants inoculated with the AMF NT4 or a combination of NT4tPB 101 were not different
from the uninoculated control. Irrespective of the inoculation aeatrnents, plants fertilized
with 10 or 20 ppm of P krtilizer had significantly higher levels of the root biornüss
compared to those in the non-fertilized soil (Appendix Tables B.5 and B.6).
Al1 of the inoculants Sgnificantly enhanced the total root dry weight of plants in
the non-fertiiized soil (Table 6.3.5). Application of P fertilizer sipificantly increased the
root dry wzight of control piants and plants inoculated with the rhizobial strain LX77.
whereas variable responses were noted for plants inoculated with other treatments. For
example, the application of P fertilizer had no siegificant effect on the root biomass of
NT4tLX77-inoculated plants. but a 10 pprn P amendment significantly increascd the root
dry weight of lentil inoculated with the NT4+PB 101 treatment
The effect of the microbial inoculants on the grain yield of lentil varied
sipîfïcantly. irrespective of the P level (Appendix Tables B.5 and B.6). For example,
the N'T'&LX77 treatrnent sib@5cantly increased the grain yield of lentil over the
NT4tPB 101 treatment. However. the AMF NT4 had no effect on the grain yield of lentil
compared to the control. The application of 10 or 20 ppm of P fertilizer sipifcantly
increased the grain yield of lentil over plants not receiving P fertilizer, when averaged
over the inoculation treatments (Appendix Tables B.5 and B.6).
There was no significant difference in the grain yield of control and NT3
inoculated plants at any P level. However, the rhizobia and the AMF+rhizobia treaiments
significantly increased the grain yield of lentil by at least 131%over the uninoculated
plants in the non-fertilized soi1 (Table 6.3.6). Furthemore. this increase in grain yield by
rhizobia and the AMF+rhizobia treatments in the non-fertïiized soi1 was s i ~ c a n t l y
greater than that of uninoculated plants fertilized with up to 20 ppm of P fertilizer. In
general, the grain yield response of lentil inoculated with the rhizobia varied with the
application of P-fefllizer. but usually was positive. For exarnple. 20 ppm of P fertilizer
signifcantly increased the grain yield of plants inoculated with LX77 cr NT4+U(77 over
diat of plants in the non-fertilized soil. Only 10 ppm of P fertilizer w u required ro elicit a
similar yield increase in lentil inoculated with PB 1O 1 or NT4+PB 101. and additional P
was detrimental. This increase in the grain yield of the NT4+PB101 tremnent was Ca.
87% more than the respective treatment in the non-fertilized soil.
Irrespective of the added P level. the rhizobial strains LX77 and PB101 alone n d
in combination wÎth the AlMF NT4 significantly increased the harvest index of plants
compared to the control plants or plants inoculated with only NT4 (Appendix Tables B.5
and B.6). Imspective of the inoculation aeatments, plants receiving 10 or 20 ppm of P
fertilizer had a higher harvest index than those not receiving P f e f i z e r (Appendix Tables
13.5 and B.6). The microbial inoculants did not siedcantly
alter the harvest index of
lentil compared to the control in the non-fertilized soil. The application of P fertilizer had
no effect on the harvest index of plants inoculated with the NT4. LX77 or hT4+LX77
treatments (Table 6.3.6). In contrat. amending the soil with only 10 ppm of P fertilizer
signiticantly increased the harvest index of lentil inoculated with PB 101 or NT4+PB 10 1.
6 . 3 . 5 Effect of P on the nutrient content of ientii
Inoculation of lentil ~ 4 t the
h microbial inoculants had very different effects on the shoot
N content of plants. irrespective of the P level (Appendix Tables B.5 and B.7). For
exarnple, plants inoculated with the rhizobia main LX77 alone and in combination with
the AW NT4 sipificantly increased the shoot N content compared to those inoculated
with the rhizobia ssain PB 101 alone and in combination with the AMF NT4. The shoot
N content of plants inoculated with only NT4 was not si_&cantly
different from that of
the uninoculated control. Application of high levels of P fertibzer did not benefit the
shoot N content of lentil, irrespective of the inoculation treatment (Appendix Tables B.5
and B.7). For example. plants receiving no P fertilizer had significantly higher levels of
shoot N than plants receiving 20 ppm of P fedizer.
The AMF and rhizobial inoculants simcantly
increased the shoot N content of
lentil grown in the non-feràlized soi1 (Fig. 6.3.3). Furthemore, the shoot N content of
plants inoculated with the microbial inoculants was siConificantlyhigher than that of
uninoculated plants fertilized with up to 20 ppm of P fertilizer. The rhizobial inoculants
were more effective than the AMF NT4, but not diffèrent from one another. The
application of P fertilizer had no effect on the shoot N content of conaol plants or plants
inoculated with LX77, but reduced that of plants inoculated with only NT4. However.
the shoot N content of the PB 101 treatment was significantly reduced b y the application
of 10 ppm of P fertilizer. The NT4 inoculant modified the shoot N response of
Rhizobium-inoculated plants to P addition. For exarnple, the addition of 10 pprn P
simcantly
increased the shoot N yield of lentil CO-inoculatedwith NT4 and LX77. In
contrast, the addition of 20 ppm of P fefllizer signitïcantly reduced the shoot N content
of the NT4iPB 101 treatments mg. 6.3.3).
The shoot P content of lentil was sitmcantly
increased by rnicrobial inoculation
compared to the control. irrespective of the P level (Appendix Tables B.5 and B.7). For
example. lentiI inoculated with LX77, NT4+LX77, PB 101 and NT4+PBlOI had
sipificantly higher levels of shoot P than the control plants or plants inoculated with only
NT4. However, in contrast to the shoot N content, the application of 20 ppm of P
fertilizer enhanced the shoot P content of lentil, whereas plants not receiving P fertilizer
had the l e u t shoot P. irrespective of the inoculation treatment (Appendix Tables B.5 and
B.7).
Added P (ppm)
-- 11
Y
O
e
Io
LSD (0.05)
Added P (ppm)
Fig. 6.3-3.
Mean (n=5) shoot N and P content of lentil inoculated with the AMF
species Glornus clarum NT4 andfor the Rhizobium leguminosarurn bv.
viceae strains PB 101 and LX77 and grown for 110 d in soi1 amended with
0,10 or 20 mg kg-* of P and containing indigenous AMF.
AU treatrnent
means within a parameter were separated using the l e s t signifiant
difference (LSD) test at pc0.05.
Inoculation of Ientil with NT4. PB IO 1 and NT4cPB 101 significantly increased
the shoot P uptake in the non-fertilized soil. whereas the inoculants LX77 and
NT4+LX77 had no effect. Application of 20 ppm P f e d z e r signifcantly increased the
shoot P content of control plants. but application of P fertilizer had no etiect on N T 4
inoculated plants. There was no clear response in the other inoculation treatments to
application of P fertilizer. For example, application of 10 ppm of P feràlùer significandy
increased the shoot P content of the LX77- and NT4+LX77-inoculated plants compared
to plants in the nor.-fefized soil (Fig 6.3.3). However. application of 10 ppm of P
fertilizer significantly reduced the shoot P content of PB 101-inoculated plants. and
application of 20 ppm of P fertilizer reduced the shoot P content of the NT4+PB 101
treatment,
The grain N content of lentil was sigmficantly increased by inoculating with
LX77- NT4+LX77, PB 101 and NT4+PB 101 relative to the control. irrespective of the P
level (Appendix Tables B.5 and B.7). Howevzr, the grain N content of lentil inoculated
with WC77 or NT4+LX77 was sipificantly higher than pIants inoculated with PB 101 or
NT4+PB 101. Irrespective of the inoculation treatment, application of 10 or 20 pprn of P
fertrlizer siC@?icantlyincreased the gain N content of lentil relative to plants not receiving
P fertilizer (Appendix Tables B.5 and B.7).
All of the inoculation treatments. except the hT4 treatment. sitonificantiy increased
the grain hTcontent of lentil in the non-fertilized soi1 (Fig. 6.3.4). This increase in the
grain N content of plants in the non-fertilized soi1 was significantly higher than
uninoculated plants fertilized with up to 20 pprn of P. Furthemore, the grain N content
of plants inoculated with LX77 or NT4+LX77 was significantly higher than the PB IO1
or NT4tPB 101 treatments. Application of P fertilizer did not alter the grain N content of
control plants or those inoculated with only NT4 (Fig. 6.3.4). However, the application
1
LSD (0.05)
n
O
1O
20
Added P (pprn)
Added P (ppm)
Fig. 6.3.4.
Mean (n=5) grain N and P content of lentil inoculated with the AMF
species Glomus clarum NT4 andor the Rhizobium leguminosarurn bv.
viceae strains PB 101 and LX77 and g r o m for 110 d in soi1 arnended with
0, 10 or 20 mg k g 1 of P and containhg indigenous AMF. AU treatment
means within a parameter were separated using the l e s t si@cant
difference (LSD) test at pcO.05.
of 20 ppm P f e d i z e r signifcantly increased the grain N content of the LX77-inoculated
plants over that of plants in the non-fedized s d . but P-ferblizer application had no effect
on the hT4+LX77 treatment. In contrast, arnending the soil with 10 ppm of P fertilizer
si@cantly
increased the grain N content of the PB 101and NT4tPB 101 treatments.
over the respective treamients in the non-fertilized soil.
Irrespective of the P level. the rhizobia and AMF+rhizobia treatments significantly
increased the grain P content of plants compared to the control and plants inoculated with
only NT4 (Appendix Tables B.5 and B.7). When averaged over all the inoculation
treatments. plants receiving 10 or 20 pprn of P fenilber had sipificantiy higher levels of
grain P than plants not receiving P fertdizer (Appendix B.5 and B.7).
AU of the inoculation treatments, except the NT4 treatment. significantly increased
the grain P content of lentil in the non-fedïzed soil (Fig. 6.3.4). The grain P content of
plants inoculated with the LX77, NT4+LX77 and NT4+PB 101 was significantly higher
than that of uninoculated plants fertilized with up to 20 pprn of P femlizer. Application of
P-fertilizer had no effect on the grain P content of control plants or those inoculated with
only NT4. Similarly, application of 10 ppm of P fertilizer had no sign.ifïcant effect on the
grain P content of LX77 and NT4+LX77-inoculated plants, but a 20 pprn P amendment
significantly increased the grain P content compared to plants in the non-fertilized soil. In
contrast. the grain P content of plants inoculated with PB 101 and NT4+PB 101 was
siEonificantlyincreased with the application of only 10 pprn of P fermizer. but additional
P had no significant effect (Fig. 6.3.4).
Irrespective of the P level, the rhizobia and AMF+rhizobia treatments enhanced
the shoot PUE of lentil, compared to the control plants and plants inoculated with only
NT4 (Appendix Tables B.5 and 5.8). Irrespective of the inoculation treatmenc plants not
receiving P fertilizer had a significantly higher shoot PUE than those receiving P
fertilizer. Furthemore. a progressive decrease in the shoot PUE levels was evident with
increases in the amount of P f e d i z e r added (AppendLx Tables B.5 and B.8).
The rhizobial inoculants enhanced the shoot PUE of lentil plants in the nonfertilized soil. compared to the control plants or plants inoculated with only NT4 (Table
6.3.7). Application of P fertilizer had no effect on the shoot PUE of the controI. PB IO1
or NT4+PB 101 treatments. However. application of 20 ppm of P fectilizer significantly
reduced the shoot PUE of the NT4 and LX77 treatments over the respective treatrnents in
the non-fertilized soil, and application of only 10 pprn of P fertiLizer reduced the shoot
PUE of the NT4+LX77 treatment (Table 6.3.7).
The rnicrobial inoculants did not drarnatically alter the grain PUE of IentiI relative
to the control plants. irrespective of the P level (Appendix B.5 and B.8). For example.
only plants inoculated with NT4+LX77 significantly increased the grain PUE of lentil
over the conuol, whereas the other treatrnents had the same effect as that of the control,
Irrespective of the inoculation treatments, the application of P fiitilizer reduced the grain
PUE of lentil (Appendix B.5 and B.8). As noted for the shoot PUE. a progressive
decrease in the gain PUE was observed with increases in the application of P fertilizer.
The rhlzobia strain LX77. alone and in combination with the AMF NT4.
signïfïcantly increased the grain PUE of lentil plants in the non-fertilized soil. whereas
other inoculation treaiments had no effect (Table 6.3.7). The application of P fertilizer
had no significant efiect on the grain PUE of the control. NT4 or NT4+PB 101 treatrnent.
However, application of 10 ppm of P femhzer significantly reduced the grain PUE of
plants inoculated with LX77 or NT4+LX77, and a 20 ppm P amendment fùrther reduced
it (Table 63.7). The application of 20 ppm P fertilizer reduced the grain PUE of PB IO 1-
inoculated plants compared to that of these plants in the non-fertilized soil.
6 . 3 . 6 Effect of P on A M F colonization and nodulation of lentil
There were siC@ficant difièrences in the AMF colonization of plants inoculated with the
rhizobial strains done or in combination with the AhlF NT4 irrespective of the P Ievels
(Appendix Tables B.5 and B.8). For example. lentil inoculated Mth the rhizobial strain
LX77 alone and in combination with the AMF NT4 had significantly higher Ievels of
AMF colonization than plants inoculated with PB 101 or NT4+PB 101. Howevei-. when
averaged over the inoculation treatments, the application of P fertilizer had no effect on
the AMF colonization of lentil (Appendix Tables B.5 and B.8).
Inoculation of lentil with NT4 or the rhizobial main PB 101 had no effect on the
AMF colonization of lentil roots in the non-fertilized soil cornpared to the uninoculated
plants. In contrast, inoculation with LX77 enhanced the AMF colonization with or
without the NT4 inoculant in the non-fertdized soil. In general. the application of P
fertilizer had no eEect on AiMF colonization of lentil roots (Table 6.3.8). For example.
application of P fertilizer had no effect on the AMF colonization of the control, NT4.
PB 101 or NT4+LX77 treatments. However, a 10 pprn P amendment significantly
decreased the AMF coIonization of N T W B 1O 1-inoculated plants. and a 20 pprn P
amendment restricted AMF colonization of lentil inoculated with only LX77, compared to
the respective treatrnents in the non-fertilized soil.
The rhizobial inoculants alone or in combination with the NT4 inoculant increased
nodulation of lentil comparzd to the control or NT4 treatment in the non-fenilized soil
(Table 6.3.8). Application of P Rrtilizer had no effect on the nodulation of the control.
NT4 or PB 101 treatment compared to plants in the non-fertilized soil. However.
application of 10 ppm P fertilizer increased nodulation of the LX77. M4+LX77 and
NT4+PB 101 treatrnents (Table 6 -3.8).
6.4
Discussion
The response of legumes to AiMF and rhizobia depends on the specific combination of the
symbiotic microorganisms (Azcon et al. 1991: Ahmad. 1995). indicating that not all AMF
interact equally well with aU rhizobia. Incornpatibiliq between AMF and rhizobia in the
tripartite association can be manifested as yield depressions (Bethlenfalvay et al. 1985).
These yield depressions may be the result of com~etitionbetween the host and the
endophytes for nutrients such as P (Barber and Lorighman. 1967; Medina et al- 1988). or
cornpetition between the endophytes for carbohydrates (Bethlenfalvay et al. 1985).
Alternatively. the inability of the endophytes to supply adequate levels of N or P to the
host could lead to poor plant growth and yield (Stamford et al. 1997).
The NT4 inoculant had no effect on the growth, yield or nutrition of pea
compared to uninoculated plants in the non-fertilized soil. However. NT4 si,aificantly
increased the shoot biornass, shoot N and P content of lentil compared to uninoculated
pIants in the non-fertilized soil. This difference in the performance of the NT4 inoculant
between pea and lentil rnay be explained by the greater dependency of lentil than pea on
rnycorrhizae. The effecü of AMF inoculants on plant growth and yield c m be compared
to that of P fedizers beczuse AMF often enhance host P nutrition. For example. Menge
et al. (197Sa) demonstrated using citrus plants, that AMF inoculants can be substituted
for P fertilizers, because AMF have the same effect as that of P fertilizers on plant
growth. The nutritional benefits of inoculaMg plants with AMF include not only
enhanced hyphal uptake of P and micronutrients. but also N fiom soi1 (Barea et al. 1987,
1989). This is because the AMF hyphae are able to acquire P and N fiom soil resources
not available to non-mycorrhizal plants. In my study. the grain yield of pea inoculated
with NT4 in the non-feràlized soil was equivalent to that of uninoculated plants receiving
20 pprn of P fertilizer. In addition. the NT4 inoculant si,.nificantly increased the shoot
biomass and shoot N content of lenbl over uninoculated plants receiving 20 ppm of P
fertilizer. These positive growth or yield and N and P uptake rzsponses reflect the
benefits of inoculating pea and lenol with G. clarum NT4 compared to application of high
IeveIs of P fertilXzers.
The addition of P fertihzer generally e b a t e s the benefits a host derives from the
AMF-host association (Thomson et al. 1986; Amijee et al. 1993). On the other hand. the
growth and yieid of mycorrhizal plants receiving moderate arnounts of P fertiiïzer was
significantly greater than mycorrhizal plants not receiving P (Medina et al. 1988: Xavier
and Germida, 1997). However. in the present study. the application of up to 20 ppm P
fertilizer had no effect or was detrimental to both pea and lentil plants inoculated with only
NT4, compared to NTCinoculated plants not receiving P fertilizer. Jasper et al. (1979)
demonstrated using two adjacent field sites. that the AIvlF in a virgin non-fertilized soi1
were more sensitive to P fertilizer application than AMF in the adjacent fertiiized soil. It
is possible that the AMF (including NT41 associated with pea and t e d l were sensitive ta
P fertilizers. This was confïmed by the significant reduction in the AMF colonization of
pea roots following P application. However. no si,oniticant effect was observed in lenàl.
Nonetheless, the AMF endophytes in the non-fertilized soil were as effective as the P
fertilizer at enhancing plant growth and yield.
Several workers have studied the influence of P on the mpartite association
between AMF, rhizobia and legumes (Smith and Daft. 1977: Asirni et al. 1980:
Bethlenfalvay et al. 1982: Brown et al. 1988: Morton et al. 1990; Azcon and Barea. 1992:
Stamford et al. 1997). S o m of these studies suggest that AMF substituted for the effect
of P fertifizers on plant growth. For example, Asimi et al. (1980) reported that CO-
inoculation of soybean plants with G. mosseae and a B. japonicarm striain enhanced the
yield and percentage P content of soybean. and that P fertilization eliminated mycomhizd
effects on plant growth, nodulation. nitrogenase acaivity and rnycorrhizal colonization of
soybean r=iots. Smith and Daft (1977) reported that plant growth. nodulation. nitrogen
kation. and the N and P concentration of Medicogo sotiva plants CO-inoculatedwith G.
mosseae and R. meliloti were sit&cantly
higher in myconhizal plants than non-
mycorrhizal plants fertilized with P. 1too found that inoculation cf pea with the rhizobia
or AMF+rhizobial inoculants sipificantly increased the grain yieid. grain N content and
grain PUE of pea in the non-fertilized soil more than uninoculated plants fertilized with 20
ppm of P fertilizer. Similarly, inoculation of lentil with the rhizobia or AMF+rhizobia
significantly increased the growth, grain yield, shoot and g a i n N content of lentil in the
non-fertdized soil more than uninoculated plants fertilized with 20 ppm of P fertilizer.
These observations suggest that the NT4 +rhizobia combinations had the same or a
greater effect on plant yield and nutrition than that of high levels of P fertilizers.
Bethlenfalvay et al. (1982) found that the growth and nitrogen fixation of bean
plants CO-inoculatedwith G.fascicnlatzm and a R. phaseoli strain were inhibited at the
lowest (i.e.. O mg P hydroxyapatite per pot) and highest (Le.. 200 mg hydroxyapatite per
pot) added P level. but the best effect on plant growth and P uptake was noted at the
medium P level (Le.. 50 mg hydroxyapatite per pot). They further proposed that growth
inhibition at the low P level was due to intersyrnbiont cornpetition for P and
photosynthates. In rny study. the grain yield. grain N and P content of pea CO-inoculated
with the combination of NT4 and LX43 and fertilized with 20 ppm of P fertilizer was
significandy greater than pea CO-inoculatedwith NT4 and LX43 in the non-fertilized soil.
Similady, the shoot biomass. shoot N and P content of lentil CO-inoculatedwith KT4 and
LX77 and ferulized with 10 ppm of P fertïiizer, and the grain yield, grain N and P content
of lentil CO-inoculatedwith NT4 and PB 101 and fertilized with 10 ppm cf P fertilizer
were sipificantly greater than respective treatments in the non-fertilized soil. Contrary to
the fmdings of Bethlenfalvay et al. (1982). even the highest P level (i-e.. 20 ppm P)
testcd in this study did not inhibit the growth. yield or nutrition of pea or lentil coinoculated with some AMF+rhizobia combinations. However. the actual arnount of
avadable P in soil (i-e.. after P fertilizer application) was not derermined. Therefore. it is
possible that the actud amount of available P was not high enough to deter the activity of
the different AMF+rhizobia combinations. In addition, the responses obtained in this
study could not be compared to those reported by Asirni z t al. (1980) and Bethlenfalvay et
al. (1982) who assessed the response of dual symbiotic plants to P levels which were
either too high (i-e., 250-1000 ppm of KH2PO.4or ca. 57-228 pprn of P-Asimi et al.
1980) or not clearly defined (Le...O to 200 mg of hydroxyapatite per 1.25 L of a 1:2
perlitcsand mix-Bethlenfalvay et al. 1982). The added P levels tested in my study were
selected based on the recomrnended level of P fertilizer applied to pea and lentil in
Saskatchewan.
Studies evaluating the effect of different P levels on the performance of
AMF+rhizobia combinations on plant growth and yield have not included more than one
AMF+rhizobia combination. However, 1evaluated the effect of two combinations of
AMF and rhizobia on pea and lentil growth. yield and nutrition over a range of P levels.
