REPORT #
RRTAC 88-3
Revegetation Of Oil Sands Tailings:
Growth Improvement Of Silver-Berry And
Buffalo-Berry By Inoculation With
Mycorrhizal Fungi And Na-Fixing Bacteria
Heritage Fund
Jbcsna
LAND CONSERVATION AND
RECLAMATION COUNCIL
Reclamation Research
Technical Advisory Committee
Report No. RRTAC 88-3
REVEGETATION OF OIL SANDS TAILINGS:
GROWTH IMPROVEMENT OF SILVER-BERRY AND BUFFALO-BERRY
BY INOCULATION WITH MYCORRHIZAL FUNGI
AND N2-FIXING BACTERIA
BY
Suzanne Visser
and
Robert M. Danielson
Kananaskis Centre for Environmental Research
The University of Calgary
Prepared for
The Oil Sands Reclamation Research Program
of
THE LAND CONSERVATION AND RECLAMATION COUNCIL
(Reclamation Research Technical Advisory Committee)
1988
Digitized by the Internet Archive
in 2015
https://archive.org/details/revegetationofoiOOviss
STATEMENT OF OBJECTIVE
The recommendations and conclusions in this report are those of the
authors and not those of the Alberta Government or its
representati ves.
This report is intended to provide government and industry staff
with up-to-date technical information to assist in the development of
guidelines and operating procedures. The report is also available to
the public so that interested individuals similarly have access to the
best available information on land reclamation topics.
TV
ALBERTA'S RECLAMATION RESEARCH PROGRAM
The regulation of surface disturbances in Alberta is the
responsibility of the Land Conservation and Reclamation Council. The
Council executive consists of a Chairman from the Department of
Forestry, Lands and Wildlife. Among other functions, the Council
oversees programs for reclamation of abandoned disturbances and
reclamation research. The Reclamation Research Program was established
to provide answers to the many practical questions which arise in
reclamation. Funds for implementing both the operational and research
programs are drawn from Alberta's Heritage Savings Trust Fund.
To assist in technical matters related to the development and
administration of the Research Program, the Council appointed the
Reclamation Research Advisory Committee (RRTAC). The Committee first
met in March 1978 and consists of eight members representing the Alberta
Departments of Agriculture, Energy, Forestry, Lands and Wildlife,
Environment and the Alberta Research Council. The Committee meets
regularly to update research priorities, review solicited and
unsolicited research proposals, arrange workshops and otherwise act as a
referral and coordinating body for Reclamation Research.
Additional information on the Reclamation Research Program may be
obtained by contacting:
Dr. G.A. Singleton, Chairman
Reclamation Research Technical Advisory Committee
Alberta Environment
4th Floor, Oxbridge Place
9820 - 106 Street
Edmonton, Alberta T5K 2J6
(403) 427-5868
This report may be cited as: Visser, S. and R.M. Danielson, 1988.
Revegetation of Oil Sands Tailings: Growth Improvement of Silver-Berry
and Buffalo-Berry by Inoculation with Mycorrhizal Fungi and N^-Fixing
Bacteria. Alberta Land Conservation and Reclamation Counci r Report
#RRTAC 88-3. 98 pp.
Additional copies may be obtained from:
Publication Services
Queen's Printer
11510 Kingsway Avenue
Edmonton, Alberta T5G 2Y5
V
RECLAMATION RESEARCH REPORTS
** 1. RRTAC 80-3:
The Role of Organic Compounds in Salinization of
Plains Coal Mining Sites. N.S.C. Cameron et al .
46 pp.
DESCRIPTION:
This is a literature review of the chemistry of
sodic mine spoil and the changes expected to
occur in groundwater.
** 2. RRTAC 80-4:
Proceedings: Workshop on Reconstruction of
Forest Soils in Reclamation. P.F. Ziemkiewicz,
S.K. Takyi, and H.F. Regier. 160 pp.
DESCRIPTION:
Experts in the field of forestry and forest soils
report on research relevant to forest soil
reconstruction and discuss the most effective
means of restoring forestry capability of mined
lands.
N/A 3. RRTAC 80-5:
Manual of Plant Species Suitability for
Reclamation in Alberta. L.E. Watson, R.W.
Parker, and P.F. Polster. 2 vols, 541 pp.
DESCRIPTION:
Forty-three grass, fourteen forb, and thirty-
four shrub and tree species are assessed in terms
of their fitness for use in Reclamation.