The yield and nuvition rzsponse of pea inoculated with the NT4+LX43 cornbination to
application of P fertilizer was generally positive, whereas that of the NT4+-175P4
cornbination was rarely positive, or mostly negative. Factors such as changes in the P or
N use eEciency (Brown and Bethlenfalvay, 1987; Brown et al. 1988). carbon allocaaon
(Fang and Paul. 1980). or moisture and salinity stress (Sprent. 1986: Azcon et al. 1988:
Pena et al. 1988; Azcon and El-Atrach. 1997) c m influence the eftect of AMF on
Rhizobium activity. Analysis of the parameters measured in this study reveaied that the
shoot and grain PUE of the NT4+l75P4 treatment was unchanged (grain PUE- 10 ppm
P) or si,dicantly reduced (shoot PüE-10 and 20 ppm. gnio PUE-20 ppm) compared to
plants in the non-fertilized soil. Therefore, it is possible that this nautral or negative
response to P application in the NT4+175P4 treatment was probably due to the low P use
efficiency of plants, compared to the hT4tLX43 treatment.
The groowth, yield and nutrient content response of pea and lentil inoculated with
AMF andor rhizobia varied with P fertilizer application. However. the AMF root
colonization response of the different treatments to the different P levels was seldom
enhanced. and mostly unaffected or reduced. It is possible that the different plant
responses to P feralizer application were mediated by changes in the activiry of the
external mycelium and not necessarily the AMF colonization of pea or lentil roots. as
suggested by Miller et al. (1995)-Jakobsen et al. (1992) and Lu et al. (1994). This is
because the extemal mycelium is the nutrient absorbing organ of the AMF, and whose
activity is directly afferted by the fluctuations in the soil nutrient levels.
Although the AMF and rhizobial inoculants and inoculant combinations in the
non-fertilized soil were as effective on plant productivity as the highest P level tested. the
application of rnode~ateto high levels of P fertilizers m e r enhanced the productivity of
some AMF+rhizobial combinations. The response of pea and Ientil treated with the
microbid inoculants to the different P levels may be explained by differences in the
mycorrhizal dependency of the hosf and changes in host activity (e-g.. change in the
PUE) following inoculation with AMF+Rhizobium. This study aiso demonstrated that
different Rhizobium strains can influence the activity of the sarne AMF speties in
different ways or vice versa, and ultimately alter plant response to both rnicrosymbionts.
7.0
EFFECTS OF BACTERIA ASSOCLATED WI'ILTH ARBUSCULAR
MYCORREIZAL FUNGAL (AMF) SPORES ON TEE GLOMUSRHIZOBIUM-PISUM SYTvLBIOSPS
7.1
Introduction
The colonization of roots by AMF and the subsequent benefits derived by a host plant
depend on the sunival of propagules. especially the AMF spore. AMF spores in the soi1
are subjected to v ~ o u physical
s
and biotic stresses which often reduce their viability
and/or function. Several workers have shown that bacteria and fun@ are intimately
associated with AMF spores, either as intiracellular entities (MacDonald and Chandler.
1982 ; MacDonald et al. 1982; Tilak et al. 1989; Bianciotto et al. 1996) or as organisms
that colonize AMF spore wall layers (Schenck and Nicholson, 1977: Daniels and ~Menge,
1980; Mayo et al. 1986: Azcon, 1987; Hass et al. 1994: Walley and Germida. 1996).
Reports on the presence of intracellular organisms within AMF spores concluded that no
cytopathic effects were observed within the spores i.e., the spore intemal contents
(MacDonald and Chandler, 1981; MacDonald et al. 1982; Bianciottû et al. 1996). T i l k et
al. (1989) detected nitrogenase activiq @y acetylene reduction) in surface-sterilized
spores of vanous Glomus spp. However, the authors failed to confirm whether the
surface-sterïiization procedure effectively eliminated the spores of tightly adhering
extemal bacteria More recenùy ,usiog molecular ph ylogenetic analysis. Bianciotto and
CO-workers(1996) reported that Burkholderia cepcia was the sole occupant of the
cytoplasm of Gigaspora margarita spores. Although the authors did not speculate on the
role of these bactena within Gigaspora spores. they did not report any adverse effects due
to the presence of these bacteria
Many workers report that bacteria and hngi occur naturally on the surface of
A M F spores (Schenck and Nicholson. 1977: Daniels and Menge. 1980: Azcon-Aguilar et
al. 1986a. b: Mayo et al. 1986; Walley and Germida. 1996). For example. Daniels and
Menge (1980) found that sporocarps of Glomusfascicula~sand G. epigaeus were ofien
hyperparasitized by soil-borne fungi. Walley and Germida (1996) could not obcain sterile
and viable G. clarum spores despite 60 min. of decontamination with 5% chloramine-T
due to the presence of bacteria associated with AMF spore walls. Mayo et al. (1986)
found that the gemiination of G. versiforme spores was stimulated in the presence of
Pseudomonas and Coryrzebacterium which were isolated from "non-disinfested" spores.
Mugnier and Mosse (1987) reported that G. mosseae spores germinated in vitro only in
the presence of contaminants such as Sporoîhrir schenckii or Streptomyces orientalis.
Others have evaluated the response of AMF spores or hyphae to rhizosphere bactena or
fun@ (Krishna et al. 1982; Meyer and Linderman. 1986: &con, 1987: Mugnier a d
Mosse, 1987; Gonzalez, 1988; Will and Syl-via, 1990; Rousseau et al. 1996). Azcon
(1987) found that the germination and hyphal growth of G. mosseae spores increased i
n
response to the presence of whole bactenal cultures and cell-free supematants. although
the bacterhm was isolated from the rhizosphere soil. Mu,onier and Mosse (1987)
observed that Strepromyces orientalis stimulated the germination of G. mosseae spores.
Gonzalez (1988) reported that Rhizobium spp. enhanced the hyphal grcwth of G.
mossene spores under axenic conditions. Will and Sylvia (1990) found that the
germination and hyphal growth of G. deserticola spores was enhanced by Klebsiella
pneumoniae.
To date. the information generated on AMF spore wall bacteria (SWB) and their
effect on plant-AMF interactions is at best scattered and highiy frapented. The aim of
this study was to (i) isolate, identie, and study the effect of the SWB on the germination
of AMF spores and (ii) assess the effect of select SWB on the pea-AMF-Rhizobir[rn
symbiosis.
7.2
Materials and Methods
7 . 2 . 1 AMF spores
Spores were obrained from a 14- month old monospecific culture of G. clancm NT4
produced in a sterilized (1: 1)soil: sand mix with maize as the host (Talukdar and
Germida. 1993a). Spores were retrieved by wet sieving and decantirq (Gzrdeman and
Nicholson, 1963) foLlowed by a 20:60 sucrose density gradient cennifugation step ( M e n
et al. 1979). Spores at the interface were removed using a Pasteur pipette and washed
thoroughly with tap water. Any remaining debns in the NT4 spore inocuium was
rnanually rernoved with a pair of forceps under a microscope (x47) and spores were then
stored in three milliliters of sterilized tap water at 4OC.
7 . 2 . 2 AMF spore decontamination
The NT4 spores were decontaminated with 5% chloramine-T (BDH, Toronto. ON) for
30 (S30), 45 (S45) and 60 (S60) min. in a water bath maintained at 30°C. according to
the procedure of Walley and Germida (1996). The control treatment (SO) consisted of
spores treated identically to the a l l other treatments except without chloramine-T.
Unaeated (SO) and decontaminated spores were incubated on water agar ('WA). 0.02%
(w/v) yeast extract agar (YEA), 0.3% (w/v) nyptic soy agar USA). 0.8% (w/v) nutrient
agar (NA) and Luria-Bertani agar (LBA) for up to two weeks. Five replicate plates were
maktained per growth medium.
7.2.3 IsoIation of SWB
Bacteria were isolated from untreated spores and spores decontarninated for 30 and 60
min. Bacterial colonies from unaeated spores were selected based on rnorphological
characteristics, whereas all the bacterial colonies frorn decontminated (i-e.. 30 and 60
min.) spores were selected. Bacterial colonies isolated on NA. T'SA and LBA plates were
transferred to the respective fresh agar plates and restreaked for purity. Punfied isolates
were stored on 1.5% TSA slants at 4OC. The S m isolates which could not be cultured
on NA, TSA and LBA afkr the initial isolation from these media, and those isdated from
YEA and WA plates were puscd
on 0.38 TSA and stored on 0.3% TSA slants at 4OC.
All the cultures were dso rnaintained in a stede 5050% (v/v) TSB:gIycerol mixture at
-70°C.
7 . 2 . 4 Identification of §WB
The SWB isolated from untreated (SO) 2nd decontaminated (530 and S60) NT4 spores
were identified based on their FAME profiles. Fatty acids were extracted from CU. 40 mg
of a 24 or 48-h old bacterial culture and esterifed according to the procedure of Sasser
(1990). and as described in section 3.2.5. FAME profiles of the SWB cultures generated
by a gas chromatograph (GC) (Hewlea Packard 5890 Series II) equipped with the
Microbial Identification Software (version 1.2) were compared to and matched with
entries of the TSBA library based on a similarity index.
7 . 2 . 5 In vitro bioassays
7.2.5.1
Response of rhizobia to SWB
In order to assess the response of rhizobia to the SWB and vice versa. and to select for
S M isolates that influence the tripartite symbiosis. the SWB were cross-streaked against
10 R. leguminosarum bv. viceae snains (which Vary in their effectiveness on pea) on
duplicate tryptone yeast extract (TYJ5) agar plates. Plates were incubated at 27OC for
seven days, and growth enhancement or inhibition of the rhizobial strains was recorded
as + or -, respectively.
7.252
Response of AiMF spores to SWB
The response of G. clarum NT4 spores to select SWB was evaluated according to Walley
and Germida (1997). G. clarum NT4 spores were retrieved from a monospecific culture
as described in section 7.2.1. In order to obtain "clean" spores (Le.. spores devoid of
adhering bacteria) and to mabtain the Mability of the spores. NT4 spores were
decontaminated with 5% chloramine-T for 30 min. However. since this treatrnent did not
completely eliminate bacterial contamination, spores were also treated with a solution of
300 pg of streptomycin ml-1 for 30.60.90 and 120 min. Thirty minutes of spore
decontamination with 5% chloramine-T and 300 pg of streptomycin ml-1 provided
"clean" NT4 spores which geerminaated on 0.3% and 3.0% TSA &ter seven days of
incubation at 27OC. Using this method of decontamination. G. clarum NT4 spores were
decontaminated and stored in three milliliters of sterile tap water at 4OC. The SWB that
evoked a positive or negative response from the rhizobia in the previous assay (section
7.2.5.1) were seiected for ibis assay. Using a sterile Cotton swab, SWB cells from a
stock culme were streaked on tnplicate 1.58 TSA plates to obtain a uniform lawn of
bacterial growth. Plates were incubated at 27OC for 24 h before the start of the bioassay.
The bioassay assessed the effect of directly released, diffusible and volatile SWB
chernicals on NT4 spores (Fig. 7.2.1). All the materials used in the assay were
autoclaved pnor to use. For the direct assay. a 25-mm dia 0.22 p m pore size filter
(Millipore Corporation, Bedford, MA) was placed on the 24-h bacterial lawn.
Decontaminated NT4 spores were pipetted onto a 13-mmdia. 0.22 pm pore size filter and
placed on the 25-mm dia filter. For the diffusible assay, decontaminated NT4 spores
were pipetted ont0 a 13-mm dia. 0.22 pm pore size filter manged over a 1.5% agarose
block (cut using a IO-mm dia. steïile auger) which was placed on a 25-mm dia. 0.22 pm
pore size fllter laid on the bacterial lawn. The volatile assay consisted of decontaminated
NT4 spores pipetted onto a 13-mm dia. 0.22 pm pore size filter manged on a 1.5%
agarose block which was placed on a 22 x 30 mm g l a s cover slip (Coming. USA) laid
Fig. 7.2.1.
Tiyptic soy aga- ( 1.5%)plates containhg the direct ciiffusible and volatile
bioassays. Plates were streaked wiii-t the SWB isolütes and incubated for
24 h behre the difièrent bioassüys were assernbled.
on the bacterial lawn. Plates were sealed with parafilm and incubated at 27'C for 10 d.
d e r which plates were examined for NT4 spore germination and hyphd r o w t h or
inhibition.
7.2.6 Plant assay
To evaluate the effect of SWB on the pea-AMF-Rhizobium tripartite symbiosis. a
bioassay was conducted using pea (cv. Trapper), G. clarum NT4 spores, Rhizobium
legurninosarurn bv. vicerte LX43. and two SWB isolates which had a positive (Bacillus
pubdi L M ) or negative (B. chitinosporus LA6a) impact on NT4 spores. The following
treatments were included: (1) Cmtrol; (2) G.clarum NT4: (3) R. leguminosanrm bv.
viceae LX43: (4) Bacillus chitinosporus LA6a; (5)B. pabuli LM: (6) NT4 + LX43; (7)
NT4 + LA6a: (8) NT4 + LA3; (9) LX43 + LA6a; (10) LX43 + LA3; (1 1) NT4 t LX43 +
LA6a; (12) NT4 t LX43 + L A I AU the treatments were replicated five rimes.
The AMF inoculum consisted of G. clamm NT4 spores decontaminated widi 5%
chloramine-T and 300 yg of streptomycin ml-1at 30°C for 30 min. The decontaminated
NT4 spores were stored in 50 ml of sterile tap water at 4OC until used. In order to
enurnerate the number of NT4 spores/ml, one milliliter sampIe (n=3) of the
decontaminated NT4 inoculum was vacuum-fïltered onto a 47 mm dia. 0.8 pm pore size
Millipore fdter and counted with a microscope (~47). One milliliter of the NT4 inoculum
contained 403k4 NT4 spores. The Rhizobium sp. culture was produced by inoculating a
loopful of R. legurninosarurn bv. viceae LX43 cells from a stock culture into 100 ml of
YEM broth and gronring on a gyrotory shaker (150 rev min-') at 28OC for 72 h. The titre
of the Rhizobium sp. culture as d e t e d n e d by senai dilution and plating on YEM agar
was ccr. 108 c h ml-1. The SWB cultures were prepared by inoculating one Ioop of a
TSA stock into 100 ml of 1.5% tryptic soy broth in an Erlenmeyer flask which was
incubated on a gyrotory shaker (150 rev min-1) at 28OC for 72 h. The SWB cultures
contained ca. 108 cfu ml-' as determined by serial dilution and plating on 1.5% T'SA.
The nutrient content of the 1:2 soi1:sand mix (wfw)used in this study was as
follows (pg g-1):
N. 1.1; P. 3.8; K, 84.4: S .
1.5: Cu. 0.2; Mn. 1.4: Zn. 0.1: B. 0.3:Fe.
3.2; Cl. 0.6. One kilogram of the thoroughly blended soi1:sand mixture was weighed
into 12-cm d i a pots and autoclaved at 17l0Cfor one hour for w o consecutive days with
a 24-h interval. Pea seeds were immersed in 70% ethano1 for three minutes followed by
soakiag in a 2.5% sodium hypochlorite solution (20% commerciaI Javex Bleach) for
seven mioutes. Seeds were washed thoroughly in several changes of sterile tap water.
placed on 0.3% TSA and incubated at 24OC for up to five days for germination.
Two hedthy, uniform seedlings were placed at 3-cm depth from the soi1 surface
and one milliliter of sterile tap water contaioing ca. 403 decontaminated NT4 spores was
added. For the rhizobial treatments. two milliliters of the LX43 culture were added, and
for the SWB treatments. two milliliters of the appropnate culture were added. For the CO
inoculation treatments. the AMF inoculum was placed on the pea seedlings foIlowed by
the Rhizobium sp. culture andor the S m cultures. Pots were placed in a growth
chamber with the following growth conditions: 25OC 16 h day, 20°C 8 h night. CU. 6 0 8
relative humidity and 375-400 pM m-2 sec-1 of irradiance. Plants were grown for six
weeks. harvested and the shoot and root dry weights. root:shoot (R:S) ratio. number of
nodules, %AMI?-colonized root lengh, the shoot N and P content, and P use efficiency
(Raju et al. 1990) detemiuied.
7.2.7 Statistics
AU percentage values were subjected to angular transformation before statistical analysis.
Quantitative data were analyzed using the ANOVA procedure and means separated u s h g
the LSD test in SAS (SAS, 1997)- Unless mentioned otherwise. al1 treament rnems
were tested for signifcant differences at p-cO.05.
7.3
ResuIts
7.3-1 AMF spore decontamination
Nutrient-rich media, such as TSA, NA and LBA supported growth of spore-associated
bacteria whereas YEA and WA supported growth of si,&cmtly
fewer, if any, bacteria.
Thus. 1006 of non-treated spores yielded bacterial growth when incubated on nunientrich media Fig. 7.3.1). It was noted that it required ca. four to five weeks for all NT4
spores incubated on YEA and WA to yield contaminant growth. This suggests that the
tîme taken for the SWB to proliferate on poor media was much longer than those
incubated on nutrient-rich media. The percentage of "decontaminated spores" s a
contaminated was si@cantly
reduced (Le., from 100% to 1549%) by 60 min.
treament with chloramine-T, indicating that a longer decontamination time was required
g
were
to eliminate the SWB. This observation also suggests that these s u ~ v i n bacteria
either protected inside the spores or else they formed resistant. resting structures.
7 . 3 . 2 Identification of SWB
A total of 65 bacterial colonies were recovered from non-treated NT4 spores incubated on
the different media For exarnple, non-treated NT4 spores incubated on nutrient rich
media such as TSA, LBA and NA yielded 17. 14 and 22 bacterial isolates. respectively.
whereas spores incubated on YEA and WA yielded five and seven bacterial isolates.
respectively. Ten of the 65 bacterial isolates obtained from non-treated spores did not
match entries in the TSBA library. The number (n=51) of bacterial isolates obtained from
NT4 spores decontaminated for 30 min. was not reduced considerabiy from that of
untreated spores (n=65). However, the number of bacterial isolates recovered from the
decontaminated spores incubated on each of the different media varied from that of
30
45
60
Decontamination time (min)
Decontamination time (min)
Fig. 7.3.1.
Mean (n=5) percentage contamination of G. clarum NT4 spores treated
with chloramine-T at 30°C for 0,30,45 and 60 min. as detected on
various media. Spore contamination was measured after (A) 24 h and (B)
72 h incubation on water agar (WA). 0.02% yeast extract agar (YEA),
0.3% üyptic soy agar (TSA), Luria-Bertani agar (LBA) and 0.8% nutrient
agar (NA). Verticd bars represent the least siCpZicantdifference (LSD) at
p<O.OS.
untreated spores. For example. five. seven and five bacterial isolates were recovered
from NT4 spores incubated on TSA, LBA and NA. respectively. whereas. 22 and 12
bacterid isolates were recovered frorn NT4 spores placed on YEA and WA. The number
of bacterid isolates obtained fiom NT4 spores decontamùiated for 60 min. was 35. of
which eight were obtained from TSA plates, 12 were obtained from LBA. six were
obtained from NA, five were obtained from YEA and four were obtained from WA
plates.
The SWB were identified based on cellular FAME profiles. AIthough most of the
S W B isolates were identified, some isolates did not match entries in the TSBA library.
For example. 10 SWB recovered from untrcated NT4 spores and one SWB each from
NT4 spores decontaminated for 30 and 60 min. could not be identified.
A number of different bacterial genera were found on untreated NT4 spores
(Table 7.3.1). For example. soi1 and rhizosphere rnicroorganisrns such as Alcaligenes,
Bacillus spp.. Burkholderia, Flavobacterium and Pseudomonas spp. were found on
untreated NT4 spores (Table 7.3.1). However, when hT4 spores were decontaminated
with chloramine-T for 30 min.. ca. 80% of the SWB were Bacillus spp., suggesting that
the SWB produced resistant, resting structures. Simrlarly, Ca. 89% of the SWB isolates
recovered from NT4 spores decontaminated for 60 min. were Bacillus spp. Interestingly.
no G- bactena were isolated from decontaminated (i-e., 30 and 60 min) NT4 spores.
These Bacillus spp. probably susvive on AMF spores and spore walls by forming
endospores.
7 . 3 . 3 Response of rhizobia to SWB
About 34% (22 of 65) of the bacteria isolated from non-treated NT4 spores stimulated or
inhibited the growth of at least one of the 10 Rhizobium strains or isolates tested. In
some cases. interactions between the SWB and Rhizubi~rmstrains resulted in enhanced
growth of rhizobia (Table 7.3.2). For exarnple. Flavobacteri~rntdei*orarzsTAI b
stimulated the growth of nine of the 20 Rhizobium strains tested. indicating a non-specific
growth stimulation. On the other hand. some of the bacteria ùihibited the growth of the
Rhizobium strains (Table 7.3.2). For exarnple, B. licheniformis T.49 inhibited the
growth of six of the 10 Rhizobium strains tested. Some of the rhizobia strains also
C
influenced the growth of the SWB. For exarnple, the rhizobial strains LX13 positively
(n=52) or negatively (n=3) influenced the growth of 55 SWB isolates. and the rhizobia
strain LX48 positively (n=57) or negatively (n=2) influenced the growth of 59 SWB
isolates. suggesting the involvement of a chemical released by these rhizobial s ~ a i n in
s
the growth response of the SWl3 or vice versa.
The response of the rhizobia to C a . 40% (13 of 35) of the SWB resistant to a 60
min decontamination treatment was both positive and negative. although most of the
interactions had no effect on the gowth of the rhizobia (Table 7.3.3). One of the S m
isolates, Arthrubacter ilicis WA2 enhanced the growth of five of 10 rhizobia strains
tested. On the other hand. some Rhizobium isolates responded to most of the SWB
(e.g., L X 3 md LX48). Interestingly, the Rhizobium isolate LX13 was ineffective on
the growth of pea. whereas isolate LX48 was very effective at enhancing pea growth and
N nutrition under gnotobiotic conditions (see Table 4.3.1).
7.3.4 Response of AMF spores to SWB
A majority of the SWB recovered from non-treated spores did not affect NT4 spores as
profoundly as SWB from decontarninated (Le.. S60 treatment) spores (Table 7.3.4). The
response of NT4 spores to SWB recovered fiom decontaminated spores ranged from
inhibition of spore germination to enhmced hyphal growth. For exarnple. the SWB
isolate B. chitinosporzis LA6a inhibited the germination of NT4 spores, whereas the
Table 7.3.4. Response of Glomus clal-rrm NT4 spores to direct contact. and diffusible
and volatile chernicals produced by spore wall bacteria (SWB). as assessed in a plate
assay.
aResponse of NT4 spores to SWB
Chloriunine-
Bacterial species and isolate ID.
Direct
Diffusible Volatile
Alcaligenes xyZusoxydans TA8a
O
O
I
Bacillus alvei TAla
1
2
1
B. brevis TA22
3
1
1
B. laterosporus LA4
O
O
O
B. lichen$omis TA9
2
1
O
B. licheniformis TA15
Z
O
1
B. macerans NA14
O
I
O
B. megatelium YA6
2
1
1
B. polymyxa TA7a
I
Z
2
Burkholderia pickettii W a
O
f
1
B. solanaceancm NA17
I
2
2
Flavobacreriurn devorum T Al b
1
1
2
Methylobacterium radiotolerans LA7
O
1
1
No Match TA8b
2
2
1
Pseudomonas comgata NA2
O
O
O
P. 9uorescens NA6
O
1
1
P. marginalis NA2Oa
1
1
1
P. sy-ingae TA1 l b
O
1
O
Salmonella choleraesius WAS
O
O
1
P. saccharophila YAS
2
2
O
Variovoraxparacloxus LA1 l a
O
O
O
T matment
Untreated
Bioassays
Table 7.3 -4 (contimed)
aResponse of NT4 spores to SWB
Cldoramine-
Bacterial species and isolate ID.
T treatment
Bioassays
Direct
Diftùsible Volatile
Untreated
Vibrioparahaemolyticus NA%
O
3
2
60 &.
Arthobacter ilicis WA2
2
1
O
B. brevis WA4
O
2
2
B. cfrculans WA3
B. firmus YA5
B. larerosporus LA6b
B. longisporus LA4
B. megaterium LA1
B. megaterium YA3
B. pabulr' LA3
B. pabuli LA5
Listeria monocvtoaenes LA8
2
2
2
T h e effects of direct contacf and diffusible and volatile chemicals produced by SWB
recovered from untreated (NS) and decontaminated (i-e.,60 min.) spores on the
germination m d hyphal elongation of G. clarum NT4 spores after 10 d incubation at
2 7 T . Scores assigned: no spore germination (inhibition), O; genn tube emegence, 1;
slight growth, 2: moderate growth. 3: moderate to good growth. 4: and hyphal g o w t h
more than the diameter of the spore, 5.
SWB isolate B. pab~rliLA3 enhanced the hyphai growth of NT4 spores (Fi:. 7.3.2).
The germination response of NT4 spores to volatile a d o r non-volatile substances (i.e..
direct, diffusible and volatiie assays) released by the SWB appeared to vary significantly.
For example. non-volatile substance(s) (i.e.. direct and diffusible assay) released by the
SWB isolates B. pabuli LA3 and B. firmrrs YA5 s i e g c a n t l y increased the hyphal
.=orowth of NT4 spores. In conuast. volatile and non-volatile substances released by the
SWB isolate B. laterosparus LA6b inhibited NT4 spore germination. Although NT4
spore germination was inhibited by the diftùsible andor voIatile substances produced by
the SWB isolates, stimulation of NT4 spore germination was observed only when spores
were placed in contact with the SWB (direct assay) or when SWB chernicals were
allowed to diffuse to the spores (diffusible assay), indicating that the stimulation of NT4
spore germination involved non-volatile substances.
7 . 3 . 5 Effect of SWB on the tripartite symbiosis
An evaluation of the response of pea to CO-inoculationwith G. clarum NT4 and 20
different Rhizobium strains in a growth chamber study (Section 5.0) revealed that
interactions between the Rhizobium isolate LX43 and the AMF NT4 were very specific,
and resulted in a sie&5cant increase in the g a i n yield and N and P nutrition of pea over
other NT4+rhizobia combinations tested (Section 5.0). ft was hypothesized that the
SWB isolates W and LA6a which positively (e.g., LA3) or negatively (e.g.. L M a )
influenced the germination and hrphal growth of NT4 spores would si,pifïcantly alter the
effectiveness of the NT4 inoculant, done or involved in a tripartite association with
rhizobia and pea. Therefore. the efTect of the SWB isolates LA6a and LA3 was assessed
on the tripartite symbioses between pea (cv. Trapper), G. clarum NT4, and the rhizobial
isolate LX33. in a sterilized soii:sand ( 1 2 ) mix in a growth chamber.
:t
of dift'usible chernicals excreted by the SWB isdates (,A)B. p,
and (B) B. chitiizo.spo~-~r.s
LA6a on the pmiination o f Gioniots c
spores. Spores were observed for germination or hyphal dong
after incubation on 1.5% TSA plates.
Inoculation of pea wïth the AMF NT4 or either SWB isolate had no effect on the
shoot dry weight compared to the uninoculated plants. However. the SWB isolate LA3
si,@fÏcantly
increased the shoot dry weigiit of plants in the presence of the NT4 inoculant
compared to the NT4tLA6a treatment m d control (Table 7.3.5). This suggested that an
enhancernent in A
iMF activity by LA3 enhanced pea shoot growth- Inoculation of pea
with the rhizobial isolate LX43 resulted in the highest shoot biomüss cornpared to al1
other treatrnents (Table 7 - 3 3 . However. CO-inoculationof pea with the rhizobia LX43
and the SWB isolates LA6a or LA3 sipificantly reduced the shoot biomass of plants.
compared to the LX43-inoculated plants. The shoot dry weight of the LX43+LA6a
treatment was siEgnificantIy higher than that of the uninoculated plants. whereÿs the shoot
dry weight of the LX43+LA3 treatment was simrlar to that of the uninoculated plants.
This suggests that interactions between LA3 and LX43 may not be syner=@tic. The
effect of the SWB isolates LA6a and LA3 on the response of pea to CO-inoculationwith
the AMF NT4 and the rhizobia isolate LX43 varied sit;nificantly. For example, pea
inoculated with the combination of NT4+LX43+LA6a yielded CU. 19% more shoot
biomass than the NT4;LX43+LA3 combination (Fig. 7.3.3). Furthemore, the shoot
biomass of the NT4+LX43+ LA6a combination was Ca. 22% signifïcantly higher than the
uninoculated plants, whereas the shoot biomass of the NT4+LX43tLA3 was similar to
that of the uninoculated plants.
Inoculation of pea with the AMF NT4. the rhizobia LX43. or either SWB isolate
had no significant effect on the total root dry weight compared to uninoculated plants
(Table 7.3.5). Co-inoculation of pea with NT4 and LX43 sigmficantly reduced the total
root dry weight of plants compared to the control. The effect of the SWB isolates LA6a
and LA3 on the root dry weight of NT4-inoculated plants was si,pificantly different frorn
one another and the uninoculated connol plants. For example. the SWB isolate LA6a
sipificantly reduced the root biomass of NT4-inoculated plants, whereas the SWB
Fig. 7.3.3.
Effect of the AMF Glomus clarum NT4, the rhizobia isolate LX43 and the
spore wall isolates Bacillus chitinospom LA6a or B. pabuli LA3 on the
growth of pea at six weeks after planting. Plants were grown in a
sterilized 112 soil:sand mix.
isolate LA3 sipiticantly increased the root biomass of plants (Table 7.3.5). Similarly.
the S m isolate LA6a significantly reduced the root dry weight of LX43-inoculated
plants compared to the uninoculated plants, whereas the root biomass of pea inoculated
with the combination of LX43tLA3 was similar to that of the control. Nthough the
effect of the SWB isolates on the NT4 and LX43-inoculated plants was signiiïcüntly
different fiom one another. the root dry weight of plants CO-inoculatedwith NT4. LX43
and the S W isolate M a was not different from that of plants inoculated with the
NTr4+LX43+LA3 combination (Table 7.3 -5)-
The R:S ratio of plants inoculated with only NT4 was similar to that of the
uninoculated plants. indicating that inoculation with NT4 did not reduce host allocation of
carbon to producing roots (Table 7.3.5). Plants inoculated with LX43 or CO-inoculated
with NT4 and LX43 had a similar R:S ratio, but si@llfcantly smaller than the
uninoculated plants. The R:S ratio of pea inoculated with the SWB isolate LA6a. alone or
in combination with the AMF NT4, was not si@cantly
different from that of the
respective treatments involving the SWB isolate L A I In contrast, with or wirhout the
NT4 inoculant, the R:S ratio of plants CO-inoculatedwith the rhizobial isolate LX43 and
the SWB isolate LA6a was significantly smaller thm that of plants inoculated with
LX43+LA3. suggesthg that the SWB isolates altered plant response to the rhizobial
isolate LX43 (Table 7.3.5).
Nodulation was observed on dl the plants inoculated with the Rhizobium isolate
LX43. The NT4 inoculant did not sigdicantly alter the nodulation of pea by LX43
(Table 7.3.5). The SWB isolates LA6a and LA3 did not influence the growth of rhizobid
isolate LX43 in vitro (Table 7.3.3). However, the SWB isolates altered the number of
nodules formed by LX43. For example, ail nodulated plants CO-inoculatedwith the SWB
isolate LA6a had consistently higher levek of nodulation compared to the respective
treatments with LA3 (Le.. LX43 + LA6a vs. LX43 + LA3 or NT4 -r LX43 t LA6a vs.
NT4 + LX43 + LM). sugesting that the effect of the SWB isolates on the rhizobia
isolate LX43 may be related to the noddation process.
Al1 the plants inoculated with the NT4 inoculant were mycorrhizal. However. the
Ievek of A M . colonization were low (Le.. 40%). In contrast to nodulation. inoculation
of pea with the NT4 and rhizobia isolate LX43 significantly increased the % A m colonized root compared to the NT4 treatment reflectinp the positive effect of LX43 on
NT4 (Table 7.3.5). The positive effect of the SWB isolate LA3 on die germination and
hyphd growth of NT4 spores was not only noted in vitro, but also extended to the
intraradical colonization of pea roots by NT4. Thus. the NT4+LA3 combination
signifïcantly increased the AMF colonization of pea compared to the NT4 + LA6a
combination. However, when the AMI? NT4, rhizobia isolate LX43 and the S m
isolates were combined, there were no differences between combinations involving LA6a
and LA3 (Table 7 - 3 3 ,suggesting that the presence of rhizobia isolate LX43 altered the
effect of the SWB isolates on NT4.
Inoculation of pea with the AMI? NT4 had no si3@ficant effect on the shoot N
content However, the rhizobia isolate LX43.alone and in combination with NT4
sipficantly increased the shoot N content, compared to the uninoculated plants. The
effect of the SWB isolates LA6a and LA3 on the shoot N content of plants was not
sitonificantly different from one another. or from the uninoculated plants (Table 7.3.6).
Inoculation of plants widi N T 4 + W increased the shoot N content more rhan the
NT4+LA6a combination, suggesting that LA3 enhanced the uptake of N by NT4 fiom the
soi1 N pool. This also suggests that the means of N availability to the pea plants werz not
resaicted to nitrogen fixation alone. As noted for nodulation, the effect of the SWB
isolates on the shoot N content of LX43-inoculated plants was very different For
Table 7.3.6, Mean (n=5) shoot N and P content and P use efficiency of pea plants coinoculated with the AMF species Glornus dai-um NT4, the Rhizobium legr~rninosamm
bv. viceae strain LX43,and the SWB isolates Bacillus chirinospoius LA6a or B.pabuli
LA3 after 6 weeks of growth in a sterile 1:2 soil: sand mix. LSD- Least Significant
Difference
Treatment
Shoot nutrient content (&pot)
N
P use efficiencya
P
Control
NT4
LX43
NT4 + LX43
LA6a
LA3
NT4 + LA6a
NT4 + LA3
LX43 + LA6a
LX43 + LA3
NT4 + LX43 + LA6a
NT4 + LX43 + LA3
LSD (0.05)
3.3
0.02
222
aP use efficiency is expressed as g P absorbed per g of shoot dry weight
exarnple, plants inoculated with the combination of LX43+LA6a yielded ca. 2 1 52 more
shoot N than the LX43+LA3 combination. and CU, 72% more shoot N chan the control
plants (Table 7.3.6). In contrast. the SWB isolates did not sipificantly alter the shoot N
content of plants from one another in the presence of NT4 and LX43. aithough both
treatments (i.e.. NT4+LX43+LA6a and NT4+LX43+LA3) were noted to have at least
40% more shoot N compared to the control.
The shoot P content of plants inoculated with only NT4 was significantly lower
than that of the uninoculated plants. Inoculation of pea with LX43 with or without the
h T 4 inoculant had a similar effect on the shoot P content. The STVVB isolate LA3
siYglificantlyincreased the shoot P content of pea. compared to the SWB LA6a and the
control. However. the SWB isolates did not benefit the shoot P content of pea in the
presence of the NT4 inoculant In contrast. the SWB isolate LA6a significantly increased
the shoot P content of tX43-inocuiated plants by Ca. 24% over that of the LX43+LA3
combination, and eu. 18% over the control. Similady, the SWB isolate LA6a
significantly increased the shoot P content of plants CO-inoculatedwith NT4 and LX43 by
ca. 215% over the NT4+LX43+LA3 combination, and ca. 11% over the controI.
The PUE of plants inoculated with only NT4 was not different from that of the
uninoculated plants (Table 7.3.6). In contrast, inoculation of pea with LX43. with or
without the NT4 inoculant significantly increased the PUE. The SWB isoiate LA6a
sipificantly ùicreased the PUE of plants compared to the isolate W. but the effect of
these isolates on the PUE was not significantly dit'ferent from that of the control.
enhanced the PUE of NT4-inoculated plaats
However. the SWB isolate LA3 ~i~gnificantly
compared to the NT4+LA6a combination and the control (Table 7.3.6). This su,,~ l ~ e s t s
that the positive effect of LA3 on NT4 spore germination and hyphaI elongation favored
enhanced NT4 hyphal activity in the rhizosphere. There were no sipificant differences
in the PUE between the SWB isolates when CO-inoculatedwith LX43. However. COinoculation of pea with NT4+LX43+LA6a resulted in a Ca. 1 6 8 ùicrease in PUE
compared to the NT4+LX43+LA3 combination. and a ca. 35% increase in PUE
compared to the control treamient.
7.4
Discussion
Several workers have studied the in vina response of AMF spores to rhizosphere bactena
(Azcon, 2987; Rousseau et al. 1996) or spore wall associated microflora (Schenck and
Nicholson. 1977: Daniels and Menge. 1980: Mayo et al. 1986: Walley and Germida.
1996). Others have studied the response of rnycorrhizd plants to SWB (Andrade et aI.
1995; Bethlenfdvay et al. 1997). The present study represents a comprehensive
examination of the presence of bacteria on AMF spores. their effect on the germination
and hyphal growth of AMF spores. and their effects on the tripartite symbiosis between
AMF, rhizobia and a lemme host. In addition. this study dernonstrates that SWB c m
influence crucial processes such as AMF spore germination and hyphal elongation.
contrary to the suggestions of MacDonald and Chandler (198 1).
Occlswence of SWB on AMF spores
The growth of the bacterial contaminants tiom NT4 spores was unrestricted on TSA.
LBA and NA due to the availability of hi& levels of nutrients. In contrat, the growth of
bacterial contarninants from NT4 spores was highly restricted on YEA and WA because
of the low level of readily-available nutrients. These results are in agreement with the
observations made by Walley and Germida (1996) for G. clanrrn NT4 spores.
Furthemore. the percentage contamination of M4 spores incubated on the different
growth media was higher after 72 h than after 24 h. This high level of NT4 spore
contamination by bacteria despite decontamination for up to 60 min. may be explained by
the age of the NT4 culture, (i.e.. 56 weeks). Because of the physical proximity of
bacteria to AMF spores in the soil. AMF cultures stored for a long penod of timz were
probably extensively colonized by bacteria-
Few workers have reportai the occurrence of SWB on AMF spores (Azcon-
Aguilar et al. 1986a: Mayo et al. 1986). For exarnple. Azcon-Aguilar et al. (1986a)
found that a Bacillus sp. isolate was associated with surface-stenlized G.mosseae
spores. Mayo et al. (1986) found that G. versiforne spores were natutdy associated
with Pseudomonas sp. and Corynebacterium sp. n i e bacteBa1 species isolated from
untreated NT4 spores in my study included both Gram positive and Gram negative types.
The Gram positive bacteria included Bacillus spp., whereas the Gram negative bacteria
included many rhizosphere bacteria such as Pseudomonas spp., Burkholderia spp. and
Flavobacterium sp. (Hicks and Loynachan, 1989; Hoflich et al. 1995). In contrast, a i l of
the bactena isolated fiom decontaminated NT4 spores (Le., S30 and S60) were Gram
positive. Of these bacteria, ca. 91 (60 min)-92% (30 min) were endospore-faming
Bacillus spp. The non-spore forming bacteria isolated from decontaminated NT4 spores
included Arthrobacter cin-eus (30 min) and Liste ria monocytogenes (60 min).
Response of rhizobiu ta SWB
In order to fully understand the effect of SWB on the AMF-rhizobia association, the
response of rhizobia to SWB was assessed. Results revealed that some of the SWB
isolates positively influenced the growth of some rhizobial saains tested, whereas some
others inhibited the growth of rhizobia. However, mcst of the SWB isolates had no
effect on the growth of rhizobial strains. Fuhrmann and WoIlurn (1989a) evaluated the
effect of 115 soybean rhizosphere bactena on the growth of five bradyrhizobial strains in
vitro, and found that 23 rhizosphere bacteria inhibited one or more bradyrhizobial strains,
whereas most of them did not have an effect on the rhizobial strains. These authors
suggested that the inhibition of rhizobial strains was caused by siderophore-induced iron
deprivation. Sirnilarly. Hicks and Loynachan ( 1989) assessed the effect of non-specifc
bacteria. actinornycetes. G- bacteria. enterobacteria and pseudomonads isolated from
soybean rhizosphere on two bradyrhizobial sîrains in vitro using an agar-overlay
technique. They found that 31 9 of the isolates were inhibitory. whereas 88 stimulated
bradyrhizobial growth. However, as these different effects on the bradyrhizobial strains
were not observed in situ, the authors described the inhibition or stimulation of
bradyrhizobial growth in vitro as an artefact created by growth on synthetic. energy-nch
media,
Stimulation of N-fixersr gowth by rhizosphere bacteria may be due to growth
factors or oganic acids produced by the rhizosphere bacteria. For example. Holguin and
Bashan (1996) found that aspartic acid produced by a Sîuphylococcus sp. isolate obtained
from mangrove rhizosphere stirnulated nitrogen fixation by Azospirillum sp. in vitro.
Bhalla and Sen (1973) obtauied 102 isolates from the rhizosphere of chickpea plants that
were inoculated with Rhizobium sp. (Cicer) or Ieft uninoculated. and tested the effect of
these rhizosphere bacteria on the growth of Rhizobium sp. (Cicer)in vitro. The authors
found that 39% of the isolates obtained from the inoculated chickpea rhizosphere
sàmulated rhizobial growth, whereas 20% of isolates from the non-inoculated chickpea
rhizosphere sbmulated rhizobial growth. They also found that about 50% of the isolates
requiring amino acids, growth factors, and yeast extract stimulated Rhizobium growth in
v i r a although up to 25% of the isolates inhibited rhizobia. The authors also found that
ceUulose decomposers stimulated rhizobial growth. whereas ammonifiers were inhibitory
to rhizobia. Using a bio tin auxotroph and a wild type strain, Streit et al. (1996)
demonstrated that growth factors such as biotin and other water soluble vitamins altered
the competitiveness of rhizobia colonizing alfaifa roots, and that the addition of vitamins
significantly enhanced the growth and root colonization by R. meliloti. In my study.
most of the bacteria isolated from unîreated spores were cornmon rhizosphere bacteria. In
keeping with the observations of Fuhrmann and Wollum (1989a. b). Holguin and Bashan
(1996) and B h d a and Sen (1973). it is possible that the positive or negative growth
response of some Rhi'obium strains to SWB isolates or vice versa in the present study
rnay be due to the release of arnino acids and/or growth factors by the SWB or the
rhizobia. or alternatively. siderophore production. On the other band. the inhibition of
rhizobial growth by SWB may be due to the production of anti-microbial. or in particular.
substances deleterious to rhizobial growth. Altematively. this effect may be due to
siderophore-induced iron deprivation. Whatever the mechanism(s) cmployed. the results
indicated synergistic and antagonistic interactions between some of the SWB isolates and
the rhizobia tested.
Response of AMF spores to SI.VB
Most of the SWB isolates in my study did not have a marked effcct, whereas some
bacteria completely inhibited the germination of G. clarum NT4 spores. and others
stimulated the germination and hyphal growth of NT4 spores. The germination of &MF
spores can be hastened by the presence of soi1 microorganisms. These organisms rnay
include bacteria (Azcon-Aguilar et al. 1986a; Mayo et al. 1986). fiingi (Schenck and
Nicholson, 1977; Azcon-Aguilar et al. l986b) or actinomycetes (Mumer and Mosse.
1987: Carpenter-Boggs et al. 1995). Many of these microorganisms were isolated from
the rhizosphere (Mosse, 1959; Schenck and Nicholson. 1977: Azcon-Aguilar et al.
1986a: Mugnier and Mosse, 1987: WaUey and Gemlida. 1997), although some are
closely associated with outer layers of AMF spores (Tommerup. 1985: Azcon-Aguilar et
al. 1986a; Mayo et al. 1986). In contrat to other studies, the present study assessed the
effect of bacteria isolated from the spore surface or deep-seated within ,4MF spore walls.
In considering previously published reports on the effect of microorganisms on AMF
spore germination. it is not totally surprising that some of these bacteria isolated from
non-treated spores which are common rhizosphere bacteria. influenced the germination of
NT4 spores.
Azcon-Aguilar and Barea (1992) proposed three dBërent mechanisms for the
stimulatory effects of microorganisms on AMF spore prmhation and hyphal elongation.
These include (i) removaI of toxic material (i-e.. high Ievels of toxic elernents such as Mn.
Zn, etc.) from the medium, (ii) the unlization of the self-inhibitors (Watrud, 1978)
produced by AMF by other microorganisms, and (iii) the production of stimulatory
compounds. Stimulatory compounds may include volatile (Mugnier and Mosse. 1987:
Carpenter-Boggs et al. 1995) or non-volatile compounds (Azcon, 1987). For example,
Mugnier and Mosse (1987) found that a volatile substance produced by Streptomyces
orientalis stunulated the gemiination of G. mosseae spores in vino. Io contrast Mayo et
al. (1986) and Azcon (1987) found that germination of AMF spores was stimulated in the
presence of bacterial cell-free supematants, which suggest a stimulatory effect by
diffusible chernicals produced by bactena. Azcon-Aguilar et al. (19 86a) found that the
number of vegetative spores formed by G. nosseae was sunilar to that obtained for G-
caledonicrrm supplied with organic substances such as vitamins and amino acids by
Hepper (1979), which are also excreted by soi1 microorganisms (Lynch, 1976). In my
snidy, the stimulation of NT4 spores occurred only when the spores were in direct
contact (i-e.. direct assay). or were allowed contact with diffusible chernicals from the
bacteria (i.e.. diffusible assay). This suggests that the stimulation of NT4 spore
eermïnation and hyphal eiongation occurred as a result of amino acids or other growth
V
factors such as vitamuis excreted by the spore wall bactena.