Range maps, growth habit, propagation, tolerance,
and availability information are provided.
N/A 4. RRTAC 81-2:
1980 Survey of Reclamation Activities in Alberta.
D.G. Walker and R.L. Rothwell. 76 pp.
DESCRIPTION:
This survey is an update of a report prepared in
1976 on reclamation activities in Alberta, and
includes research and operational reclamation,
locations, personnel, etc.
N/A 5. RRTAC 81-3:
Proceedings: Workshop on Coal Ash and
Reclamation. P.F. Ziemkiewicz, R. Stien, R.
Leitch, and G. Lutwick. 253 pp.
DESCRIPTION:
Presents nine technical papers on the chemical,
physical and engineering properties of Alberta
fly and bottom ashes, revegetation of ash
disposal sites and use of ash as a soil
amendment. Workshop discussions and summaries
are also included.
VI
N/A 6» RRTAC82-1:
Land Surface Reclamation: An International
Bibl iography. H.P. Sims and C.B. Powter. 2
vols, 292 pp.
DESCRIPTION:
Literature to 1980 pertinent to reclamation in
Alberta is listed in Vol . 1 and is also on the
University of Alberta computing system. Vol . 2
comprises the keyword index and computer access
manual .
N/A 7. RRTAC 82-2:
A Bibliography of Baseline Studies in Alberta:
Soils, Geology, Hydrology and Groundwater. C.B.
Powter and H.P. Sims. 97 pp.
DESCRIPTION:
This bibliography provides baseline information
for persons involved in reclamation research or
in the preparation of environmental impact
assessments. Materials, up to date as of
December 1981, are available from the Alberta
Environment Library.
N/A 8» RRTAC 83-1:
Soil Reconstruction Design for Reclamation of Oil
Sand Tailings. Monenco Consultants Ltd.
185 pp.
DESCRIPTION:
Volumes of peat and clay required to amend oil
sand tailings were estimated based on existing
literature. Separate soil prescriptions were
made for spruce, jack pine, and herbaceous cover
types. The estimates form the basis of field
tri al s .
N/A 9. RRTAC 83-3:
Evaluation of Pipeline Reclamation Practices on
Agricultural Lands in Alberta. Hardy Associates
(1978) Ltd. 205 pp.
DESCRIPTION:
Available information on pipeline reclamation
practices was reviewed. A field survey was then
conducted to determine the effects of pipe size,
age, soil type, construction method, etc. on
resulting crop production.
N/A lOo RRTAC 83-4:
Proceedings: Effects of Coal Mining on Eastern
Slopes Hydrology. P.F. Ziemkiewicz. 123 pp.
DESCRIPTION:
Technical papers are presented dealing with the
impacts of mining on mountain watersheds, their
flow characteristics and resulting water quality.
Mitigative measures and priorities were also
di scussed.
N/A n. RRTAC 83-5:
N/A n. RRTAC 83-5:
DESCRIPTION:
vi i
Woody Plant Establishment and Management for Oil
Sands Mine Reclamation. Techman Engineering Ltd.
124 pp.
This is a review and analysis of information on
planting stock quality, rearing site preparation,
planting and procedures necessary to ensure
survival of trees and shrubs in oil sand
reclamation.
***12. RRTAC 84-1:
Land Surface Reclamation: A Review of
International Literature. H.P. Sims, C.B.
Powter, and J.A. Campbell. 2 vols, 1549 pp.
DESCRIPTION:
Nearly all topics of interest to reclamation
including mining methods, soil amendments,
revegetation, propagation and toxic materials are
reviewed in light of the international
1 iterature.
** 13. RRTAC 84-2:
Propagation Study: Use of Trees and Shrubs for
Oil Sand Reclamation. Techman Engineering Ltd.
58 pp.
DESCRIPTION:
This report evaluates and summarizes all
available published and unpublished information
on large-scale propagation methods for shrubs and
trees to be used in oil sand reclamation.
* 14. RRTAC 84-3:
Reclamation Research Annual Report - 1983. P.F,
Ziemkiewicz. 42 pp.
DESCRIPTION:
This report details the Reclamation Research
Program indicating priorities, descriptions of
each research project, researchers, results and
expenditures.
** 15.- RRTAC 84-4:
Soil Microbiology in Land Reclamation. D.
Parkinson, R.M. Danielson, C. Griffiths, S.