Parasitization of f u n p l spores Ieads to a reduction in spore germination (Old and
Wong. 1976; Sneh et al. 1977: Daniels and Menge. 1980; Toyota and Kimura, 1993).
The presence of certain groups of microorganisrns can inhibit AMF spore gemination
(Stouky. 1972: Tommerup. 1985: Wilson et al. 1989: Walley and Germida. 1997). For
example. Stoaky (1972) suggested that the inhibition of fungal spore germination is due
to 'mycostatis'. a biological phenornenon. Wilson et al. (1989) noted that AMF spore
germination was suppressed in non-sterile soil. and suggested that it rnay be due to the
presence of microorganisms in a suppressive soil. In my study. the germination of NT4
spores was inhibited by some bacteria (e.g., the SWB isolates LA6a or LA6b resistant to
60 mui. decontamination). Using an identical plate assay. Walley and Germida (1997)
found that G. clarum NT4 spore germination was inhibited in the presence of a
Pseudomonas putida straui. and suggested that a non-volatile substance released by the
pseudomonad rnay have inhibited germination. Le Tacon et al. (1983) and Becard and
Piché (1989) reported that s m d increases in the atmospheric CO2 concentration (i-c.. up
to 0.58) stimulated the germination and hyphal gowth of AMF spores. but higher leveis
were detrimental. increases in CO2 concentration rnay be simply due to respiration. In
keeping with these hdings. it is possible that the deleterious effect of some SWB isolates
in rny study, may have been due to high levels of CO2 merely as a result of microbial
respiration, or alternatively, due to the release of toxic volatile chernicals by the SWB
isolates, although the latter appears to be more plausible.
E-fect of SWB on the AMF-rhkobia-pea syrnbiasis
Due to their effect on the germination of AMF spores, SWB c m influence the growth of a
legume host colonized by AMF and rhizobia. Andrade et al. (1995) found that a
rhizobacterium most closeiy resembling Bacillus simplex isolated from surface-stenlized
G. mosseae spores (Azcon-Apilar et al. 1986a). did not significantly impact pea biomass
production. However. the authors found that when the rhizobacterium was combined
with G. mosseae, plant growth was reduced by 30%. suggesting that it may be a
deleterious rhizobacterium. In my study. the SWB isolates LA6a and LA3 had no effect
on pea shoot biornass production. compared to uninoculated plants. However.
CO-
inoculation of pea with the S m isolate LA3 and the AMF N T 4 significantly increased
the shoot biomass. percentage AMF-colonized root. shoot N content and P use efticiency
of plants over the NT4+LA6a combination. The increase in the shoot biomass of the
NT4tLA3-inocdated plants is probably the result of an increase in the percentage AMFcolonized root, shoot N content and enhanced PUE, Barea et al. (1987. 1989)
demonstrated using mycorrhizal alfalfa plants that the A I hyphae c m absorb N from
the soi1 N pool, which is not readily available to non-mycorrhizal plants. It is possible
that the NT4+LA3-inoculated plants not only enhanced the PUE of the plants. but also
enhanced the N uprake frorn soil, resulting in the enhancement of shoot biornass. This
was probably due to positive effect of LA3 on G.clarzcm NT4 spore germination and
hyphai elongation.
The positive effect of LA3 on NT4 spores was eliminated when plants were coinoculated with the AMF NT4 and the rhizobia LX43. In fact, the combination of
NT4+LX43+LA6a significantly increased the shoot biomass, nodulation. shoot N and P
content and PUE of pea cornpared to the W4+LX43+LA3 combination. The effect of
LA6a on the tripartite symbiosis between the 14PvIF NT4. the rhizobia strain LX43 and pea
may be explained as either the effect of LA6a on NT4 spores or on LX43. The SWB
isolate LA6a inhibited the germination of NT4 spores, and had no effect on the growth of
LX43 in vitro. However, the nurnber of nodules and the shoot N content of plants
inoculated with the combination of NT4+LX43+LA6a were significantly higher than the
NT4+LX43+LA3 combination. Kucey and Paul (1982) found that G. rnosseae enhanced
the nodule biomass of bean pIants and nitrogenase activity of R. phaseoli. Louis and Lim
(1988) and Kaur and Singh (1988) found that effects of AMF on nodulation were
correlated with plant growth responses. In my study, the shoot biomass. nodulation.
shoot N or P content of the NT4+LX43 tteatment was not significantly different with or
without the SWB isolate LA6a. Therefore, it appears that the negative effect of LA6a on
h l 4 spores NI vin-Owas not significant enough to impact on NT4 spore germination in
sinr,or in the presence of the rhizobial straîn LX43.
On the other hand, it is possible that synergistic interactions between the rhizobial
strain LX43 and the SFVB isolate LA6a in situ (as confirmed by enhanced nodulation and
N-fixation) may have enhanced the growth and nutrition of pea in spite of the negative
effect of LA6a on NT4 spores. compared to the NT4+LX43+LA3 combination. The lack
of an effect of i A 6 a on LX43 in vitro, but a positive effect on the nodulation and nitrogen
fixation by LX43 indicates that the positive effect on LA6a on LX43 was through the pea
host or on the nodulation and nitrogen fixation process. For example, growth factors or
organic acids such as hormones (e-g., IAA) produced by microorganisms c m
sipificantly increase the number of root hairs, which in tum can enhance sites for
nodulation, resulting in enhanced nitrogen fixation. Altematively. it is possible that
metabolites released by LA3 were deaimental to the Rlzizobium strain, resulting in the
reduction of nodulation and shoot N content of pea compared to LA6a. which resulted in
the reduction of shoot biomass.
UntiI a few decades ago. A M . spores were considercd as independent enaties.
However, many workers have s h o w that bacteria or bacteria-like organelles are
intimately associated with AMF spores, and have the capacity to stimulate or inhibit the
germination of AMF spores. My snidy has shown that different types of bacteria are
intimately assocïated with AMF spores, some of which not only have a significant impact
on AMF spore germination and hyphal elongation, but also influence interactions benveen
AMF. rhizobia and their host plants.
8.0
GENERAL DISCUSSION AND CONCLUSIONS
Several groups of microorganisms interact with plants in vanous ways. Some organisms
reniain on the root exterior or in the rhizosphere. whereas some inhabit the root system.
Some organisms enhance plant growth. whereas othen are causal agents of various plant
diseases. The AMF are a special g o u p of fun@ which inhabit the roots of compatible
host plants and 5 pically enhance plant growth. Mechanisms of plant growth
enhancement include enhanced host nutrition, enhanced disease resistance and protection
against saluiity and drought A popular theory among mycorrhizologisists is that the AMF
are not capable of biosynthesis of severd important enzymes which precludes them from
saprophytic growth. Therefore. AMF are termed obligate biotrophs. The AMF meet all
of their C requirements through host photosynthates which are received mostiy through
the arbuscules (tree-2ike branched intracellulx hyphae). It is widely bebeved that the
AMF originated approximately 450 million years ago. and were instrumental in plants
coloniting land (Simon et al. 1993a). Although the AMF were observed over 100 years
ago (Frank, 1885). the interest in AMF research is still very high, because the full
potential of these fun@ for plant growth enhancement is not knom. Difficulties
encountered in the accilrate interpretation of studies involving A M F are because of their
oblîgately biotrophic status on plants. Nonetheless, the AMF are a large component of
the ecosystem and are capable of sigificantly influencing nutrient cycling. plant
community succession and survival.
Various grain and specialty crops such as legumes are often inoculated with
diazotrophs such as rhizobia, PGPR. and PSB, to reduce high inputs of firtilizers. or to
enhance the availabiliv of readily-available nutrients. and enhance plant productivity (Raj
et al. 1981; Rennie and Hynes, 1993; Brockwell et al. 1995: Hynes et al. 1995).
However. field-grown legumes typically form nipartite symbioses with A M F and
rhizobia. It has been proposed that the AMF symbiont provides phosphorus required for
the rhizobia-mediated nitrogen fixation process. whereas the rhizobid symbiont provides
adequate N for the host to support both rnicrosyrnbionts (Azcon-Aguilar and Barea
1992). Therefore. an effective tripartite symbiosis between AMF. rhizobia and a legume
host results in the enhancement of growth. yield and nutrition of the host. and suggests
that the three partners in the tripartite symbiosis m u t be compatible. This compatibility
has been proven using pea mutants by Duc et al. (1989). Balaji et al. (1994) and Sagan et
al. (1995). These workers found that Nod- pea mutants were also Myc-. suggesting that
both traits are controlled by one gene. Althou& several workers have CO-inoculated
legumes with AMF and rhizobia (Azcon et al. 1991: Ianson and Linderman. 1993:
Ahmad, 1995: Saxena et al. 1997; Ibijbijen et al. 1997). very few workers have studied
the importance of the individual microsyrnbionts or the interactions between the host and
microsyrnbionts in the outcome of the tripartite symbiosis flanson and Linderman. 1993:
Ahmad, 1995). My thesis assessed factors such as the efficacy of the microsymbionts
(Le., AMF and rhizobia), the influence of phosphorus and the effect of other associated
microorganisms on the functioning of the tripartite symbiosis. in order to detemine if
specific combinations of AMF and rhizobia influenced the productivity of pea and lentil.
These objectives were accomplished in a series of growth chamber studies.
The AMF colonization of plants is thought to be regulated by the host plant
(Gianinazzi-Pearson. 1996). On the other hand. this can be interpreted as host
susceptibility to AMF colonization. Therefore. in order to assess whether AMF
specitically interacted with pea and lentil. seven lentil and five pea field sites were selected
across Saskatchewan, and soi1 and intact plant samples were collected. The average
number of viable AMF spores at the lentil field sites was higher (i.e.. 8 g Lsoil) than that
at the pea field sites (i.e., 5 g-1soil). Similady, the average AMF colonization of roots
was slightly higher for lentil than pea This difference behveen the lentil and pea sites
was probably because of the higher dependency of lentil dian pea on mycorrhizae.
Similar results confirming enhanced spomlation under a mycotrophic host rather than ü
facultatively mycomhizal host have been documented (E3oyetchko and Tewari. 1990:
Simpson and Daft, 1990).
The affinity of a host for AMF is generally refened to as AMF-host selectivity or
A m - h o s t specificity. This implies that the host allows preferential colonization of roots
by some AMF, which may enhance plant gowth or yield. In spite of its importance.
AMF-host selectivity has rai-ely been studied (Rosendahl et al. 1989: Bever et al. 1996).
In order to examine whether pea and lentil exhibit AMF-host selectivity, AMF in the
rhizosphere of pea and lentil (SOStrap) and the AMF colonizing pea and lentil roots (root
trap) were trapped using a sorghum-sudangrass host. The number of AMF spores in the
soi1 traps was higher than that in root traps. Furthemore. the oumber of AMF spore
morphotypes was generally higher in the soil traps than in the root traps. indicating that
the pea and lentil hosts had selected AMF from the rhizosphere to preferentially colonize
their roots. In addition, more AMF spore morphotypes were observed in one of the 12
root trap cultures than in the soi1 trap cultures, suggesting that certain AiMF species were
enriched in the endorhizosphere of the host This study demonstrates that pea and lentil
interact specifically with AMF, and that lentil and pea exhibit selectivi~towards M F .
This is an important trait that determines the use of the best available AMF species for
inoculation purposes.
The most suitable AMF species ideally would colonize host roots and enhance
host growth and nutrition. It was determined that one AMF morphotype with bright
yellow spores and a diameter of 130 pm was found in a l l of the root trap cultures. and
that this morphotype was apparently identical to the AMF species identifiéd previously by
Talukdar and Germida (1993a) as most closely resembling Glomrs c [ u r ~ m
NT4 (INVAM
no. SA10 1). Furthemore. Talukdar and Germida ( 1993a) found that the G. ch-wn NT4
spore type was found in dl the Saskatchewan qricultural soils they sampled. suggesting
that this AMF species was prevalent in Saskatchewan soils. Talukdar and Gemida
(I993a) also reported that in addition to G. clanim NT4. G. mosseae NT6 was found in
alL the soils they sampled. These AMF species si@cantly
increased the growth and
yield of wheat ard lentil in a stenlized soil:sand mix (Taiukdar and Germida. 1994).
Furthemore. the NT4 inoculant increased the yield of wheat and Ientil in non-sterile soil
containing indigenous AMF with or without the application of P fertilizer (Xavier and
Germida. 1997. 1998). Hoivever. the effect of these AMF species on pea is not known.
Therefore, because of their prevalence in Saskatchewan soils and their positive effect on
host plants. G. clarum NT4 and G. mosseae NT6 were selected as the AMF species to be
studied in association with rhizobia and, pea or lentil. Similar to the finduigs of Talukdar
and Germida (19941, G. clamm NT4 and G. mosseae NT6 influenced the growth. yield
and nutrition of pea and lentil in my study. However, Talukdar and Germida (1994)
reported that NT4 was more effective than NT6. 1found that the NT6 inoculant was
more effective on pea and lentil than NT4 when inoculated alone. Talukdar and Germida
(1994) assessed the effect of NT4 and NT6 in a stenlized soil: sand mix, whereas I
assessed the effect of NT4 and NT6 in a soil:sand mix. It is possible that cornpetition
between the introduced and indigenous AMF in soi1 altered the efficacy of the NT4
inoculant in my study. However, the NT4 inoculant was more effective on lentil whçn
CO-inoculatedwith different rhizobia saains. This suggests that interactions between G.
clarum NT4 and the rhizobia strains were very specific and influenced the productivity of
peü and lentil. A positive interaction between AW and rhizobia is a desirable trait,
because seldom is a legume host not colonized by AMF and rhizobia
The presence of AMF structures such as hyphae and arbuscules within host roots
provides ample evidence that the ANIF is compatible wïth the host. and vice versa.
Although the presence of M c m be verified using various techniques such as
isoenzyme analysis (Sen and Hepper. 1986). diagnostic enzyme analysis (Rosendahl et
al. 1989) and DNA analysis (Simon e t al. 1993b). AMF presence in roots is usually
deterrnined using rnicroscopy. However. recently. FAME biomarkers have been used for
the identification of various groups of rnicroorganisms (Yang et al. 1993: J m i s and
Tighe, 1994; Bentivenga and Morton. 1996; Olsson et al. 1995. 1997). For example.
Olsson et al. (1995) estimated the biornass of AMF in soi1 ushg rnicroscopy. ATP
measurements and fatq- acid analysis, and found that the amount of FAME 16:1 o5c was
correlated with the length of mycorrhizd hyphae and the amount of ATP in soil. The
FAME biomarker technique was used in my study (i) to examine the relationship between
the presence of AMF within roots as determined ushg microscopy and the FAME
biomarker and (ü) to determine if the 16: 1 w5c could be used as a biornarker for AMF
identification. Sorghum-sudangrass roots from the soil trap cultures were readily
colonized by the AMF in the rhizosphere soil. Analysis of the roots from the soil aaps
also revealed the presence of the FAME biomarker. It was determined that four of the 12
second cycIe maize root trap cultures contained AMF using rnicroscopy. but eight of these
12 root traps contaïned the FAME biomarker. Furthermore, a significant positive
correlation (ra.79; pc0.01) between AMF cotonization and the FAME biornarker was
noted for the second cycle rnaize root trap cultures. Similar to the fmdings of Olsson et
al. (1995. 1997). these results suggest that the FAME biomarker 16:l o 5 c rnay be
effectively used to determine the presence of AMF within roots without using a
microscope. Altematively. the FAME biomarker may be used as a suppleme~tarytool to
determine the level of AMF colonization witiiin roots.
As an important component that provides N for the o v e r d functioning of the
tripartite symbiosis. the selection of suitabIe rhizobial strains is crucial. The rationale for
the selection of rhizobial strains in CO-inoculationstudies has been strain avdability or
effectiveness of the rhizobial strain (Azcon et al. 1991:Ahmad. 1995). However. under
fieid conditions. the efficacy of an effective rhizobial strain may be reduced by various
biotic and abiotic factors (Gibson, 1967: Richardson and Simpson. 1989: Zahran and
Sprent. 1986; O'Hara et al. 1988: Thies et al. 1991). Therefore. it is important to
understand the interactions between an infenor rhizobial strain and other partners in the
tripartite symbiosis- However, there are no reports on the effectîveness of a tripartite
association between AMF, a legurne host and an inferior rhizobial strain. To this end.
two commercial rhizobial inoculants. eight reference stmïns and 40 root nodule bacteria
isolated from field-grown pea were evaluated on pea (cv. Trapper) under gnotobiotic
conditions. Similarly, two commercial rhizobid inoculants, 8 reference strains and 44
root nodule bacteria isolated from field-grown lentil were evaluated on Ientil (cv. Laird)
under gnotobiotic conditions. Based on their effectiveness on pea and lentil growtli and
nutrition, 10 rhizobial strains for pea and nine rhizobial strains for lentil were selected.
Of these rhizobial strains, fourfor pea and three for lentil were ineffective.
Several workers have shown that there are specific interactions between AMF and
rhizobia which influence the outcome of the h i p h t e syrnbiosis (Azcon et al. 1991:
Ahmad, 1995). Therefore, compatibility between the partners in a tripartite association
usually results in enhanced host growth and yield. I found that (i) interactions between
the AMF species and the rhizobial sîrains were specific, (ü) the effectiveness of an
inferior rhizobial strain was enhanced by a superior AMF species and vice versa. and (iii)
growth and yield increases in an effective aipartite symbiosis were associated with
increases in host N and P nutrition, but not AMI? colonization of roots. This study also
confkned the findings of Azcon et al. (199 1) and Ahmad (1995) that AMF and rhizobia
hteract very i n h a t e l y with the host and one another. and that these interactions c m
~i~onif-~cancly
alter the host productivicy. However. it appeared that dthouph the
productivity of pea or lentil inoculated with an ineffective rhizobial snain was higher
when CO-inoculatedwith an effective AMF species. plant productivity was not enhanced
over that of plants inoci:lated with an effective rhizobid strain. This suggests that the
contribution of the rhizobial symbiont in the pea and lentil aipartite symbioses was more
thm that of the AMF symbiont The use of rhizobial or plant mutants in an effective and
an ineffective tripartite symbiosis may provide additional information on the role of the
individual partners involved in the association.
Phosphorus is an important element which influences the rhizobia-legrne and
AMF-plant symbioses (Azcon and Barea, 1992). The importance of P in the nitrogen
fixation process c m not be overstated, because 16 ATP molecules are requircd for the
reduction of one molecule of nitmgen to arnmonia (Atkins and Rainbird. 1982). On the
other han& very low or high P levels deter AMF activity. but medium P levels enhance
the AMF-mediated benefits (Same et al. 1983; Amijee et al. 1993; Thomson et al. 1986;
Medina et al. 1988). Various workers have studied the effect of P f e d i z e r application on
the tripartite syrnbiosis (Smith and Daft 1977: Asimi et al. 1980; Bethlenfalvay et al.
1982). The key findings in these studies are (i) application of P fertilizer enhances the
productivity and niaogen fixation of dual symbiotic plants and (ii) high or low P levels do
not benefit the tripartite symbiosis. but medium P levels enhance the productivity of a
dua' symbiotic host. 1assessed the influence of P levels on the pea and lentil tripartite
symbiosis in a growth chamber study. The results of my study agree with the
observations made by Smith and Daft (1977). Asimi et d.(1980), and Bethlenfalvay et
d.(1982). However. in addition, I found that rhizobia-inoculated or AMF-trhizobia
inoculated pea plants yielded a similar amount of grain as that of plants inoculated with 20
mg P kg-1 soil, whereas rhizobia-inoculated or AMF+rhizobia-inoculated lenhl plants
yielded more grain than plants receiving 20 pprn of P fertilizer. Furthemore. I found that
application of P fermizer had no effect on the growth of pea inoculated with either of the
two AMF+rhizobia combinations. suggesting that factors other than P availability such as
the PUE of the host influenced the outcome of this study. In contrast. the application of
20 pprn of P fertilizer increased the grain-jield and N and P nutrition of die lentil plants
inoculated with the efièctive NT4tLX77 combination. whereas only 10 ppm of P
tèrmizer was required for the same response in NTJ+PB 101-inoculated lentil plants.
These results suggest that the application of P fertilizer enhanced the productivity of lentil
plants. but not pea plants.
Lentil has thicker root hairs than pea, and therefore. is more dependznt on
rnycorrhizae than pea for nutrient absorption. In mycotrophic hostç. the AMF extemal
rnycelium acts as the nutrient a b s o r b a organ or an extension of the root hair. Although
the same level of P fertilizer is recommended for pea and lentil grown in Saskatchewan
(i.e., 12.5 pprn), the addition of P fertilizer in my snidy benefited the dual symbiotic lentil
host and not the pea host. Furthemore, the lentil host inoculated with the effective or
ineffective AMF+rhizobia combination utilized the applied P more efficiently than the pea
host. Therefore. becausz Iegumes are invariably colonized by indigenous AMF and
rhizobia of varyhg effectiveness in the field. it may be important to consider the effective
utilization of applied P fertilizers by the host on a legume-by- legurne bais.
It is well accepted that AMF spores are in contact with various rhizosphere
microorganisms when present in soil. Depending upon the interactions between the A V F
spores and the microorganisms, AMF spore germination may be inhibited or stimulated
(Schenck and Nicholson, 1977: Mayo et al. 1986; Mugnier and Mosse. 1987; Walley and
Gemida, 1997). These effects are usually mediated by volatile or non-volatile chernicals
released by the associated organism (Mayo et al. 1986; Mugnier and Mosse. 1987;
Wdley and Germida. 1997). Therefore. AMF spores may not be considered ÿs
independent entities. When the germination of an AMF spore is affected by the
associated organisrn. the potential contribution of the AMF to the tripmite symbiosis may
be altered. This was my rationale for studying the effect of AMF spore wall bacteria
( S m ) on the tripartite symbiosis.
Some of the SWB bacteria isolated from NT4 spores stimulated the germination
of "dean" NT4 spores, whereas others inhibited NT4 spore germination. Two SWB
isolates which stimulated or inhibited AMF spore germination were combined with an
effective AMF+rhizobiatpea association. The stimulatory SWB isolate signif5cantly
enhanced the growth, N content and PUE of NT4-inoculated pea, compared to the
inhibitory SWB isolate. suggesting that the in vitro stimulatory effect of the SWB isolate
benefited the host. In contrast, the response of NT4-inoculated pea to the stimulatory
SWB isolate was altered when plants were CO-inoculatedwith the rhizobial simin LX43.
This sugeests that either the interactions becween the stimulatory SWB isolate and the
rhizobial strain were antagonistic or the interactions between the inhibitory SWB isolate
and the rhizobial strain was synergistic. Since synergistic interactions between ihe
inhibitory SWB isolate and the rhizobial strain resulted in significmtly higher levels of
nodulation and N and P content, the latter hypothesis appears to be more plausible.
These results also imply that the S m can influence the outcome of the tripartite
symbiosis. and by various means, Le.. through AMF or rhizobia.
L e p r n e growth and yield in the field can be altered by various abiotic factors such
as temperature. pH. drought, salinity and nutrient levels. On the other hand, biotic
factors such as inwractions between the host and soil rnicroorganisms also siC$nificantly
influence plant growth and yield. The results of this snidy show that AMF also interact
intimately with both pea and lentil, and Se@5cantly alter the interactions between rhizobia
and the legume host Interiistions between AMF. rhizobia and legumes are also modified
by the application of P fertilizer. Whereas the AMF+rhizobia+lentil association benetïted
from P fertilizers. the application had no effect on the AiMF+rhizobiatpea association.
This indicates that legume productivity can be further enhanced by not only selecting
appropriate combinations of AMF and rhizobia. but also applying appropriate mounts of
P f e a z e r s . Furthemore. as shown in this study. legume hosts respond very differently
to root-colonizing endophytic microorganisms which influence their productivity. The
magnitude of growth and yield increases due to CO-inoculationwas greater in Lentil than in
p e a This difference between the pea and lentil hosts in their response to AMF and
rhizobia was probably because lentil was more dependent on mycorrhizae than pea
The a x a under pea and lentil cultivatiori in Saskatchewan has been increasing
steadily over the years (Agriculniral Statistics-1996, 1997). Pea and lentil are invariably
inoculated with rhizobial inoculants in Saskatchewan to enhance their yield and grain N
content (Hjmes et al. 1995). Kucey and Paul (1983) and Talukdar and Germida (1993a)
have reported that Saskatchewan soils contain active AMF communities that colonize the
roots of various grain and le,we crops. Although Saskatchewan crops are not
inoculated with AMF inoculants, the hi& levels of AMF propagules interact with hosts
and alter their productivity (Xavier and Germida, 1997, 1998). Since field-grown
legumes intimately associate with AMF and rhizobia which drain host carbon for their
growth requirements. an ineffective tripartite association may lead to severe yield ioss.
Therefore. it is important to assess the effect of microsymbiont eKicacy on host
productivity. Furthemore. as shown in this study. the efficacy of the AMF can
signficantly alter the response of the legume host to the rhizobial inoculant. This has
direct implications for the commercial rhizobial inoculants applied to legume crops.
It has been hypothesized that the AMF-legume and rhizobia-legume symbioses are
controlled by one plant geoe (Duc et al. 1989; Gianinaui-Pearson. 1996). For example.
Duc et al. (1989) and Balaji e t al. (1994) using pea mutants showed that a functional
rhizobial symbiosis was necessary for rnycorrhizae formation. In contrast. Wyss et ai.
(1990) and Xie et al. (1995) using soybean mutants demonstrated that a f'unctiond
rhùobial symbiosis is not essential for rnycorrhizae formation. It is not clear whether
these contlicting hypotheses were the result of the use of different plant genotypes (Le..
pea vs soybean). In my study. interactions between rhizobia and the AMF were very
specific, and were not influenced by the effectiveness of either microsymbiont. This
suggests that rnicrosymbiont interactions and microsymbiont interactions with the host
were probably regulated at the genetic level. Additional studies conducted under
controlled conditions Le., in systerns without indigenous AMF or rhizobia. uniform
growth conditions, and multiple host-microsymbiont ccmbinations. using rhizobia md/or
plant mutants may provide conclusive answers to whether the AMF-legume and rhizobia-
Legume symbioses are regulated by one gene.
9.0
REFERENCES
Abbott, L, K. and Robson. A. D. 1977. The distribution and abundance of vesiculararbuscular endophytes in some western Australian soils. Australim Journal of
Botany 25: 515-522.
Anonymous, 1997. &ricultural Statistics- 1996, Saskatchewan Agriculture and FoodStatistics Branch, 3085, Albert Street. Regina, SK. 146 pp.
Ahiabor, B. D. and Hirata. H. 1995. Influence of growth stage in the association
between some tropical legurnes and two variant species of Glumus in an Andosol.
Soil Science and Plant Nutrition 41: 481-496.
Ahmad, M.H. 1995. Compatibdity and CO-selectionof vesicular-arbuscular mycorrhizal
fun@ and rhizobia for tropical legumes. Critical Reviews in Biotechnolom 15: 229239.
Allen, O. N. and Allen. E. K. 1981. The Leguminosae. A Source Book of
Characteristiccs. Uses and Nodulation. University of Wisconsin Press, Madison.
WI. 812pp.
Ames, R. N.. Thiagarajan. T. R., Ahmad. M. H. and McLaughlin. W. A. 1991. Coselection of compatible rhizobia and vesicular-arbuscular mycorrhizal fun$ for
cowpea in sterilized and non-sterilized soils. Biology and F e m t y of Soils 12: 112116.
Amijee, F., Tinker, P. B. and Stribley, D. P. 1993. The development of
endornycorrhizal root systems. W. A detailed snidy of effects of soil phosphorus
on colonization. The New Phytologist 123: 297-306.
Anand. R. C. and Dogra. R. C. 1997. Comparative efficiency of Rhizobium/
Bradyrhizobium spp. suains in nodulating Cujurzus cujun in relation to chuacteristic
metabolic enzyme activities. Biology and Fertility of Soils 24: 283-287.
Andrade, G., Azcon. R. and Bethlenfalvay. G. 1. 1995. A rhizobacterium modifies
plant and soil responses to the mycorrhizal fungus Glomi~smsseue. Applied Soil
Ecology 2: 195-202.
Asimi. S., Gianinazzi-Pearson. V. and Giariinazzi. S. 2980. Influence of increasing soi1
phosphorus levels on interactions between vesicular-arbuscular mycorrhizae and
Rhizobi~mzin soybeans. Canadian Journal of Botany 58: 2200-2205.
Atkins, C. A. and Rainbird. R. M. 1982. Physiology and biochemistry in biological
nitrogen fixation in legumes. In: Advances in Agncultural Microbiology (N. S.
Subba Rao ed.), Butterworth. London, pp. 26-52.
Azcon, R. 1987. Germination and hyphal growth of Glorn~~srnosserre
in vitro: effects of
rhizosphere bacteria and ceU-free culture media Soil Biology and Biochemistry 19:
417-419,
Azcon, R. 1989. Selective interaction between free-li~lngrhizosphere bacteria and
vesicular-arbuscular mycorrhizal fun@. Soi1 Biology m d Biochemisny 21 : 639644.
Azcon, R. and Barea, J. M- 1975- Synthesis of auxins. gibberefins and cytokinins by
Azotobacter vinelandii and Azotobacter beijerinckii related to effects produced on
tomato plants. Plant and Soil 43: 609-619.
Azcon. R. and Barea, J. hl. 1992. Nodulation. Nî fixation ( I ~ N ) and N nuuition
relationships in rnycorrhizal or phosphate-arnended alfaifa plants. Symbiosis 12:
33-41.
Azcon, R. and El-Atrach, F. 1997. Influence of arbuscular mycorrhizae and phosphoms
fernlization on gowth. nodulation and N2 fixation (l%) in Medicago sativa at four
salinity levels. Biology and Feaility of Soils 24: 8 1-86.
Azcon, R.. Barea, J. M. and Callao. V. 1973. SeIection of phosphate-solubilizing and
nitrogen-fixing bacteria for using as biological fertilizers. Cuad Circular of Biology
2: 23-30.
Azcon. R,. El-Atrach. F. and Barea. J. M. 1988. Influence of mycorrhiza vs. soluble
phosphate on growth, nodulation and IV2 fixation (15N) in alfalfa under different
levels of water potential. Biology and Fertility of Soils 7: 28-3 1.
Azcon. R., Rubio, R- and Barea. J. M. 1991. Selective interactions between different
species of mycorrhizd fun@ and Rhizobi~mmeliloti strains. and their effects on
growth, N2-fixation (15N) and nutrition of Medicago sunvu L. The New Phytologist
117: 399-404.
Azcon-Aguilar, C. and Barea, J. M. 1992. Interactions between mycorrhizal fun$ and
other rhizosphere microorganisms. In: Mycorrhizal Functioning: An Inteptive
Plant-Fungal Process (M. F. Men. ed.). Chapman and Hall. New York. pp. 163198.
Azcon-Aguilar, C., Barea. J. M.. Azcon. R. and Olivares. J. 1982. Effictiveness of
Rhizobium and VA mycorrhiza in the introduction of Hedysarurn co>vnariumin a
new habitat. Agriculture and Environment 7: 199-206.
Azcon-Aguilar. C.. Diaz-Rodriguez, R. M. and Barea, J. M. 1986a Effect of soi1
microorganisms on spore germination and growth of the vesicular-arbuscular
mycorrhizal fungus Glomus mosseae. Transactions of the British Mycological
Society 86: 337-340.
Azcon-Aguilar, C., Diaz-Rodriguez. R. M. and Barea. J. M. 1986b. Effect of free-living
fiingi on the germination of G. mosseue soil extract In: Physiological and Genetical
Aspects of Mycorrhizae (Gianinazzi-Pearson and S. Gianinaui eds.). INRA. Pans.
pp. 515-519.
Azcon-Aguilar, C.. Gianinazzi-Pearson. V.. Fardeau, J. C. and Gianinazzi. S. 1986c.
Effect of vesicular-arbuscular rnyconhizal fun3 and phosp hate-solubilizing bac tena
on growth and nutrition of soybean in a neutrd-calcareous soil amended with 32P45~a-uicalciumphosphate. Plant and Soi1 96: 3- 15.
Baas. R. 1990. Effects of Glomusfasciculatum and isolated rhizosphere
microorganisms on growth and phosphate uptake of Planrugo major ssp.
pleiosperma. Plant and Soil 124: 187493.
Bagyaraj, D. J. and Menge. J. A. 1978. Interaction between vesicular-arbuscular
mycorrhizal fungi and Azotobacter and their effecü on rhizosphere rnicroflora and
plant growth. The New Phytologist 80: 567-573.
Bakerspigel, A. 1956. Endogone in Saskatchewan and Manitoba. Amencan Journal of
Botmy 43: 471-474.
Bdaji. B.. Ba. A. M.. LaRue. T. A-. Tepfer. D. and Piche. Y. 1994. Pis~rrnsarivmn
mutants insensitive to nodulation are also insensitive to invasion in vitro by the
mycorrhizal fungus. Gigaspor-rimargarirn. Plant Science 102: 195-203.
Baldani. J, f., Caruso, L., Bddani. V. L. D.. Goi, S. R. and Dobereiner. J. 1997.
Recent advances in BNF with non-lepme plants. Soil Biology and Biochemistry
29: 91 1-922.
Barber. D. A. and Loughrnan. 3. C. 1967. The effect of microorganisrns on the
absorption of inorganic nutrients by intact plants. II. Uptake and uàlization of
phosphate by barley plants grown under sterile and non-sterile conditions. Journal
of Experimenral Botaly 18: 170-176.
Barea. J. M. and Brown. M. G. 1974. Effect on plant growth produced by Atotobucrer
paspali related to synthesis of plant growth repdating substances. Applied
Bacteriology 37: 583-593.
Barea, I. M., Azcon-Aguilar, C . and Azcon, R. 1987. Vesicular-arbuscular mycorrhiza
improve both syrnbiotic N2-fvtation and N uptake from soil as assessed with a 15N
technique under field conditions. The New Phytologist 106: 7 17-725.
Barea J. M., Bonis, A. F. and Olivares, J. 1983. Interaction between Azospirillum and
vesicular-arbuscular mycorrhizae and the? effects on growth and nutrition of rnaize
and ryegrass. Soi1 Biology and Biochemistry 1:705-7 10.
Barea, J. M,, El-Atrach, F. and Azcon, R. 1989. Myconhiza and phosphate
interactions as affecting plant development, N2-fixation .N - ~ m s f e rand N-uptake
from soil in legume-gras mixtures by using a I ~ dilution
N
technique. Soi1 Biology
and Biochemistry 21: 58 1-589.
Barea, J. M., Escudero. J. L. and Azcon-Aguilar. C. 1980. Effects of introduced and
indigenous VA mycorrhizal fun@ on nodulation, growth and nutrition of Medicago
sutivu in phosphate-fixing soils as affected b y P feflizers. Plant and Soil 54: 283296.
Barton, L. L.. Johnson. G. V., Schitoskey, K. and Wertz, M. 1996. Siderophoremediated iron rnetabolism in growth and nitrogen fixation by alfaEa nodulaied with
Rhizobium meliloti. Journal of Plant Numtion 19: 1201- 1210.
Batzli. J. M., Graves. W. R. and van Berkum. P. 1992. Diversity among rhizobia
effective with Robinia psetrdoacuciu L. Applied and Environmentai Microbiology
58: 2137-2143.
Baylis. G. T. S. 1972. Fungi. phosphoms and the evolution of root systems. Search 3:
257-258.
Becard. G. and Piché. Y. 1989. Fun@ growth-stimulation by CO2 and root exudates in
vesicular-arbuscular mycorrhizal symbiosis. Applied and Environmental
Microbiology 55: 2320-2325.
Becard. G., Douds, D. D. and Pfeffer. P.E. 1992. Extensive in vin-u hyphal growth of
vesicular-arbuscular mycorrhizal fun$ in the presence of CO2 and flavanols.
Applied and Environmental Microbiology 58: 82 1-825.
Bentivenga, S. P. and Morton' J. B. 1996. Congruence of fatty acid methyl ester
profiles and morphologie! characters of arbuscular mycorrhizal fun$ in
Gigasporaceae. Proceedings of the National Academy of Sciences USA 93: 56595662.
Berraho, E., Lesueur, D., Diem. H. G. and Sasson. A. 1997. Iron requirement and
siderophore production in Rhizobium ciceri during gowth on an iron-deficient
medium. World Journal of Microbiology and Biotechnology 13: 50 1-5 10.
Bethlenfalvay, G. J., Andrade, G. and Azcon-Aguilar, C. 1997. Plant and soi1
responses to mycorrhizal fûngi and rk~obacteriain nodulated and nitrate-krtilized
peas (Pisum sativum L.). Biology and Fertility of Soils 24: 164-1 68.
Bethlenfalvay, G. J., Brown. M. S. and Stafford, A. E. 1985. The Glycine-GlornusRhizobium symbiosis. II. Antagonistic effects between mycorrhizal colonization
and nodulation. Plant Physiology 79: 1054-1058.
Bethlenfalvay, G. J., Pacovsky. R. S., Bayne, H. G. and Stafford. A. E. 1982.
Interactions between nitrogen fixation, mycorrhizal colonization. and host-plant
growth in the Phaseulus-Rhizobium-Glornus symbiosis. Plant Physiolog 70: 446450.
Bever, J. D.. Morton. J. B.. Antonovics. J. and Schultz. P. A- 1996. Host-dependent
sporulatïon and species diversity of arbuscular rnycorrhizal fungi in a mown
vrassland. Journal of Ecolûgy 84: 7 1-82.
z
Bhalla. H. and Sen. A. N. 1973. Effect of rhizosphere bacteria of gram (Cicerarietinum) of different morphological. nutritional and physiological groups on
Rhizobium nodulating the same host. Science and Culture 39: 191- 193.
Bianciotto. V.. Bandi. C.. Minerdi. D., Sironi. M.. Tichy. H. V. and Bonfante. P.
1996. An obligately endosymbiotic mycorrhizal fungus itrelf harbors obligately
uitracellular bactena. Applied and Environmental Microbioiogy 62: 3005-3010Black. R. and P. B. Tinker. 1979. The development of endomycorrhizal root systems.
II. Effect of agronomie factors and soil conditions on the development of vesiculararbuscular mycorrhizal infection in barley and on the endophyte spore density. The
New Phytologist 83: 401-413.
Boivin, C.. Ndoye. I., Moloi?ba. F.. de Lajudie. P., Dupuy. N. and Dreyfus. B. 1997.
Stem nodulation in legumes: Diversity. rnechanisms and unusual characteristics.
Critical Reviews in Plant Sciences 16: 1-30.
Bowen. G. D. 1987. The biology and physiology of infection and its development. In:
Ecophysiology of VA Mycorrhizal Plants (G.R. Sdi'ir ed. ). CRC Press. Boca
Raton. Florida. pp. 27-57.
Boyetchko. S. M. and Tewari. J .P. 1990. Root colonization of different hosts by the
vesicular-arbuscular mycorrhizal fungus Glomus dimorphic~im. Plant and Soil 129:
131-136.
Brockwell. J., Bottomley, P. J. and ?oies. J. E. 1995. Manipulation of rhizobia
microflora for improving legume productivity and soil fern'lity: A critical assessrnent
Plant and Soil 174: 143- 180.
Bronstein. J. L. 1994. Our cunent understanding of mutualism. The Quarterly Rcview
of Biology 69: 3 1-51.
Broughton. W. J. and Dilworth, M. J. 1971. Control of leghaemoglobin synthesis in
snake beans (Vigna sinensis, sesquipedalis). Biochemistry Journal 125: 10751080.
Brown. M. S. and Bethlenfdvay. G. 3. 1987. The Glycine-GZomils-Rhizobiunz
symbiosis. VII. Photosynthetic nutrient use efficiency in nodulated. rnycorrhizd
soybean. Plant Physiology 86: 1292-1297.
Brown. M. S.. ThamsuraliuI. S. and Bethlenfalvay. G. J. 1988. The Glycine-Glorn~rsBI-adyrhkobiurnsymbiosis. Vm- Phosphorus-use efficiency of CO? and Nî_
fixation in mycorrhizal soybean. Physiologia Plantarum 74: L59-163.
Brundett, M. C. 1991. Mycorrhizas in natural ecosystems. Advances in EcologicaI
Research 21: 171-183.
Burkholder. P. R. 1952. Cooperation and conflict among primitive organisrns.
Amencan Scientist 40: 60 1-631.
Camprubi. A., Calvet. C. and Estaun. V. 1995. Growth enhancement of Citrrrs reshni
after inoculation with Glomus intramdices and Trichodema aureoviride and
associated effects in microbial populations and enzyme activity in potting mixes.
Plant and Soil 173: 233-238.
Caron, M. 1989. Potential use of mycorrhizae in control of soil-borne diseases.
Canadian Journal of Plant Patholoa 11: 177-179.
Carpenter-Boggs, L.. Loynachan, T. E. and Stahl, P. D. 1995. Spore germination of
Gigaspora margarita shulated by volaales of soil-isolated actinomycetes. Soil
Biology and Biochemistry 27: 1445-1451.
CavipeLli. M. A.. Robertson. G. P. and Klug, M. J. 1995. Fatty acid methyl ester
(FAME) profiles as measures of soi1 microbial community structure. Plant and Soil
170: 99-113.
Chen,W.X..Li,G.S.,Qi,Y.L..Wang.E.T..Yuan,H.L.andLi.J.L.
1991.
Rhizobiitm huakii sp. nov. isolated frorn the root nodules of Asmulugris sinicus.
International Journal of Systemahc Bactenology 41 : 275-280.
Cress. W. A.. Johnson, G. V. and Barton, L. L. 1986. The role of endomycorrhizal
fun@ in ïron uptake by Hilaria jamesii. Journal of Plant Nutrition 9: 547-556.
Cmsh. J. R. 1995. Effect of VA rnycorrhizas on phosphoms uptake and growth of
white cloi~er(Tr$olium repens L.) growing in association with rj-egrrtss (Lolirrm
perenne L.). New Zealand Journal of A,giculmral Research 38: 303-307.
Daniels, B. A. and Menge- 5. A. 1980. Hyperparasitization of vesicular-arbuscular
mycorrhizal fun$ Pkytopatholog 70: 584-58 8.
Daniels. B. A. and Trappe, l. M. 1980. Factors affecting spore germination of the
vesicular-arbuscular mycorrhizal fungus Glornus epigaeus. Mycologia 72: 457471.
Daniels-Hetnck, B. A., Wilson, G, T.. Kitt, D. G . and Schwab. A. P. 1988. Effects of
soil microorganisms on mycorrhizal contribution to growth of big bluestem g r a s in
non-stede soil. Soi1 Biology and Biochemistry 20: 501-517.
Darvill, A. G. and Albersheirn, P. 1984. Phytoalexins and their elicitors - A defense
a g a k t microbial infection in plants. Annual Review of Plant Physiology 35: 243-
de Almeida, D. L.. Pessanha, G. G. and de Penteado, A. F- 1973. Effects of liming
and phosphoms and nitrogen fertilizer on nodulation and yield of beans (Phaseolus
vrrlgaris). Pesquisa-Agropecuaria Brasiliera-Agronomia 8: 127-130.
DeMars, B. G. and Boerner. R. E. 1996. Vesicular arbuscular mycorrhizal developmerit
in the Brassicaceae in relation to plant life span. Flora 191: 179- 189.
Dixon. R. K., Rao, M. V. and Gag, V.K. 1993. Inoculation of Leucaena and Prosopis
seedlings with Glomus and Rhizobium species in saline soil: rhizosphere relations
and seedling gowth. And Soi1 Research Rzhabilitation 7: 133-144.
Douds. D. D. and Schenck. N.C . 1990. Relationship of colonization and sporulation
by VA mycorrhizal fun& to plant nuaient and carbohydrate contents. The New
Phytologist 116: 62 1-627.
Douds. D. D.Jr. 1994. Relationship between hyphal and arbuscular colonization and
sporulation in mycorrhiza of Paspalum notamm Flugge. The New Phytologist 126:
233-237.
Douds, D. D. Jr- and Chaney, W, R- 1986. The effect of high nutrient addition upon
seasonal patterns of mycorrhizal development. host growth. and root phosphorus
and carbohydrate content in Frarinzrspennsyli,anica March. The New Phytologist
130: 91-106.