Visser, and J.C. Zak. 2 vols, 676 pp.
DESCRIPTION:
This is a collection of five reports dealing with
re-establishment of fungal decomposers and
mycorrhizal symbol nts in various amended spoil
types.
** 16. RRTAC 85-1:
Proceedings: Revegetation Methods for Alberta's
Mountains and Foothills. P.F. Ziemkiewicz.
416 pp.
DESCRIPTION:
Results of long-term experiments and field
experience on species selection, fertilization,
reforestation, topsoiling, shrub propagation and
establishment are presented.
VI 1 1
* 17, RRTAC 85-2: Reclamation Research Annual Report - 1984. P.F.
Ziemkiewicz. 29 pp.
DESCRIPTION: This report details the Reclamation Research
Program indicating priorities, descriptions of
each research project, researchers, results and
expenditures.
** 18.
RRTAC 86-1 :
A Critical Analysis of Settling Pond
Alternative Technologies. A. Somani .
Design and
372 pp.
DESCRIPTION:
The report examines the critical
settling pond design and sizing and
technologies.
issue of
alternative
** 19.
RRTAC 86-2:
Characterization and Variability
Reconstructed after Surface Mining
Alberta. T.M. Macyk. 146 pp.
of Soi 1
in Central
DESCRIPTION:
Reconstructed soils representing
different
materials handling and replacement techniques
were characterized and variability in chemical
and physical properties was assessed. The data
obtained indicate that reconstructed soil
properties are determined largely by parent
material character! sties and further tempered by
materials handling procedures. Mining tends to
create a relatively homogeneous soil landscape in
contrast to the mixture of diverse soils found
before mining.
* 20. RRTAC 86-3: Generalized Procedures for Assessing Post-Mining
Groundwater Supply Potential in the Plains of
Alberta - Plains Hydrology and Reclamation
Project. M.R. Trudell and S.R. Moran. 30 pp.
DESCRIPTION: In the Plains region of Alberta, the surface
mining of coal generally occurs in rural ,
agricultural areas in which domestic water supply
requirements are met almost entirely by ground-
water. Consequently, an important aspect of the
capability of reclaimed lands to satisfy the
needs of a residential component is the
post-mining availability of groundwater. This
report proposes a sequence of steps or procedures
to identify and characterize potential
post-mining aquifers.
IX
** 21. RRTAC 86-4:
Geology of the Battle River Site: Plains
Hydrology and Reclamation Project. A Maslowski-
Schutze, R. Li, M. Fenton and S.R. Moran. 86 pp.
DESCRIPTION:
This report summarzies the geological setting of
the Battle River study site. It is designed to
provide a general understanding of geological
conditions adequate to establish a framework for
hydrogeological and general reclamation studies.
The report is not intended to be a detailed
synthesis such as would be required for mine
planning purposes.
** 22. RRTAC 86-5:
Chemical and Mineral ogi cal Properties of
Overburden: Plains Hydrology and Reclamation
Program. A. Maslowski-Schutze. 71 pp.
DESCRIPTION:
This report describes the physical and
mineral ogi cal properties of overburden materials
in an effort to identify individual beds within
the bedrock overburden that might be
significantly different in terms of reclamation
potenti al .
* 23. RRTAC 86-6:
Post-Mining Groundwater Supply at the Battle
River Site: Plains Hydrology and Reclamation
Project. M.R. Trudell, G.J. Sterenberg and S.R.-
Moran. 49 pp.
DESCRIPTION:
The report deals with the availability of water
supply in or beneath cast overburden at the
Battle River Mining area in east-central Alberta
to support post-mining land use. Both
groundwater quantity and quality are evaluated.
* 24. RRTAC 86-7:
Post-Mining Groundwater Supply at the Highvale
Site: Plains Hydrology and Reclamation Project.
M.R. Trudel 1 . 25 pp.
DESCRIPTION:
This report evaluates the availability of water
supply in or beneath cast overburden to support
post-mining land use, including both quantity and
quality considerations. The study area is the
Highvale mining area in west-central Alberta.
* 25. RRTAC 86-8:
Reclamation Research Annual Report - 1985.
P.F. Ziemkiewicz. 54 pp.
DESCRIPTION:
This report details the Reclamation Research
Program indicating priorities, descriptions of
each research project, researchers, results and
expenditures.
X
c
** 26. RRTAC 86-9:
Wildlife Habitat Requirements and Reclamation
Techniques for the Mountains and Foothills of
Alberta. J.E. Green* R.E. Salter and D.G.
Walker. 285 pp.
DESCRIPTION:
This report presents a review of relevant North
American literature on wildlife habitats in
mountain and foothills biomes* reclamation
techniques, potential problems in wildlife
habitat reclamation, and potential habitat
assessment methodologies. Four biomes (Alpine,
Subalpine, Montane, and Boreal Uplands) and 10
key wildlife species (snowshoe hare, beaver,
muskrat, elk, moose, caribou, mountain goat,
bighorn sheep, spruce grouse, and white-tailed
ptarmigan) are discussed.
** 27. RRTAC 87-1:
Disposal of Drilling Wastes. L.A. Leskiw, E.
Reinl -Dwyer, T.L. Dabrowski , B.J. Rutherford and
H. Hamilton. 210 pp.
DESCRIPTION:
Current drilling waste disposal practices are
reviewed and criteria in Alberta guidelines are
assessed. The report also identifies research
needs and indicates mitigation measures. A
manual included provides a decision-making
flowchart to assist in selecting methods of
environmental ly safe waste disposal.
** 28. RRTAC 87-2:
Minesoil and Landscape Reclamation of the Coal
Mines in Alberta's Mountains and Foothills. A.W.
Fedkenheuer, L.J. Knapik, and D.G. Walker.
174 pp.
DESCRIPTION:
This report reviews current reclamation practices
with regard to site and soil reconstruction and
re-establishment of biological productivity. It
also identifies research needs in the
Mountain-Foothills area.
** 29. RRTAC 87-3:
Gel and Saline Drilling Wastes in Alberta:
Workshop Proceedings. D.A. Lloyd (compiler).
218 pp.
DESCRIPTION:
Technical papers were presented which describe:
the mud systems used and their purpose;
industrial constraints; government regulations,
procedures and concerns; environmental
considerations in waste disposal; and toxic
constituents of drilling wastes. Answers to a
questionnaire distributed to participants are
included in an appendix.
XT
* 30. RRTAC 87-4:
Reclamation Research Annual Report - 1986.
50 pp.
DESCRIPTION:
This report details the Reclamation Research
Program indicating priorities, descriptions of
each research project, researchers, results and
expenditures .
* 31. RRTAC 87-5:
Review of the Scientific Basis of Water Quality
Criteria for the East Slope Foothills of
Alberta. Beak Associates Consulting Ltd.
46 pp.
DESCRIPTION:
The report reviews existing Alberta guidelines
to assess the quality of water drained from coal
mine sites in the East Slope Foothills of
Alberta. World literature was reviewed within
the context of the east slopes environment and
current mining operations. The ability of coal
mine operators to meet the various guidelines is
di scussed.
** 32. RRTAC 87-6:
Assessing Design Flows and Sediment Discharge on
the Eastern Slopes. Hydrocon Engineering
(Continental) Ltd. and Monenco Consultants Ltd.
97 pp.
DESCRIPTION:
The report provides an evaluation of current
methodologies used to determine sediment yields
due to rainfall events in well-defined areas.
Models are available in Alberta to evaluate
water and sediment discharge in a post-mining
situation. SEDIMOT II (Sedimentology Disturbed
Modelling Techniques) is a single storm model
that was developed specifically for the design
of sediment control structures in watersheds
disturbed by surface mining and is well suited
to Alberta conditions.
* 33. RRTAC 87-7:
The Use of Bottom Ash as an Amendment to Sodic
Spoil. S. Fullerton. 83 pp.
DESCRIPTION:
The report details the use of bottom ash as an
amendment to sodic coal mine spoil. Several
rates and methods of application of bottom ash
to sodic spoil were tested to determine which
was the best at reducing the effects of excess
sodium and promoting crop growth. Field trials
were set up near the Vesta mine in East Central
Alberta using ash readily available from nearby
coal -fired thermal generating station. The
research indicated that bottom ash incorporated
to a depth of 30 cm using a subsoil er provided
the best results.
34. RRTAC 87-8: Waste Dump Design for Erosion Control. R.G.
Chopiuk and S.E. Thornton. 45 pp.
DESCRIPTION: This report describes a study to evaluate the
influence of erosion from reclaimed waste dumps
on downslope environments such as streams and
rivers. Sites were selected from coal mines in
Alberta's mountains and foothills, and included
resloped dumps of different configurations and
ages, and having different vegetation covers.