Duc, G.. Tonivelot. A-. Gianinazzi-Pearson. V. and Gianinazzi. S. 1989. First report
of non-mycorrhizal plant mutants (Myc-) obtained in pea (Pisnrn sutivcrm L.) and
fababean (Viciufaba LJ. Plant Science 60: 215-222.
Edwards, C. A.. Stinner, B. R., Stinner, D. and Rabatin, S. 1988. Biologicd
Interactions in Soil. Elsevier Science Publishing Company Inc. W .USA.
El-Auach. F., Vierheilig, H. and Ocampo, J. A. 1989. Influence of non-host plants on
vesicular-arbuscular mycorrhizal infection of host plants and on spore germination.
Soil Biology and Biochemistry 21: 161- 163.
El-Hassan, G. A.. Hemandez. B. S. and Focht. D. D. 1986. Cornparison of 'Hup' trait
and intrinsic mtibiotic resistance for assessing rhizobial competitiveness axenically
and in soil. Applied and Environmental Microbiology 5 1: 546-551.
El-Raheem. A.. El-Shanshoury. R.. Hassan, M. A. and Abdel-Ghaffar, B.A. 1989.
Synergistic effect of vesicular-arbuscular mycorrhizas and Azotobacter chroococcum
on the growth and the nutrient contents of tomato plants. Phyton (Austria) 29: 2032 12.
El-Sheikh. E. A. E. and Wood. M. 1989. Response of chickpea and soybean rhizobia
to salt: osmotic and specifc ion effects on salts. Soil Biology and Biochemistry 21:
889-895.
El-Sheikh. E. A. E. and Wood, M. 1990. Salt effects on survival and multiplication of
chickccea and soybean rhizobia Soil Biology and Biochemistry 22: 343-347.
Ekan. G . H. 1992. Biological nitrogen fixation systems in tropical ecosystems: an
o v e ~ e w In:
. Biological Nitmgen Fixation and Sustainability of Tropical
Agriculture (K. Mulongoy, M. Gueye and D. S. C. Spencer, eds.) John Wiley and
Sons, Chichester, UK. pp. 27-40.
.
Fahraeus, G. 1957. The identification of clover root hairs by nodule bacteria, studied by
a simple g l a s slide technique. Journal of General Microbiology 16: 374-38 1.
Fischer. H. M. 1994. Genetic regulation of nitrogen fixation in rhizobia.
Microbiological Reviews 58: 352-386.
Fitter. A. H. 1986- Functioning of vesicular-arbuscular mycorrhizas under field
conditions. The New Phytologist 99: 257-265.
Fitter. A. H. 1989. The role and ecological sigificance of vesicular-arbuscular
rnycorrhizas in temperate ecosystems. Agriculture. Ecosysterns and Environment
29: 137-151.
Franco. A. A.. Serpa. A. and Souto. S. M. 1973. Symbiosis ofstrains homologous
with lines of Centrosemu prtbescens. Pesquisa Agropecuaria Brasdeira-Zootecnia 8:
Frank, A. B. 1885. über die auf W'drzelsymbiose begninde Ernahning gewisser Baume
durch unterirdische Pilze. Ber. Dtsch, Bot- Ges. 3: 128-145.
Franke, M. and Morton, J. B. 1994. Ontogenetic cornparisons of the endomycorrhizal
fun@ Scutellospora heterogama and Scutellospora pellucida: revision of taxonomie
character concepts, species descriptions. and phylogenetic hypotheses. Canadian
Journal of Botany 72: 122-134.
Frankenberger. W. T. and Arshad. M. 1995. Microbial biosynthesis of auxins. In:
Phytohormones in Soil (W. T. Frankenberger and M. Arshad eds.). Marcel Dekker.
New York pp. 35-7 1.
Freiberg, C.. Fellay, R., Bairoch, A., Broughton. W. J.. Rosenthal. A. and Perret. X.
1997. Molecular basis of symbiosis between Rhizobium and legumes. Nature 387:
394-40 1.
Frey. B. and Schuepp, H. 1993. A role of vesicular-arbuscular mycorrhizal f u n 9 in
facilitating interplant nitrogen transfer. Soi1 Biology and Biochcmistry 25: 65 1-658.
Friedencks, J. B., Hagehom. C . and Vanscoyok, S. W. 1990. Isolation of Rhizobium
leguminosarurn (biovar nifolii)suains from Ethiopian soils and symbiotic
effectiveness on African annual clover species. Applied and Environmental
Microbiology 56: 1087-1092.
/
Fuhmann, J. and Wollum II, A. G. 198Ça. In vitro growth responses of
Bradyrhizobium japonicum to soy bean rhizosphere bacteria. Soi1 B iology and
Biochemistry 21: 131-135.
Fuhrmann. J- and Wollurn II, A, G. 1989b. Nodulation cornpetition among
Bradyrhizobium japonicum strains as infiuenced by rhizosphere bacteria and iron
availability. Bioloa and Fertilïty of Soils 21: 131-135.
Gange. A. C. and West, H.M. 1994. Interactions between arbuscular myconhizal fungi
and foliar-feeding insects in Plantago lunceolata L. The New Phytologïst 128: 7987.
Gazey, C., Abbott, L. K. and Robson, A. D. 1992. The rate of deveiopment of
mycorrhizas affects the onset of sporulation and production of extemal hyphae by
two species of Acaulospora. Mycological Research 96: 643-650.
George. M. L. C. and Robert, F. M. 1992. Cornpetition among Rhizobium
leguminosarum bv. phmeoli strains for nodulation of common bean. Canadian
Journal of Microbiology 38: 157-160.
Gerdernann. J.W. and Nicholson, T. H. 1963. Spores of mycorrhizal Endogone species
extracted from soi1 by wet sieving and decanting. Transactions of the British
Mycological Society 46: 235-244.
Geurts, R. and Franssen, H. 1996. Signal transduction in Rhizobium-induced nodule
formation. Plant Physioloa 112: 447-453.
Gianinazzi-Pearson, V. 1996. Plant cell responses to arbuscular mycorrhizal fun@ :
Gemiig to the roon of the symbiosis. The Plant Cell 8: 1871-1883.
Gianinazzi-Pearson, V., Gianinazzi. S., Guillemin. J. P.. Trouvelot A. and Duc, G.
1991. Genetic and cellular analysis of resistance to vesicular-arbuscular (VA)
mycorrhizal fun@ in pea mutants. In: Advances Ui hlolecular Genetics of PlantMicrobe Interactions (H. Hennecke and D. P. S. Verma eds.). Kluwer Academic
Publishers. London. pp. 336-342.
Gibson, A. H. 1967. Physiczl environment and symbiotic nitrogen fixation. IV.
Factors affecthg the early stages of nodulation. Ausualian Journal of Biological
Sciences 20: 1087-1104.
Gildon. A. and Tinker. P. B. 1983. Interactions of vesicular-arbuscular mycorrhizal
infection and heavy rnetals in plants. 1. The effects of heavy metals on the
development of vesicular-arbuscular mycorrhizas (Glomus rrzosseae). The New
Phytologist 95: 247-26 1.
Gillis, M., Kersters. K.. Hoste. B., Janssens, D.. Kroppenstedt. R. M.. Stephan. M.
P., Teixeim, K. R. S., Dobereiner. J. and de Ley. J. 1989. Acerobacter
diazotrophicrrs sp. nov.. a nitrogen-fixing acetic acid bacterium associated with
sugarcane. International J o m a l of Systematic Bactenology 39: 36 1-364.
Giovanetti, M. and B. Mosse. 1980. An evaluati~nof techniques for measuring
vesicular-arbuscular mycorrhizd infection in roots. The New Phytologist 84: 489500.
Giovannet& M.. Avio, L., Sbrana, C. and Citemesi. A. S. 1993a. Factors affecting
appressorium development in the vesicular-arbuscular mycorrhizal fungus Glomus
rnosseae (Nicol. &Gerd.) Gerd.&Trappe. The New Phytologist 123: 114422.
Giovannetti, M.. Sbrana, C., Avio, L.. Citernesi. A. S. and L o g . C. 1993b.
Diffirential hyphal rnorphogenesis in arbuscular mycorrhizal fungi during preinfection stages. The New Phytologist 125: 587-594.
Giovannetti, M-, Sbrana. C., Citemesi, A. S., Avio, L., Gollotie, A-. GianinazziPearson, V. and Gianinaui. S. 1994. Recognition and infection process. b a i s for
host specificity of arbuscular mycorrhizal
In: Sustainable A,gicul ture and
Natural Ecosystems (S. Gianinavi and H. Scheupp eds.), Birkhauser Verlag, Basel.
Switzerland. pp. 61-72.
fima.
Gonzalez. S . B. 1988. Ecologia y biotecnologia de micomzas en leguminosas (soja y
alfalfa). Ph. D. Thesis. University of Granada.
Graham, I. H.. Hodge, N.C. and Morton, J . B. 1995. Fatty acid methyl ester profdes
for characterization of glomalean fun@ and their endomycorrhizae. Applied and
Environmental Microbiology 6 1: 58-64.
Graham, J. H., Leonard, R. T. and Menge, J. A. 1981. Membrane-mediated decrease
in root exudation responsible for inhibition of vesicuIar-arbuscular mycorrhiza
formation. Plant Physiology 68: 548-552.
Green, N. E.. Graham, S. 0. and Schenck. N. C. 1976. The influence of pH on the
germination of vesicular-arbuscular mycorrhizal spores. Mycologia 69: 929-934.
Gryndler, M. and Vosatka. M. 1996- The response of Glomusfist~clos~tm-maize
mycorrhïza to treatments with culture fractions from Pseudomonas purida.
Mycorrhiza 6: 207-21 1.
Harinikuma.,M. and Bagyaraj, D. J. 1988. Effect of crop rotation on native vesiculararbuscular mycorrhizal propagules in soil. Plant and Soil 110: 77-80.
Kadey. J. L. and Harley, E. L. 1987. A check-list of rnycorrhiza in the British flora.
The New Phytologist (Suppl.) 105: 1-102.
Harris. D., Pacovsky, R. S. and Paul, E. A. 1985. Carbon economy of soybeanRhizobi~mr-Glomusassociations. The New Phyt~logist101 : 427-440.
Haselwandter, K. 1995. Mycorrhizal fungi: Siderophore production. Critical Reviews
in Biotechnology 15: 287-291.
Hass, H., Talylor, T. N. and Remy, W. 1994. Fun@ from the lower Devonian Rhynie
Chert: rnycoparasitism. Amencan Journal of Botany 8 1 :29-37.
Hayman, D. S. and Stovold, G. E. 1979. Spore populations and infectivity of
vesicular-arbuscular mycorrhid alngi in New South Wdes. Australian Journal of
Botany 27: 227-233.
Heidstra, R. and Bisseling, T. 1996. Nod factor-induced host responses and
mechanisms of Nod factor perception. The New Phytologist 133: 25-43.
Hely, F. W. 1964. Calcium carbonate as an aid to clover establishment in soils with low
winter temperatures. Field Station Circula-. Division of Plant Industly. CSIRO 3:
63-68.
Hendrix, J. W., Guo, B. 2. and An, 2. Q. 1995. Divergence of mycorrhizal fungal
communities in crop production systems. Plant and Soil 170: 131-140.
Hendrix, J. W., Jones, K. J. and Nesrnith, W. C. 1992. Control of pathogenic
mycorrhizal fun@in maintenance of soil productivity by crop rotation. Journal of
Production Agriculture 5 :383-386.
Hepper. C. M. 1979. Germination and growth of Glom~ïsmledonir~sspores: the eflects
of inhibitors and nutrients. Soil Biology and Biochemistry 1 !:269-277.
Herrera, M. A., Salamanca, C. P. and Barea, J. M. 1993. Inoculation of woody
legumes with seleçted arbuscular rnycorrhizal fun$ and rhizobia to recover
desemfied mediterranean ecosystems. Applied and Environmental Microbiology 59:
129-133.
Hetnck, B. A. D. 1984. Ecology of VA mycorrhizal fungi. In: VA Mycorrhiza. (C. L.
Powell and D. J. Bagyaraj eds.). CRC Press. Boca Raton. Florida. pp. 35-55.
Hetrick. B. A. D. and Bloom. J. 1983. Vesicular-arbuscular mycorrhizal fungi
associated with native tall grass prairie and cultivated wïnter wheat Canadian
Journal of Botany 61:2 140-2 146.
Hetrick, B. A. D. and Bloom, J. 1986. The influence of host plant on production and
~olonizationability of vesicular-arbuscular mycorrhizal spores. Mycologia 78: 3236.
Hetrick, B. A. D., Wilson. G. W. T., Gill, B. S. and Cox. T. S. 1995. Chromosome
location of rnycorrhizal responsive genes in wheat. Canadian Journal of Botany 73:
89 1-897.
Hicks. P. M. and Loynachan, T. E. 1989. Bactena of the soybean rhizosphere and their
effect on growth of Bradvrhizobiumjaponicum. Soil Biology and Biochemistry 21:
561-566.
Hirrel. M. C. and Gerdemann, J. W. 1980. Improved growth of onion and bell pepper
in saline soils by two vesicular-arbuscular mycorrhizal fun$. Soil Science Society
of America Journal 44: 654655.
Hirrel. M. C.. Mehavaran, H. and Gerdemann, J. W. 1978. Vesicular-arbusculai
mycorrhizae in the Chenopodiaceae and Cmciferae: do they occur? Canadian Journal
of Botany 56: 2813-2817.
Hirsch, A. M. 1992. Tmsley Review no. 40: Developmental biology of l e p m e
nodulation. The New Phytologist 122: 21 1-237.
Hoflich, G., Wiehe. W. and Hecht-Buchholz. C . 1995- Rhizosphere colonization of
different crops with growth promothg Pseudomonas and Rhizobirtm bacteria.
Microbiological Research 150: 139-147.
Holguin. G. and Bashan, Y. 1996. Nitrogen-fixation by ilzospiri!l~nzbrasilense Cd is
promoted when CO-culturedwith a mangrove rhizosphere bacterium (Staphylococcris
sp.). Soi1 Biology and Biochemistry 28: 165 1-1660.
Howieson, J. G., Ewing, M. A. and dlAntuono. M. F. 1988. Selection for acid
tolerance in Rhizobium meliloti. Plant and Soi1 105: 179- 188.
Hynes, R. K.. Craig, K. A., Covert, D., Smith, R. S. a n d Rennie. R. J. 1995. Liquid
rhizobid inoculants for lentil and field pea. Journal of Production Agriculture 8:
547-552.
Ianson, D. C. and Linderman, R. G. 1993. Variation in the response of nodulating
pigeonpea (Cajarzus cajan) to different isolates of myconhizal fun$. Symbiosis 15:
105-119.
Ibijbijen, J.. Urquiaga. S., Isrnail, M.. Alves, B. J. R. and Boddey. R. M. 1996.
Effect of arbuscular mycorrhizal fun@ on growth. mineral nutrition and nitrogen
fixation of three varieties of cornmon beans (Phaseolus vulgaris). The New
Phytologist 134: 353-360.
Ibrahim, K. K., Amnachalam, V., Rao, P. S. K. and Tilak, K. V. B. R. 1995.
Seasonal response of groundnut genotypes to arbuscular mycorrhizaBradyrhizobiurn inoculation. Microbiological Research 150: 2 18-224.
Jakobsen. 1. Abbott, L. K. and Robson, A. D. 1992. Extemal hyphae of vesiculararbuscular mycomhizal fun9 associated with Tnfolium subterraneum L. 1. Spread of
hyphae and phosphorus infiow into roots. The New Phytologist 120: 37 1-380.
Jarvis. B. D. W. and Tighe, S. W. 1994. Rapid identification of Rhizobium spccies
based on cellular fatty acid analysis. Plant and Soil 161: 3 1-41.
Ja.rvis. B. D. W., Pankhurst, C. E,and Patel, J. J, 1982. Rhizobium loti. a new species
of l e p m e root nodule bacteria International Journal of Systematic Bacteriology 32:
378-380.
Jasper. D. A.. Robson. A. D. and Abbott. L. K. 1979. Phosphorus and the formation of
vesicular-arbuscular mycorrhizas. Soil Biology and Biochemistry 11: 50 1-505.
Jayachandran, K.. Schwab. A. P. and Hetrick, B. A. D. 1992. Mineralization of organic
phopshorus by vesicular-arbuscular mycorrhizal fungi. Soil Biology and
Biochemistry 24: 897-903.
Jensen, A. and Jakobsen, 1. 1984. The occurrence of vesicular-arbuscular mycorrhiza in
barley and wheat g o w n in some Danish soils with different fertilizer treatments.
Plant and Soil55: 403-414.
Johansen, A.. Finlay. R. D. and Olsson, P. A. 1996. Nitrogen metabolism of extemal
hyphae of the arbuscular mycorrhizal fungus Glomus intraradices. The New
Phytologist 133: 705-722.
Johnson. N. C.. Copeland, P. J., Crookston, R. K. and Pfleger. F. L. 1992.
Mycorrhizae: possible explanation for yield decline with continuous corn and
soybean. Agronorny Journa1 84: 387-390.
Joner, E. J. and Jakobsen. 1. 1995. Uptake of 3 2 from
~
labelled organic matter by
mycorrhizal and non-mycorrhizal subterranean clover (Trifolirrm subterraneum L.).
Plant and Soil 172: 221-227.
Kaur. S. and Singh, O. S. 1988. Response of ricebean to single and combined
inoculation with Rhizobium and Glomus in a P-deficient steriLized sod. Plant and
Soil 112: 293-295Koske, R. E. and Gemma, J. N. 1989. A rnodified procedure for stainin,O roots to
detect VA mycorrhizas. Mycological Research 92: 486-488.
Kreig, N. R. and Holt, J. G. 1984. Bergey's Manual of Determinative Bacteriology.
Vol. 1, Williams and Wilkins, Baltimore- 964 pp.
Krishna, K. R., Balakrishna, A. N. and Barnaraj. D. J. 1982. Interactions between a
vesicular-arbuscular mycorrhizal fungus and Streptomyces cinnamneous and their
effects on finger miiler. The New Phytologist 92: 401-405-
Kucey, R M. N and Paul, E. A. 1983. Vesicular-arbuscular mycorrhizal spore
populations in various Saskatchewan soils and the effect of inoculation with Glomus
mosseae on faba bean growth in greenhouse and field trials. Canadian Journal of
Soil Science 63: 87-95.
Kucey, R. M. N. and Bonetti, R. 1988. Effect of vesicular-arbuscular mycorrhizal
füngi and captan on growth and N2 fixation by Rhizobium-inoculated fieid beans.
Canadian Journal of Soil Science 68: 143-149.
Kucey, R. M. N. and Paul, E. A. 1982. Carbon flow photosynthesis. and N2-fixation
in rnycorrhizal and nodulated faba beans (Vicia faba L.). Soi1 Biology and
Biochemistry 14: 407-412.
Land, S., Dauck. H. and von Alten. H. 1989. Evaluation of VA-myconhizae in
different agrïcultrrral soils. Agriculture, Ecosystems and Environment 29: 2 17-224.
Le Tacon. F., Skinner, F. A. and Mosse, B. 1983. Spore germination and hyphal
growth of a vesicular-arbuscular mycorrhizal fungus Glomus mosseae (Gerdemann
and Trappe), under decreased oxygen and increased carbon dioxide concentrations.
Canadian Journal of Microbiology 29: 1280-1285.
Lee, P. J. and Koske, R. E. 1994. Gigaspora gigantea: parasitism of spores by fungi
and actinomycetes. Mycological Research 98: 458-466.
Leonard, L. T. 1943. A simple assembly for use in the tesring of cultures of Rhizobium.
Journal of Bacteriology 45: 523-527.
Lindstrom, K. 1989. Rhizobium galegae, a new species of legume root nodule bacteria.
International Journal of Systematic Bacteriology 39: 365-367.
Lippman. G.. Witter, B. and Kegler, G. 1989. Factors controllinp VAM colonization
percentage in arable soils. Agriculture, Ecosystems and Environment 29: 257-26 1.
Louis. 1. and Lim,G. 1988. Differential response in growth and mycorrhizal
colonization of soybean to inoculation with two isolates of Glomus clarum in soils of
dBerent P availability. Plant and Soil 112: 37-43.
Lu. S.. Braunberger, P. G . and Miller. M. H. 1994. Response of vesicular-arbuscular
mycorrhizas of maize to various rates of P addition to different rooting zones. Plant
and Soil 158: 119-128.
Lupwayi, N. 2.. Haque. 1. and HoIl. F. B. 1997. Effectiveness. cornpetitiveness and
persistence of inoculant Rhizobium for perennial Afiican clovers in a highland
Vertisol. Jounral of Agricultural Science 129: 429-437.
Lynch, J. M. 1976. Products of soil microorganisrns in relation to plant growth. CRC
Critical Reviews in MicrobioIogy 5: 67-107.
Lynch. J. M. 1990. Introduction: Some consequences of rnicrobial rhizosphere
cornpetence for plant and soil. In: The Rhizosphere (J. M. Lynch ed.). John Wiley
and Sons, West Sussex, England, p. 1-10.
MacDonald, R. M. and Chandler, M. R. 1981. Bacterium-like organelles in the
vesicular-arbuscular mycorrhizal fungus Glomris culedonius. The New Phytulogist
89: 241-246.
MacDonald. R. M.. Chandler, M. R. and Mosse, B. 1982. The occurrence of
bacterium-like organelles in vesicular-arbuscular mycorïhizal fun@. The New
Phytologist 90: 659-663.
Magurran, A. E. 1988. Ecological diversity and its measurement. Princeton University
Press, Princeton, N. J.
Maier. R. J. and Tnplett, E. W. 1996. Toward more productive. efficient. and
cornpetitive nitrogen-fixing symbiotic bacteria Critical Reviews in Plant Sciences
15: 191-234.
Malibari. A. A.. Al-Fass, F. A. and Ramadan, E. M. 1988. Incidznce and infectivity of
vesicular-arbuscular mycorrhizas in some Saudi soils. Plant and Soil 112: 105-111.
Manjunath. A., Bagyaraj, D.J. and Gowda. H. S. G. 1984. Dual inoculation with VA
mycorrhiza and Rhizobium is beneficial to Leucaena. Plant and Soil 78: 445-448.
Matin, R. C., Voldeng, H. D. and Smith. D. L. 1991. Nitrogen transfer from
nodulating soybean to maize or to nomodulating soybean in intercrops: the 15N
dilution rnethod. Plant and Sorl 132: 53-63.
Mayo. K.. Davis. R.E. and Motta, J. 1986. Stimulation of germination of spores of
Glomus versiforme by spore-associated bacteria Mycologia 78: 42643 1.