The study concluded that the average annual
amount of surface erosion is minimal. As
expected, erosion was greatest on slopes which
were newly regraded. Slopes with dense grass
cover showed no signs of erosion. Generally,
the amount of erosion decreased with time, as a
result of initial loss of fine particles, the
formation of a weathered surface, and increased
vegetative cover.
35. RRTAC 87-9: Hydrogeology and Groundwater Chemistry of the
Battle River Mining Area. M.R. Trudell, R.L.
Faught and S.R. Moran. 97 pp,
DESCRIPTION: This report describes the premining geologic
conditions in the Battle River coal mining area
including the geology as well as the groundwater
flow patterns, and the groundwater quality of a
sequence of several water-bearing formations
extending from the surface to a depth of about
100 metres.
36. RRTAC 87-10: Soil Survey of the Plains Hydrology and
Reclamation Project - Battle River Project Area.
T.M. Macyk and A.H. Maclean. 62 pp. plus maps.
DESCRIPTION: The report evaluates the capability of
post-mining landscapes and assesses the changes
in capability as a result of mining, in the
Battle River mining area. Detailed soils
information is provided in the report for lands
XT 1 1
adjacent to areas already mined as well as for
lands that are destined to be mined.
Characterization of the reconstructed soils in
the reclaimed areas is also provided. Data were
collected from 1979 to 1985. A series of maps
supplement the report.
** 37. RRTAC 87-11 :
Geology of the Highvale Study Site: Plains
Hydrology and Reclamation Project. A.
Maslowski-Schutze. 78 pp.
DESCRIPTION:
The report is one of a series that describes the
geology, soils and groundwater conditions at the
Highvale Coal Mine study site. The purpose of
the study was to establish a summary of site
geology to a level of detail necessary to
provide a framework for studies of hydrogeology
and reclamation.
** 38. RRTAC 87-12:
Premining Groundwater Conditions at the Highvale
Site. M.R. Trudell and R. Faught. 83 pp.
DESCRIPTION:
This report presents a detailed discussion of
the premining flow patterns, hydraulic
properties, and isotopic and hydrochemical
character! sties of five layers within the
Paskapoo Geological Formation, the underlying
sandstone beds of the Upper Horseshoe Canyon
Formation, and the surficial glacial drift.
* 39. RRTAC 87-13:
An Agricultural Capability Rating System for
Reconstructed Soils. T.M. Macyk. 27 pp.
DESCRIPTION:
This report provides the rationale and a system
for assessing the agricultural capability of
reconstructed soils. Data on the properties of
the soils used in this report are provided in
RRTAC 86-2.
** 40. RRTAC 88-1:
Eccles, T.R., R.E. Salter and J.E. Green. A
Proposed Evaluation System for Wildlife Habitat
Reclamation in the Mountains and Foothills
Biomes of Alberta: Proposed Methodology and
Assessment Handbook. 101 pp. plus appendix.
DESCRIPTION:
The report focuses on the development of
guidelines and procedures for the assessment of
reclaimed wildlife habitat in the Mountains and
Foothills regions of Alberta. The technical
section provides background documentation
including a discussion of reclamation planning.
XIV
a listing of reclamation habitats and associated
key wildlife species, conditions required for
development, recommended revegetation species,
suitable reclamation techniques, a description
of the recommended assessment techniques and a
glossary of basic terminology. The assessment
handbook section contains basic information
necessary for evaluating wildlife habitat
reclamation, including assessment scoresheets
for 15 different reclamation habitats, standard
methodologies for measuring habitat variables
used as assessment criteria, and minimum
requirements for certification. This handbook
is intended as a field manual that could
potentially be used by site operators and
reclamation officers.
** 41. RRTAC 88"2: Plains Hydrology and Reclamation Project: Spoil
Groundwater Chemistry and its Impacts on Surface
Water. M.R. Trudell (Compiler). Alberta Land
Conservation and Reclamation Council Report
#RRTAC 88-2. 135 pp.
DESCRIPTION: Two reports comprise this volume. The first
"Chemistry of Groundwater in Mine Spoil, Central
Alberta," describes the chemical make-up of
spoil groundwater at four mines in the Plains of
Alberta. It explains the nature and magnitude
of changes in groundwater chemistry following
mining and reclamation.
The second report, "Impacts of Surface Mining on
Chemical Quality of Streams in the Battle River
Mining Area," describes the chemical quality of
water in streams in the Battle River mining
area, and the potential impact of groundwater
discharge from surface mines on these streams.
Available from: Publication Services
Queen's Printer
11510 Kingsway Avenue
Edmonton, Alberta T5G 2Y5
* A $5.00 fee is charged for handling and postage.
** A $10.00 fee is charged for handling and postage.
A $20.00 fee is charged for handling and postage.
N/A Not available for purchase but available for review at the Alberta
Environment Library, 14th Floor, 9820-106 Street, Edmonton, Alberta
T5K 2J6.
XV
EXECUTIVE SUMMARY
The ability of actinorhizal shrubs to tolerate inhospitable
conditions while improving soil fertility and organic matter status has
led to increased usage of these plants for land reclamation and amenity
planting purposes. Silver-berry and buf falo-berry are two such shrubs
which are being tested as potential candidates for the revegetation of
the oil sands tailings in northeastern Alberta.
Associated with the roots of silver-berry and buf falo-berry
are two symbionts - the N2-fixing actinomycete, Frankia. and the
vesicular-arbuscular mycorrhizal (VAM) fungi. Numerous studies have
demonstrated that, particularly in nutrient limited conditions, mycor-
rhization and nodulation can result in significantly better plant per-
formance as a consequence of improved N and P nutrition. The benefits
conferred on the host by the symbionts may assume even greater impor-
tance in the revegetation of mine tailings which are notoriously
nutrient-poor.
In addition to reducing soil fertility, the upheaval and
mixing of soil during the mining process can lower Frankia and VAM
inoculum levels. Both soil fertility and symbiont inoculum potential
can be improved by introducing an organic amendment to the minespoil.
Soil reconstruction on the oil sands tailings is facilitated by the
application of muskeg peat which is stockpiled on the site for reclama-
tion purposes. Alternatively, if woody plants are raised as containe-
rized seedlings, they can be inoculated with both their N2-fixing and
mycorrhizal symbionts prior to being outplanted. However, before
embarking on a large-scale inoculation program which will ultimately
raise the cost of producing a seedling, factors such as plant depen-
dency on the symbionts, the level of Frankia and mycorrhizal inoculum
in the outplanting soil and the nodule/mycorrhizal status of containe-
rized seedlings leaving commercial greenhouses should be considered.
With this in mind, a research program was initiated to fulfil the
following objectives:
XVI
1. To determine the mycorrhizal affinities of various
actinorhizal shrubs in the Fort McMurray, Alberta region.
2. To establish a basis for justifying symbiont inoculation
of buffalo-berry and si 1 ver-berry. Factors investigated
included: i) the dependency of the shrubs on their mycor-
rhizal and N2-fixing symbionts as expressed in plant
performance under inoculated and uninoculated conditions,
ii) the Frankia and VAM inoculum levels in the out-
planting soil including stockpiled peat on the Syncrude
lease and soils reconstructed from peat, mineral soil and
oil sand tailings; iii) rates of mycorrhi zation and nodu-
lation in undisturbed and reconstructed tailings dyke
soils, and iv) the mycorrhizal and nodule status of
containerized shrubs raised in commercial and provincial
nurseries .
3. To develop a growing regime for the greenhouse production
of mycorrhizal, nodulated silver-berry and buf falo-berry .
4. To conduct a field trial on reconstructed soil on the
Syncrude site to critically evaluate the growth
performance of inoculated silver-berry and buf falo-berry
as compared with their uninoculated counterparts .
The major findings are:
JUSTIFICATION FOR INOCULATION
1. In Alberta, silver-berry and buf falo-berry are strictly
VA-mycorrhizal . Levels of VAM colonization in roots of
field collected plants can be as high as 60% suggesting a
high degree of symbiont dependency under field conditions.
2. Silver-berry and buffalo-berry are highly dependent on
their symbionts for optimum growth as evidenced by four
(si 1 ver-berry)and nine-fold (buf falo-berry) increases in
shoot weights when seedlings are inoculated with Frankia
and VA-mycorrhizal fungi.
XVI 1
3. The VAM inoculum potential of both stockpiled and undis-
turbed muskeg peat is negligible due to the absence of
VAM hosts.