McGonigle, T. P. and Fitter. A. H. 1990. Ecological specifcity of vesicular-arbuscular
mycorrhizal associations. Mycological Research 94: 120-122.
McGonigle, T.P. and MiUer. M. H. 1996. Mycorrhizae, phosphorus absorption. and
yield of maize in response to tfiage. The Soi1 Science Society of Amenca Journal
60: 1856-1861.
Medina O. A.. Sylvia, D. M. and Kretschmer, Ir.. A. E. 1988. Response of Siratro to
vesicular-arbuscular mycomhuial fun@: II. Efficacy of selected vesicular-arbuscular
fungi at different P Ievels. Soil Science Society of America Journal 52: 420-423.
Menge, J. A-. Labanauskas, C. K., Johnson, E. L. V. and Platt. R. G. 1978a. Partial
substitution of mycorrhizd fun@ for phosphorus fercilization in the greenhouse
culture of citrus. Soil Science Society of America Joumal 42: 926-930.
Menge, J. A., Stei.de, D., Bagyaraj, D. J. and Johnson, E. L. V. 1978b. Phosphorus
concentrations in plants responsible for inhibition of m ycorrhizal idechon. The
New Phytologist 80: 575-578.
Menz, S. M., Heithaus, J. J. and Bush. R. L. 1979. Mass production of axenic spores
of the endomycorrhizal fungus Gigaspora marganta. Transactions of the British
Mycological Society 72: 167-169.
Meyer, J. R. and Lindeman, R G. 1986. Response of subterranean clover to dual
inoculation with vesicular-arbuscular rnycorrhizal fun@ and a plant growth
promothg bactenum Pseudomonas putida. Soi1 Biology and Biochemisûy 18: 185190.
Michelsen. A. and Rosendahl, S. 1990. The effect of VA mycomhizal fun$,
phosphoms and drought stress on the growth of Acaciu nilotica and Leucaena
leucocephala seedlings. Plant and Soil 124: 7-13.
Michiels, J. and Vanderleyden, J. 1994. Molecular basis of die establishment and
functionïng of a N2-fWng root nodule. World Journal of Microbiology and
Biotechnology 10: 6 12-630.
Miller, R. M. and Jastrow. J, D. 1990. Hierarchy of root and mycorrhizal fungd
interactions with soil aggregation. Soil Bi0103 and Biochemists. 22: 579-584.
Miller. R. M.. Reinhardt D. R. and Jastrow. J. D. 1995. Externd hyphd production of
vesicular-arbuscularmycorrhizal fun$ in Pasture and tallgrass prairie communities.
Oecologia 103: 17-23.
Miranda, J. C. C . and Harris, P. J. 1994. Effects of soil phosphorus on spore
germination and hyphal growth of arbuscular mycorrhizal fun$. The New
Phytologist 128: 103-108.
Morton. J. B.. Bentivenga, S. P. and Bever. J. D. 1995. Discovery. measurement. and
inrerpretation of diversity in arbuscular endomycorrhizal fungi (Glomales.
Zygomycetes). Canadian Journal of Botany 73: S25-S32.
Morton, J. B.. Bentivenga, S. P. and Wheeler, W. W. 1993. Germ plasm in the
htemational Collection of Arbuscula. and Vesicular-arbuscular mycorrhizal fùngi
(INVAM) and procedures for culture development, documentation and storage.
Mycotavon X L W : 49 1-528.
Morton. J. B.. Yarger. J. E. and Wright, S. F. 1990. Soil solution P concentrations
necessary for nodulation and nitrogen Fixation in rnycorrhizal and non-mycorrhizal
red clover (Tnfolium prarense L.). Soil Biology and Biochemistry 22: 127- 129.
Mosse. B. 1959. The regular germination of r e s h g spores and some observations on
the growth requirements of an Endogone sp. causing vesicular-arbuscu1a.r
mycorrhiza. Transactions of the British Mycological Society 42: 273-286.
MoutogLis. P. and Widden, P. 1996. Vesicular-arbuscular mycorrhizal spore
populations in sugar maple (Acer saccharum marsh. L.) forests. Mycorrhiza 6: 9 197.
Mukgnier, J. and Mosse, B. 1987. Spore germination and viability of a vesicular
arbuscular mycorrhizal fungus, Glornus mosseae. Transactions of the British
Mycological Sociev 88: 41 1-413.
Munns, D. N. 1977. Mineral nutrition and the legume symbiosis. In: A Treatise on
Dinitrogen Fixation (R. W. F. Hardy and A. H. Gibson eds.). John Wiley and
Sons, New York, pp. 353-391.
Nehl. D. B.. Allen, S. J. and Brown. J. F. 1996. Deleterious rhizosphere bacteria: an
integrating perspective. Applied Soi1 Ecology 5: 1-20.
Novak, K.. Skrdleta, V.. Nemcova. M- and Lisa. L. 1993. Behaviour of pea
nodulation mutants as affected by increasing nitrate level. Symbiosis 15: 195-206.
OIHara,G, W.. Boonkerd. N. and Dilworth. M. J. 1988. Mineral constraints to
nitrogen fixation. Plant and Soil 108: 93-1 10.
Ocampo. J. A. 1980. Effect of crop rotations involving host and non-host plants on
vesicular-arbuscular mycorrhizal infection of host plants. Plant and Soi1 56: 283291.
OZd, K. M. and Wong. J. N. F. 1976. Perforation and Iysis of fungal spores in natural
soils. Soi1 Biology and Biochemistry 8: 285-292.
Olsson. P. A., B2ith, E. and Jakobsen. 1. 1997. Phosphorus effects on the rnycelium
and storage structures of an arbuscular mycorrhizal fungus as studied in die sail and
roots by analysis of fatty acid signatures. Applied and Environmental Microbiology
63: 3531-3538.
Olsson, P. A., Baath, E., Jakobsen, 1. and Soderstrom. B. 1995. The use of
phospholipid and neutral lipid fatty acids to estirnate biomass of arbuscular
myconhizal fungi in soil. Mycolo$caI Research 99: 623-629.
Overholt. E.. Engqvist. G.. Lindblad, P., Martensson, A.. Rydberg. 1. and Zagal, E.
1996. Pea-rhizobial and mycorrhizal syrnbiotic systems: A review of dieir
commonalities with other plant-microbe systems. Symbiosis 21 : 95- 113.
Pacovsky. R. S. 1989. Metabolic differences in Zea-Glornus-Azospirillum symbioses.
Soil Biology and Biochemistry 21: 953-960.
Pacovsky, R. S.. Fuller. G. and Paul. E. A. 1985. Influence of soil on the interactions
benveen endomycorrhizae and &ospirillurn in sorghum. Soil Biology and
Biochemisuy 27: 523-531.
Pacovsky. R. S.. Fuller. G. and Stafford. A. E. 1986. Nutrient and growth interactions
in soybeans colonized with Glomns fnsciczdantm and Rhizobiltm juponicum. Plant
and Soil 92: 37-45.
Pang P. C . and Paul. E. A. 1980. Effects of vesicular-arbuscular mycorrhiza on JC
and 1sN distribution in nodulated faba beans. Canadian Journal of Soil Science 60:
24 1-250.
Paula. M, A., Reis. V. M. and Dobereiner, J. 1991- Interactions of Glornus clamm
with Acerobacrei-diazurophicus in infection of sweet potato (Ipomoea baratm).
sugarcan (Saccharum spp.) and sweet sorghum (Sorghum vulga~x+ Biology and
FertilityofSoils 11: i11-115Paula. M. A.. Urquiaga. S.. Siqueira. J. 0. and Dobereiner. J. 1992. Synergistic
effe,ctsof vesicular-arbuscular rnycorrhizal fun$ and diazotrophic bacteria on
nutrition and growth of sweet potato (Iponzoeo batutus). Biology and Fenility of
Soils 14: 6 1-66.
Pearson, J. N.and Schweiger. P. 1993. Scutellospo~ncalospora (Nicol.&Gerd.)
Walker & Sanders associated with subtzrranean clover: dynarnics of colonization.
spomlation. and soluble carbohydrates. The New Phytologist 124: 215-219.
Pena. J. I., Sanchez-Diaz. M., Aguirreolea, J. and Becana, M. 1988. Increased stress
tolerance of nodule activity in the Medicago-Rhizobium-Glornu~~
symbiosis under
drought Journal of Plant Physiology 133: 79-83.
Persad-Chuinery, S. B. and Chinnery. L. E. 1996. Vesicular-arbuscular rnycorrhizae
and micronutrient nutrition, In: Advancements in Micronutrient Research (A
Hemantaranjan ed.), Scienufic Publishers, Jodhpur, India, pp. 367-382.
Phillips. D. A., DeJong, T. M. and Williams. L. E. 1980. Carbon and nitrogen
limitations on symbiotically-grom soybean seedlings by nodular bactena Rhizobium
japonicum. Current Perspectives in Niaogen Fixation. ElsevierMorth-HoIland
Biomedical Press, Amsterdam, pp. 117-120.
Pond. E. C.. Menge, J. A. and JarreLl, W. M. 1984. Lmproved growth of tomato in
salinized soil by vesicular-arbuscular rnycorrhizal fun@ collected from saline soils.
Mycologia 76: 74-84.
Porter, W. M. 1979. The 'Most Probable Number' method for enumerating infective
propagules of vesicular-arbuscular mycorrhizal fungi in soil. Australian Journal of
Soil Research 17: 5 15-5 19.
Poupot, R.. Martinez-Romero, E. and Prome. J. C . 1993. Nodularion factors from
Rhizobium hopici are sulfated or non-sulfated chitopentasaccharides containing an
N-methyl-N-acylglucosyIarninylterminus. Biochemistry 32: 10430- 10435.
Purcino. A. A. C.. Lurlarp, C.and Lynd. T. Q. 1986. Mycorrhiza and soil fertiliy
efiècts with growth. nodulation and nitrogen fxation of LRucama grown on a typic
Eutrustox. Communications in Soil and Plant Analysis 17: 473-489.
Quigley, P. E., Cunningham, P. J.. Hannah. M.. Ward. G. N. and Morgan. T. 1997.
Symbiotic effictiveness of Rhizobium lepminosantm bv. D-ifoliicoLlected from
pastures in south-western Victoria. Australian Journal of ExperimentaI Agriculture
37: 623-630.
Rabatin, S. C.and S timer, B. R. 1988. Indirect effects of interactions between VAM
fungi and soil inhabithg invertebrates on plant processes. Agriculture Ecosystems
and Environment 24: 135- 146.
Rahman, M. K. and Parsons, J. W. 1997. Effects of inoculation with Glomus mosseae.
Azorhizobiurn caulimduns and rock phosphate on the growth of and nitrogen and
phosphorus accumulation in Sesbunia rosrrata. Biology and FertiLity of Soils 25:
47-52,
Raj. J., Bagyaraj, D. J. and Manjunath, A. 1981. Influence of soil inoculation with
vesicular-arbuscular mycorrhiza and a phosphate-dissolving bacterium on plant
growth and 32~-uptake. Soil Biology and Biochemisay 13: 105-108.
Raju, P. S., Clark, R. B.. Ellis, J. R..Duncan, R. R. and Maranville, J. W. 1990.
Benefit and cost analysis and phosphorus efficiency of VA mycorrhizal fungi
colonizations with sorghurn (Sûrghum bicolor) genotypes grown at varied
phosphorus levels. In: Plant nutrition- physiology and applications (M. L. van
Beusichem ed.), Proceedings of the EIeventh Internariond Plant Nutrition
Colloquium, 30 July- 4 August 1989, Wageningen. The Netherlands, Kluwer.
Canada. pp. 165-170.
Rao. N. S. S., Tilak, K. V. B. £2. and Singh. C. S. 1985. Synergistic effect of
vesicular-arbuscular rnycorrhizae and Azospirillurn brasilense on the growth of
barley in pots. Soi1 Biology and Biochemistry 1: 121-129.
Ravnskov. S. and Jakobsen. 1. 1995. Functional compatibility in arbuscular
mycorrhizas measured as hyphal P transport to the plant. The New Phytologist 179:
611-618.
Redecker. D.. von Berswordt-Wdrabe. P-. Beck. D. P- and Werner, D. 1997.
Influence of inoculation with arbuscular mycorrhizal fungi on stable isotopes of
nitrogen in Phaseolus vrrlga~s.Biology and FertiLity of Soils 24: 344-346.
Redente. E. F. and Reeves. F. B. 1981. Interactions between vesicular-arbusciilar
rnycoirhiza and Rhizobium and their effect on sweetvetch growth. Soil Science
232: 410-415.
Rennie, R. J. and Hynes. R. K. 1993. Scientifk and legislative quality connol of
legume inoculants for lentil and field pea. Journal of Production Agriculture 6: 565574.
Rerkasem, B., Rerkasem, K.. Peoples, M. B., Hemdge, D.F. and Bergersen, F. J.
1988. Measurernent of N2 fixation in maize { B a mays L.)-iicebean (Vigna
umbellatu [Thunb.] Ohwi and Ohashi) intercrops. Plant and Soil 108: 125-135.
Richardson. A. E. and Simpson, R. J. 1989. Acid-tolerance and syrnbiotic effectiveness
of Rhizobium mfolii associated with a T~ifoliumsubtermeum L.-based Pasture
growing in an acid soil. Soil Biology and Biochemistry 21: 87-95.
Robleto. E. A., Kmiecik. K.. Oplinger. E. S.. Nienhuis, J. and Triplett, E. W. 1998.
Trifolitoxin production uicreases nodulation competitiveness of Rhizobium etli CE3
under agriculturd conditions. Applied and Environmental Mcrobiology 64: 26302633.
Rose. S. L. 1981. Vesicular-arbuscular endomycorrhizal associations of some desert
plants of Baja California. Canadian Journal of Botany 59: 1056-1060.
Rosendahl. S.. Rosendahl. C. N. and Sc~hting.U. 1989. Distribution of VA
mycorrhizal endophytes arnongst plants from a Danish pasland community.
A_giculture. Ecosystems and Environment 29: 329-335.
Rousseau, A., Benhamou, N.. Chet, 1. and Piché. Y. 1996. Mycoparasitism of the
exmunaaical phase of Glomus intraradices by Trichodema hanianum.
Phytopathology 86: 434-443.
Ruiz-Lozano, J. M. and Azcon. R. 1993. Specificity and functional compatibility of VA
mycorrhizal endophytes in association with Bru~?rhizobirrm
strains in Ciceiatietinum. Symbiosis 15: 2 17-226.
Ruiz-Lozano, J. M. m d Azcon, R. 1994. Development and activity of the syrnbiosis
Effect of
between Bradyrhizobium strains. Glornus species and Cicet-a~fetinrrrn:
timing of inoculation and photon irradiance. S ymbiosis 16: 249-265.
Ruiz-Lozano. J. M., Gornez, M. and Azcon. R. 1995. Influence of different Glumus
species in the time-course of physiological plant responses of lemice to progressive
drought stress penods. Plant Science 110: 37-44.
Sagan, M., Messager, A. and Duc. G. 1993. Specificity of the Rhizobium-iegume
syrnbiosis obtained after mutagenesis in pea (Pisum sativlm L.). The New
Phytologist 125: 757-76 1.
Sagan, M., Morandi, D., Tarenghi, E. and Duc. G. 1995. Selection of nodulation and
mycolrhizal mutants in the mode1 plant Medicago truncanrla (Gaertn.) d e r alpha-ray
mutagenesis. Plant Science 11 1: 63-7 1.
Sarne, B. L, Robson, A. D. and Abbott, L. K. 1983. Phosphorus. soluble
carbohydrates and endomycorrhizal infection. Soil Biology and Biochemistry 15:
593-597.
SAS Institute Inc. (1997) SAS@ Procedures Guide, Version 6. Third Edition, Cary. NC.
Saxena, A. K., Rathi, S. K. and Tilak, K. V. B. R. 1997. Differentid effect of various
endomycorrhizal fun$ on nodulating abihty of green gram by Bmdyrhizobirrm sp.
(Vigna) saain S24. Biology and Fertility of Soils 24: 175-178.
Schenck, N. C. and Kinloch. R. A. 1980. Incidence of mycarrhizal h n g i on six field
crops in monoculture on a newly cleared woodland site. Mycologia 72: 229-445456.
Schenck, N. C. and Nicolson, T. H. 1977. A zoosporic fungus occurring on species of
Gigasporu margarita and other vesicular-arbuscular mycorrhizal fun@. Mycol ogia
69: 1049-1053.
Scholla. M. H. and Elkan. G. H. 1984. Rhizobirtm fiedii sp. nov.. a fast-growing
species diat effectively nodulates soybeans. International Journal of Systematic
Bacteriology 34: 484-486.
Schreiner. R. P. and Bethlenfalvay. G . J. 1995. Mycorrhizal interactions in sustainable
a,giculture. Critical Reviews in Biotechnology 15: 27 1-285.
Schreiner, R. P. and Koide, R. T. 1993a. Antifunpl compounds from the mots of
mycotrophic and non-mycotrophic plant species. The New Phytologist 123: 99105.
Schreiner, R. P. and Koide. R. T. 1993b. Mustards. mustard oils and mycorrhizas.
The New Phytologist 123: 107-213.
Schubert, A. and Hayman, D. S. 1986. Plant growth responses to vesicular-arbuscular
mycorrhizâ XVL Effectiveness of different endophytes at different leveIs of soil
phosphate. The New Phytologist 103: 79-90.
Segovia, L., Young, J. P. W. and Mdnez-Romero. E. 1993. Reclassification of
Amencan Rhizobium leguminosa~umbiovar phaseoli type 1strains as Rhizobi~trnetli
sp. nov. International Journal of Systematic Bacteriology 43: 374-377.
Sen, R. and Hepper, C. M. 1986. Characterization of vesicular-arbuscular mycorrhizal
fun@ (Glomus spp.) by selective enzyme staining following polyacrylamide gel
elecuophoresis. Soi1 Biology and Biochernistry 18: 29-34.
Serraj, R., Roy, G. and Drevon, J. 1. 1994. Salt-stress induces a decrease in the oxygen
uptake of soybean nodules and in their permeability to oxygen diffusion.
Physiologïa Plantarum 9 1: 161-168.
Shantharam. S. and Mattoo, A. K. 1997. Enhancing biological nitroten fixation: An
appraisal of current and Iltemative technologies for N input into plants. Plant and
Soil 194: 205-216.
Shirtliffe, S. I. and Vessey. I. K. 1996. A nodulanon (Nod+/Fix-) mutant of Phaseulus
vulgaris L. has nodule-like structures lacking peripheral vascular bundles (Pvb-) and
is resistant to mycorrhizal infection (Myc-). Plant Science 118: 209-220.
Simon. L.. Bousquet. J.. Levesque. R. C . and Lalonde. M. 1993a. Origin and
diversification of endomycorrhizal fun$ and coincidence with vascular land pimts.
Nature (London) 363: 67-69.
Simon, L.. Levesque. R. C. and Lalonde. M. 1993b. Identification of endomycorrhizal
fun@ colonizing roots by fluorescent single-strand conformation polymorphismpolymerase chah reaction. Applied and Environmental Microbi010gy 59: 42 114215.
Simpson. D. and Daft, M. J. 1990. Spore production and rnycorrhizd developrnent in
various tropical crop ùosts infected with Glorn~~s
clunm. Plant and Soi1 12 1: 171178.
Siqueira. J. O., Hubbell, D. H. and Schenck, N. C. 1982. Spore germination and germ
tube growth of a vesicular-arbuscdar mycorrhizal fungus in vino. Mycologia 74:
952-959.
Smith, F. A. and Smith, S. E. 1996. Mutuahm and parasitism: Diversiîy in function
and structure in the arbuscular (VA) mycorrhizal syrnbiosis. Advances in Botanical
Research 22: 1-43.
Smith. S. E. and Dafi, M. J. 2977. interactions between growth. phosphate content and
nitrogen fixation in myconhizal and non-myconliizal Medicaga sativa. Australian
Journal of Plant Physiology 4: 403-413.
Smith, S. E. and Read, D. J. 1997. Mycorrhizal Symbiosis. Second Edition.
Academic Press, Toronto, Canada.
Sneh, B.. Humble, S. J. and Lockwood, J. L. 2977. Parasitism of oospores cf
Phvrophrhora megasperma var. sojae, P. cacrorum. Pythium sp.. and Aphanornyces
euteiches i
n soi1 by Oomycetes. Chytridiomycetes. Hyphomycetes. actinomycetes
and bacteria. Phytopatholom 67: 622-628.
Soedajo. M. and Habte, M. 1995. Mycorrhizal and non-mycorrhizal host growth in
response to changes in pH and P concentration in a manganiferous oxisol.
Mycorrhiza 5: 337-345.
Somasegaran, P. and Hoben, H. J. 1994. The Handbook for Rhizobia Methods in
Leprne-Rhizobium Technology, Springer-Verlag, New York 450 pp.
Sprent. J. 1. 1972. The effects of water stress on nitrogen fixing root nodules.
Effects of osmotically applied stress. The New Phytologist 7 1:451-469.
m.
Sprent, J. 1. 1986. Nitrogen fixation in a sussainable agriculture. Biology Agriculture 1:
153-165.
Sprent, J. 1. 1989. Tansley Review no. 15: Which steps are essential for the formation
of functional legume nodules ? The New Phytologist 111: 129-153.
St-Arnaud. M.. Hamel. C., Vimard, B., Caron. M. and Fortin, J. A. 1995. Altered
growth of Fusarium oxysponrm fsp. chrysanthemi in an in vitro dual culture systern
with the vesicular arbuscular mycon-hizal fungus Glomcrs inrraradices growing on
Daucus carota ~ansformedroots. Mycorrhiz~?5: 43 1-438.
Stafford, H- A- 1997. Roles of flavonoids in symbiotic and defense functions in legume
roots. The Botanicd Review 63: 27-39.
Stamford, N. P., Ortega, A. D., Temprano, F. and Santos, D. R. 1997. Effects of
phosphoms fermization and inoculation of Bradyrhuobium and mycorrhizal fun@ on
growth of Mimosa caesalpiniaefolia in an acid soil. Soil Biology and Biochemistry
29: 959-964.
Stotzky, G. 1972. Activity, ecology and population dynamics of microorganisms in soil.
Cntical Review in Microbiology 2: 59-137.