4. Due to the low levels of VAM inoculum in the peat, stock-
piling has no significant impact on VAM propagule
levels. Vegetating peat stockpiles with VAM hosts such
as grasses and legumes can increase VAM infectivity by
10-12% over six years. Tailings sand amended with peat
would lack VA-mycorrhi zal inoculum unless the peat had
been vegetated with VAM hosts for a substantial length of
time.
5. Soil from mixed woodlands (spruce, aspen, pine) has the
highest VAM and Frankia inoculum potential of all soils
assayed in the Ft. McMurray area. Amendation of tailing
sand with this type of soil would greatly improve
symbiont infectivity.
6. Growth of slender wheatgrass in unfertilized, stockpiled
peat is stimulated when inoculated with mycorrhizal
fungi, suggesting VAM fungi are necessary to satisfy the
nutritional demands of the plant when grown in
P-deficient peat.
7. Containerized shrubs grown in various nurseries in
Alberta and B.C. are seldom mycorrhizal and/or nodulated
if less than one year old. This means the majority of
actinorhizal shrubs are symbiont-free if shipped to the
buyer v>/ithin a year of planting. Containerized shrubs
which are more than one year old and have spent time in
the shadehouse may or may not be colonized by their
symbionts .
8. Buf falo-berry planted in reconstructed soil in the green-
house do not become mycorrhizal or nodulated until eight
weeks after planting. Since rates of colonization would
be expected to be much slower in the field than in the
xvi i i
greenhouse and since the growing season in the oil sands
region is short, it is doubtful that containerized shrubs
would obtain much benefit from the symbiosis during the
first growing season unless artificially inoculated.
9. Uninoculated silver-berry seedlings outplanted on the
Suncor dyke exhibited relatively rapid mycorrhization
(within six weeks of planting) presumably due to high VAM
inoculum levels resulting from the predominance of VAM
hosts (grasses, legumes) on the dyke. In contrast, nodu-
lation was poor caused by either a lack of Frankia inocu-
lum in the soil or poor root growth out of the planting
plug.
10. The low VAM/Frankia inoculum potential and the slow rates
of mycorrhization and nodulation in reconstructed soil on
the tailings sand dykes, combined with the high depen-
dency of silver-berry and buf falo-berry on their
symbionts, forms a strong basis for artificial
inoculation of containerized seedlings.
GROWING REGIME FOR PRODUCING MYCORRHIZAL, NODULATED SEEDLINGS
11. In order to produce mycorrhizal, nodulated silver-berry
and buffalo-berry seedlings of suitable size and quality,
fertilization should not exceed 56 mg N and 12 mg P per
application. Fertilizer concentrations in excess of this
do not totally eliminate symbiont colonization, but
mycorrhizal and nodule development is severely reduced.
The optimum fertilization regime in this study was
200 mg L~^ 28-14-14 Plant Prod Soilless Feed applied
twice weekly.
12. Silver-berry growth is significantly better in 150 cc
containers than in 65 cc containers. As soil volume is
reduced there is a concomitant decrease in symbiont
growth response so that inoculated seedlings in the
150 cc containers exhibited a significant growth response
whereas those in 65 cc containers did not.
XT X
13. Seedlings inoculated with woodland soil demonstrate
better mycorrhi zation and nodulation and a greater growth
response at 26®C than at 16®C.
14. In a Frankia inoculum trial, the best sources of inoculum
resulting in the biggest silver-berry seedlings with the
most heavily nodulated root systems were found to be wild
buffalo-berry soil, crushed silver-berry nodules, and
crushed silver-berry nodules treated with polyvinyl
pyrrolidine to reduce oxidation of phenols which inhibit
Frankia growth. Seedlings inoculated with Frankia pure
culture obtained from Rhizotec Labs in Quebec became
heavily nodulated but this was not manifested in improved
plant growth. Seedlings inoculated with a pure culture
of Frankia isolated from buffalo-berry failed to become
nodulated possibly because the Frankia strain was incom-
patible with silver-berry.
15. Mixing a highly infective soil into the planting mixture
appears to be more effective at promoting symbiont
development than applying the inoculum as a soil slurry
after plant establishment.
16. Mycorrhizal, nodulated silver-berry and buffalo-berry of
suitable size and quality can be obtained by planting
them in 150 cc containers filled with peat/vermicul ite
(1/1 v/v) which has been supplemented with high inoculum
soil (10-15% by volume), and fertilizing them twice
weekly at a rate of 200 mg 28-14-14 Plant Prod
Soilless Feed. Buffalo-berry appears to be more N-deman-
ding than silver-berry and may require a higher fertili-
zer concentration.