Streit, W. R.. Joseph, C. M. and Phillips, D. A. 1996. Biotin and other water-soluble
vitarnins are key growth factors for alfalfa root colonization by Rhizobium melilotî
1021. Molecular Plant Microbe Interactions 9: 330-338.
Stutz, J. C. and Morton, J. B. 1996. Successive pot cultures reveal high species
nchness of arbuscular endomycorrhizal fun@in arid ecosystems. Canadian Journal
of Botany 74: 1883-2889.
Swelirn. D. M.. Hashem, F. M., Kuykendall, L. D.. Hegazi. N. 1. and Abdel-Wahab,
S. M. 1997. Host specificity and phenotypic diversity of Rhizobizrrn strains
nodulating Leucaenu,Acacia and Sesbania in Egypt. Biology and Fertility of Soils
25: 224-232.
Sylvia. D. M.. Wilson. D. O.. Graham. G. H.. Maddox. J. J.. Millner. P.. Morton. J.
B.. Skipper. H. D.. Wright. S. F. and Jarstfer. A. G. 1993. Evaluation of
vesicular-arbuscular mycorrhizal fungi in diverse plants and soils. Soil Biology and
Biochemistry 25: 705-7 13.
Talukdar, N. C- and Germida, J. J. 1993a. Occurrence and isolation of vesiculararbuscular mycorrhizal fun@ in cropped field soils of Saskatchewan. Canada.
Canadian Journal of blicrobiology 39: 567-575.
Talukdar, N. C. and Germida. J. J. 1993b. Propagation and storage of vesiculararbuscular mycorrhïzal fun@ isolated from Saskatchewan soils. Canadian Journal of
Botany 71: 1328-1335.
Talukdar. N. C. and Germida. J. 1. 1994. Growth and yield of lentil and wheat
inocdated with three Glomus isolates from Saskatchewan soils. Mycorrhiza 5: 145152,
Tang, S. D. 1979. Study of the nodulation and nitrogen fixation of soybean in lessive
soils. Acta Pedologca Sinica 16: 6-16.
Thakur, A. K. and Panwar, J. D. S. 1997. Response of Rhizobi~rm-vesicular
arbuscular mycorrhizd symbionts on photosynthesis, nitrogen metabolism and
sucrose translocation in greengram (Phaseolus radiams) . Indian Journal of
Agicultural Sciences 67: 245-248.
Thiagarajan. T. R. and Ahmad, M.H. 1993. Influence of a vesicular-arbuscular
mycorrhizal fungus on the cornpetitive ability of Bradyrhizobium spp. for nodulation
of cowpea Vigna unguiculata &.) Walp in non-sterilized soil. Biolow and Fertïlity
of Soils 15: 294-296Thies. J. E.. Singleton. P. W. and Bohlool. B. B. 199 1. Influence of the size of
indigenous rhizobial populations on establishment and syrnbiotic performance of
introdriced rhizobia on field-grown lepmes. Applied and Environmental
Micro biology 57: 19-28.
Thomas. R. L.. Sheard, R. W. and Moyer. J. R. 1967. Cornpaison of conventional and
automated procedures for nitrogen. phosphorus. and potassium analysis of plant
material using a single digestion. Agronomy Journal 59: 240-243.
Thomson. B. D.. Robson. A. D. and Abbott. L. K. 1986. EfTects of phosphorus on the
formation of mycomhizas by Gigaspara culospora and Glumusfnsciculanrm in
relation to root carbohydrates. The New Phytologist 103: 75 2-765.
Tilak. K. V. B. R.. Li. C. Y. and Ho. 1. 1989. Occurrence of nitrogen-fixing
Azospirill~rrnin vesicular-arbuscular mycorrhizal fungi. Plant and Soi1 116: 38628 8-
Tommerup. 1. C. 1983. Temperature relations of spore germination and hyphal growth
of vesicular-arbuscular mycorrhizd fun$ in soil. Transactions of the British
Mycological Society 8 1: 38 1-387.
Tommerup. 1. C. 1985. Inhibition of spore germination of vesicular-arbuscular
mycorrhizd fungi in soil. Transactions of the British Mycological Sociey 85: 267278Toro. M., Azcon, R. and Barea, J. M. 1998. The use of isotopic dilution techniques to
evaluate the interactive effects of Rhizobium genotype, rnycorrhizd fun&
phosphate-solubilizing rhizobacteria and rock phosphate on nitroten and phosphorus
acquisition by Medicago sativa. The New Phytologist 138: 265-273.
Toyota, K. and Kimura. M. 1993. Colonization of chlamydospores of Fusarium
oxysporum f. sp. raphani by soil bactena and their effects on germination. Soi1
Biology and Biochemisûy 25: 193-197.
Trappe, I. M. 1987. Phylogenetic and ecologic aspects of mycotrophy in the
angiosperms from an evolutionary viewpoint. In: Ecophysiology of VA Myconhizal
Plants (G. R. Safir ed.), CRC Press, Boca Raton, FL. USA. pp. 5-25.
Trent. J. D.. Svejcar, A. J. and Bethlenfalvay, G. J. 1993. Growth and nutrition of
combinations of native and introduced plants and mycorrhizal fungi in a serniarid
r a y e . Agriculture. Ecosystem and Environment 45: 13-23.
Trinick. M. J. 1980. Relationships amongst the fast growing rhizobia of Lnblab
purpureus. Leucaena leucocephah, Mimsa spp., Acacia farnesianu and Sesbania
grandiflorci and their &inities with other rhizobial groups. Journal of Applied
Bacteriology 49: 39-53.
Tsai. S. M. and Phillips. D. A. 1991. FIavonoids released naturally from ÿIfalf-promote developmeot of syrnbiotic Glomus spores in vin-o.Applied and
Environmental Microbiolog 57: 1485- 1488.
Turco, R. F., Moonnan, T- B. and Bezdiceb. D. F, 2986. Effectiveness and
competitiveness of spontaneous antibiotic-resistant mutants of Rhizobium
leg~iminosanrrnand Rhizobium japonicum. Soil Biology and Biochemistry 18: 259262.
Vejsadova, H., Siblikova, D.. Hrselova. H. and Vancura, V. 1992. Effect of the VAV
fungus Glumus sp. on the growth and yield of soybean inoculated with
BI-adyrhizobium
japonicum. Plant and Soil 140: 121- 125.
Vincent, J. M. 1970. A manual for the practical study of root-nodule bacteria.
International Biological Programme handbook no. 15. BlackwelI Scientific
Publications, Oxford, l64pp.
Vivekanandan. M. and Fÿcen. P. E. 1991. Cropping systems effects on mycorrhizal
colonization, early gowth. and phosphorus uptake of corn. The Soil Science
Society of America Journal 55: 136-140.
Walley, F. L. and Gerrnida. J. J. 1995. Estimating the viability of the vesiculararbuscular mycorrhizal fungal spores using tetrazolium salts as vital stains.
Mycologia 87: 273-279.
Walley, F. L. and Germida, J. J. 1996. Failure to decontaminate Glomus clarum NT4
spores is due to spore wd-associated bacteria. Mycorrhiza 6: 43-49.
Walley, F. L. and Germida, J. J. 1997. Response of spring wheat (Triticum aestivum)
to interactions between Pserrdomnas species and G l o m ~clanim
s
NT4. Biology
and Fertility of SoiIs 24: 365-37 1.
Wauud. L. S., Heithaus, 3. J. III and Jaworski, E. G. 1978. Geotropism in the
endomycomhizal fungus Gigaspora rnargarita. Mycologia 70: 449-452.
Weaver, R. W. and Wright, S. F. 1987. Variability in effectiveness of rhizobia during
culture and in nodules. Applied and Enviromenta1 Microbiology 53: 2972-2974.
Weisz, P. R., Denison. R. F. and Sinclair. T. R. 1985. Response to drought stress of
nitrogen fixation (acewlene reduction) rates in field-grown soybeans. Plant
Physiology 78: 525-530.
Wfl, M. E. and Sylvia. D. M. 1990. Interaction of rhizosphere bacteria, fertilizer. and
vesicular-arbuscular mycorrhizal fun$ with sea oats. Applied and Environmental
Microbiology 56: 2073-2079.
Wilson, G. W. T.. Hetrick. B. A. D. and Kitt. D. G. 1989. Suppression of vesiculararbuscular mycorriuzd fungus spore germination by non-sterile s o i Canadian
Journal of Botany 67: 18-23.
Wyss. P.. Mellor, R. B. :md Wiemken. A. 1990. Vesicular-arbuscular mycorrhizas of
wild-type soybean and non-nodulating mutants with Glornus mosseae contain
symbiosis-specific polypeptides (mycorrhizins), ~ m u n o l o ~ c a lcross-reactive
ly
with
nodulins. Planta 182: 22-26.
Xavier, L. J. C. and Gennida, J. J. 1997. Growth response of lentil and wheat to
GZomus darurn NT4 over a range of P levels in a Saskatchewan soi1 containine
indigenous AM fun&. Mycorrhiza 7: 3-8.
Xavier, L. J. C . a d Gerrnida, J. f. 1998. Response of q r i n g wheat cultivars to
Glumus clarum NT4 in a P-deficient soil containhg arbuscular mycorrhizal fun@.
Canadian Journal of Soi3 Science 78: 481-484.
Xie. 2. P., Sraehelin. C., Vierheilig. H.. Wiemken. A.. Jabbouri. S.. Broughton. W. J..
Vogeli-Lange, R. and Boller, T. 1995. Rhizobial nodulation factors stimulate
mycorrhizal colonization of nodulating and nonnodulating soybeans. Plant
Physiology 108: 1519-1525.
Yang, P., Vauterin, L.. Vancanneyt, M., Swings, J. and Kersters. K. 1993.
Application of fatty acid methyl esters for the taxonomie analysis. Systematic and
Applied Microbioloig 16: 47-7 1.
Yanni. Y.Ge,Ri&. R. Y.. Corich, V., Squartini. A., Ninke. K., Philip-Hollingsworth.
S., Orgarnbide, G., de-Brujin, F.. Stoltzfus, J.. Buckley. D.. Schmidt, T. M..
Mateos, P. F., Ladha, J. K. and Dazzo, F. B. 1997. Natural endophytic
association between Rhizobi~mlegurniizosarum bv. tr'ifolii and rice roots and
assessrnent of its potential to promote nce growth. Plant and Soi1 194: 99-1 14.
Zahran. H.H. and Sprent. J. 1. 1986. Effects of sodium c h l o n d ~and polyethylene
glycol on root-haïr infection and nodulation of Vicia faba L. plants by Rhizobitrm
Zeguminos~nrm.Planta 167: 303-309-
Zargar. M. Y. and Kahlon. R. S. 1995. Cornparison of symbiotic efiectiveness of
Rhizobium sp. with positive hydrogen-uptake activity compared with negative
mutants in relation to nitrogen fixation in rnungbean (Vigna radiata L.). Biology and
Feaility of Soils 20: 270-274.
Zaroug, M. G. and Munns, D. N. 1980. Screening strains of Rhizobium for the tropical
legumes Clitoris tematea and Vigno b-ihbora in soils of diEerent pH. Tropical
grasslands 14: 28-33.
10.0 APPENDICES
APPENDIX A.
Effects of interactions between two Glomus species
and Rhizobium leguminosarum bv. viceae on the
growth, yield and nutrition of pea and lentil in
non-sterile soi1 containing indigenous AMlF and
shizobia
Table A. 1. ANOVA for the shoot and mot dry weight grain yield. harvest index. shoot
N and P content, grain N and P content shoot and grain PUE and C/c AMF-colonized root
length of pea inoculated with the AMF species Glomrrs clarum M4 or G. mosseae NT6
andlor 10 Rhizobium Zeguminosarum bv. viceae strains and prown for 90 d in soil
containing indigenous AMF and rhizobia.
Source
Shoot dry weight
Model (experiment)
AA4F
Rhizobia
AMF x Rhizobia
Error
Root dry weight
Model (experiment)
M
Rhizobia
AMF x Rhizobia
Error
Grain yield
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
df
Mean Square
F ratio
Probability
Table A. 1 contd.
df
Mean Square
F ratio
Probability
32
0.0076
3 -57
0.000 1
2
0.0035
1-65
O. 1979
Wzobia
10
0.0135
6.32
0.000 1
AMF x Rhizobia
20
0.005 1
2.39
0.0025
Error
99
0.0021
Source
Harvest Index
Mode1 (experiment)
AMF
Shoot N content
Model (experiment)
AMI?
Rhizobia
AMF x Rhizobia
Error
Shoot P content
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
Table A. 1 contd.
Source
df
Mean Square
99
100082.5696
Grain N content
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
Grain P content
Model (experiment)
AMF
Rhizobia
AMF x EUiizobia
Error
Shoot P m
Mode1 (experiment)
AMF
Rhizobia
AMF x Rhizo bia
Error
F ratio
Probability
Table A, 1 contd.
df
Mean Square
F ratio
Probability
32
1853.8848
1.23
0.2224
2
2349.7336
1.55
0.2 166
Rhizobia
IO
4348.4324
2.8 8
0.0034
AMF x Rhizobia
20
555.426 1
0-37
0.9936
Error
99
1512.3456
Source
Grain PUE
Model (experirnent)
AMF
Am-colonized root length
Model (experiment)
32
422.8295
13-88
0.000 1
2
87 1.7272
38-93
0.0001
Rhizobia
10
545.0545
24.3 4
0,000 1
AMF x P k o b i a
20
3 16.8272
14.15
0.0001
Error
99
22.3914
AMF
Table A.3. Meuns for shtjot and gnin N and P content of pea inoculated with the AMF species Glorws cluriiu~NT4 or G. i~io.sseueNT6
and/or 10 Rhizobiirm legrir~iiiioscrriirnbv. viceue strains and grown for 90 d in soi1 containing indigenous AMF and rliizobia. LSD- Leut
significant differençe
Source
Variables
Slioot N content
Slioot P content
Grain N content
Grain P content
(mg/po t)
(mglpot)
(ni g/po t)
(mg/pot)
AMF
Rhizobia
O
Indigenous
44.41
4.57
67.7 1
8,73
G. clurwm NT4
40.73
4.44
65.30
8.40
G.r~iosseueNT6
4.69
70.42
9.38
LSD
44.02
4.00
0.58
5.49
0.69
Indigenous
34.86
4.3 1
51.07
7.76
RGP2
41 .56
4.29
63.86
9.03
PBlOl
40.06
3.94
65.92
83 9
175P4
37.68
5.95
34.9 1
5.90
LX 1
47 ,2.8
4.4 1
90.60
10.45
L X 13
41.48
5,47
53.17
798
LX36
45,07
4.35
76.33
9.75
LX43
56.33
4,28
103.3 1
10.59
LX57
.......-....
LSD
51.16
4.83
1.1 1
70.87
9.0 1
10.50
1.32
U......~U.......~~-..................................-.......-...............-.........................,...................................................,.......................**..*....~.,...,.....................,.,..,..................
7.66
Table A.4. Meaiis for shoot and grain P use efficiency (PUE) and AMF-colonized root lengtli of pea inaçulated with the AMF species
Glo17tirscla~rri?~
NT4 or G. ~~tossecre
NT6 uiid/or 10 Rhizobiitrn legiir~tiriosui-rm
bv. viceae strains and growii for 90 d in soi1 çontaiiiirig
indigenous AMF and rhizobia. LSD- Leut significant difference
Source
AMF
Rliizobia
Shoot PUE
Grain PUE
AMF-colaiiized root lengtli
(g shoot g-1P)
(g grniri g-1 P)
(%>
Indigerious
1128
334
25
G. clur*irmNT4
1 1 14
320
31
G. rnosseue NT6
1 17 1
324
33
LSD
134
16
2
Indigenous
985
323
32
RGP2
1127
333
19
PBfOl
1345
316
23
175P4
765
31 1
30
LX 1
1262
336
25
LX13
858
302
28
LX36
1183
322
27
LX43
1349
365
42
LX48
1022
300
19
LX5 1
1483
345
23
Vai-iübles
LX57
1136
329
22
.............................................................................................................................................................................................................
<...........,...
LSD
256
32
4
Table AS. ANOVA for the shoot and root dry weight grain yield. harvest index. shoot
N and P content. grain N and P contenr shoot and grain PUE and 5% AMF-colonized root
length of ientil inoculated with the AMF species Glorn~lsclanim h i 4 or G. rnosseoe NT6
andor nine Rh&obium Ieguminosuncrn bv. viceue saains and grown for 110 d in soil
containing indigenous AMF and rhizobia.
Source
df
Mean Square
90
0.0258
Shoot dry weight
Model (expedent)
AMF
Rbizobia
AMF x Rhizobia
Error
Root dry weight
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
Grain yield
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
F ratio
Probability
Source
Harvest Index
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
Shoot N content
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
Shoot P content
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
df
Mean Square
F ratio
Probability
Table A S contd.
Source
Grain FI content
Model (experiment)
AMF
Rhizobia
A M F x Rhizobia
Error
Grain P content
Model (experiment)
AMF
Rhizobia
AMF x Rhizobia
Error
Shoot PUE
Modef (experiment)
AMF
Rhizobia
AMF x Rhizobia
df
Mean Square
F ratio
Probabiliy
Table A S contd.
df
Mean Square
F ratio
Probability
29
665-1871
1.89
0.0 123
AMF
2
543.3908
1.54
0.2199
Rhizo bia
9
482.1 180
1.37
0-2 15 1
ArvIF x Rhizobia
18
770.2546
2.18
0.0085
Error
90
352.7414
Source
Grain PUE
Mode1 (experiment)
Am-colonized root length
29
233.0747
16.78
0.0001
AMF
2
249.8083
17.99
0.000 1
Rhizobia
9
184.7407
13.30
0.000 1
AMI? x Rhizobia
18
255.3 824
18.39
0.000 1
Error
90
13.8888
Mode1 (experiment)
APBENDUL B.
The influence of phosphorus on the tripartite
symbiosis between AMF, rhizobia and le,oumes
Table B.1. ANOVA for the shoot and root dry weight. grain yield. harvest index. shoot
N and P content. grain N and P content. shoot and grain PUE and
t/c
AMF-colonized root
length of pea inoculated with the AMF species Glornus clamm NT4 andfor the Rhizobirm
legrrminosarum bv. viceae strains 1 7 F 4 and LX43 and grown for 95 d in soif arnended
with 0. 10 or 20 mg kg-1 of P and containing indigenous AMF.
Source
df
Mean Square
72
O. 1551
Shoot dry weight
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
Root dry weight
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
Grain yieId
Model (expehent)
Treatment
P Ievels
Treaûnent x P levels
Error
250
F ratio
Probability
Table B. 1 contd.
Source
Harvest Index
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
Shoot N content
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
Shoot P content
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
df
Mean Square
F ratio
Prob ability
Table B. 1 contdSource
Grain N content
Model (experiment)
Treament
P levels
Treatment x P Ievels
Error
Grain P content
Model (experiment)
Treatment
P Ievels
Treatment x P levels
Error
Shoot PUE
Model (experiment)
Treatment
P levels
Treatrnent x P levels
Error
df
Mean Square
-Fratio
Prob ability
Table BA contd.
df
Mean Square
F ratio
17
61 19,7544
4.79
0.000 1
Treatment
5
13090-0836
10.24
0.000 1
P Ievels
2
11141-6954
8.7 1
0.0004
Treatment x P levels
10
1630.2016
1.27
0.2609
Error
72
1278-7977
Source
Probability
Grain PUE
Mode1 (experiment)
AMF-colonized root length
17
6 11.8972
9.29
0.0002
Treament
5
270.6699
4.07
0.0026
P levels
2
1646.7712
24.74
0.000 1
Treatment x P levels
10
575.536 1
8.65
0.0001
Error
72
66-5675
Mode1 (experiment)
Table B.5. ANOVA for the shoot and root dry weight. grain yield. harvest index. shoot
N and P content. grain N and P contznt shoot and grain PUE and 9 AMF-colonized root
length of lentil inoculated with the AMF species Glomus clanim NT4 andfor the
Rhizobium leguminosczrum bv. viceae strains PB101 and LX77 and grown for 110 d in
soil mended with 0.10 or 20 mg kg-' of P and containing indigenous AMF.
Source
df
Mean Square
F ratio
Probability
Shoot dry weight
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
Root dry weight
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
Grain yieid
Mode1 (experiment)
17
3.1957
20.0 1
0.000 1
Treatment
5
9.09 15
56.9 1
0.000 1
P levels
2
2.0403
12.77
0.000 1
Treatment x P 1evels
10
0.4789
3.O0
0.0032
Error
72
O. 1597
Table B.5 contd,
Source
df
Mean Square
F rauo
Probability
Harvest Index
Mode1 (experirnent)
17
0.0203
6-53
O .O00 1
Treatrnent
5
0.0264
8.46
0.000 1
P levels
2
0.0333
10.66
0.000 1
Treatment x P levels
10
0.0147
4.7 1
0.000 1
Error
72
0.003 1
17
1357.4448
12-13
0.000 1
Treatment
5
3601.5124
32.18
0.000 1
P levels
2
368-4825
3 -29
0.0428
Treatment x P levels
10
433,2035
3.87
0.0003
Error
72
111.9033
17
10.0296
5.38
O .O00 1
Treatment
5
8.4375
4.53
0.0012
P Tevels
2
9.6973
5.20
0.0078
Treatment x P levels
10
10.892 1
5.84
0.0002
Error
72
1.8646
Shoot N content
Mode1 (experiment)
Shoot B content
Mode1 (experirnent)
Table B.5 contd.
Source
df
Mean Square
F ratio
Probability
17
2948.5260
18.39
0.000 1
Treaünent
5
8792.5724
54.84
0.0001
P levels
2
1011.5973
6.3 1
0.0030
Treafmentx P levek
10
413.8886
2.58
0.0099
Error
72
160.3 188
Grain N content
Mode1 (experiment)
Grain P content
Model (experiment)
Treatment
P 1eveIs
Treatment x P levels
Error
Shoot PUE
Model (experiment)
Treatment
P levels
Treatment x P levels
Error
Table B.5 contd.
Source
F ratio
df
Mean Square
17
1848.6069
4.66
Treatment
5
1085.1903
2-74
P levels
2
7654.5687
19.32
Treatrnent x P levels
10
1069.1229
2 -70
Error
72
396.286 1
Probabiiity
Grain PUE
Model (experiment)
AIVIF-colonized mot length
Model (experimentj
17
194.50 13
5.40
0.000 1
Treatment
5
484.5 164
13.45
0.000 1
P levels
2
60.7556
1.69
O. 1925
Treatrnent x P levek
10
76.2429
2.12
0.0339
Error
72
36.0360