XX
c
FIELD TRIAL TO TEST GROWTH RESPONSE OF INOCULATED SILVERBERRY
AND BUFFALO-BERRY
17. Overwinter mortality was higher for inoculated shrubs
than for uninoculated shrubs. Due to their symbiotic
condition, the inoculated shrubs may have had greater
stomatal conductance and higher rates of transpi ration
than the uninoculated shrubs when outplanted, making them
more susceptible to frost damage. It is possible that
inoculated seedlings require a longer period of hardening
off than do uninoculated seedlings, particularly if they
are to be outplanted in the fall.
18. After one growing season, shoot weights of inoculated
silver-berry were three to seven times greater than those
of the uninoculated seedlings, while shoots of inoculated
buffalo-berry were three to five times heavier than those
of their uninoculated counterparts . The much superior
growth performance of the inoculated seedlings was
continued over the second growing season.
19. The significant growth response of the inoculated shrubs
aboveground was reflected in the symbiont status below-
ground where nodule and mycorrhizal development was
significantly more extensive in the inoculated plants
than the uninoculated plants over the two growing seasons.
20. Shoot production appeared to be heavily dependent on
healthy nodule development as evidenced by the highly
significant correlations between shoot weights and nodule
weights (r = 0.91, 0.97) after one growing season. Shoot
productivity was more closely related to nodule status
than mycorrhizal status (r = 0.63 and 0.58 for shoot
weight versus mycorrhizal root length for silver-berry
and buf faloberry, respectively). Also, per cent
mycorrhizal root length was not closely correlated with
nodule number and weight suggesting that, in the field,
other factors besides mycorrhizal status may strongly
influence nodulation.
XXI
21. The much superior growth performance of inoculated seed-
lings compared with uninoculated seedlings over two
growing seasons provides unequivocal proof that pre-ino-
culation with mycorrhizal and N2-fixing symbionts can,
in the case of silver-berry and buffalo-berry, result in
more rapid revegetation of oil sands tailings. It is
strongly recommended that containerized silver-berry and
buf falo-berry seedlings, destined for reclamation and
possibly forestry sites, be inoculated with Frankia and
mycorrhizal fungi prior to outplanting.
TABLE OF CONTENTS
Page
EXECUTIVE SUMMARY ....... .......... xv
TABLE OF CONTENTS xxi 1
LIST OF TABLES xxv
LIST OF FIGURES ..... xxviii
APPENDIX TABLES xxix
ACKNOWLEDGEMENTS xxx
1. INTRODUCTION 1
1.1 Objectives 4
2. STUDY AREA 6
3. METHODS 9
3.1 Mycorrhizal Status of Woody Shrubs ......... 9
3.2 Justification for Inoculation of Containerized
Silver-berry and Buffalo-berry 9
3.2.1 The Dependency of Silver-berry and Buf falo-berry
on their Mycorrhizal and N2-Fixing Symbionts ... 10
3.2.2 Levels of Mycorrhizal Inoculum in Outplanting Soil . 11
3.2.3 Growth Characteristics and Mycorrhizal Potential of
Undisturbed Bog Peat and Stockpiled Peat 12
3.2.4 Mycorrhizal Potential of Revegetated Dyke Peat ... 12
3.2.5 Effect of VA Mycorrhizal Inoculation on Plant
Performance of Slender Wheatgrass Grown in Stock-
piled Peat under Fertilized and Unfertilized
Conditions 13
3.2.6 Mycorrhizal and Nodule Status of Containerized
Shrubs Planted on the Oil Sands Tailings
Reconstruction Plots ......... 13
3.2.7 Growth Characteristics and Symbiont Status of Woody
Shrubs Raised in Various Commercial Nurseries in
Alberta and British Columbia 14
3.2.8 Rates of Mycorrhization and Nodulation in Buffalo-
berry Grown in Woodland Soil and Amended Tailings
Sand in the Greenhouse 15
3.2.9 Rates of Mycorrhizal and Nodule Development in Silver-
berry Outplanted in an Undisturbed Woodland and the
Suncor Tar Island Dyke ................ 15
3.3 Development of a Growing Regime for Greenhouse Produc-
tion of Mycorrhizal, Nodulated Silver-berry and
Buffalo-berry. .................... 16
XX i i i
TABLE OF CONTENTS (continued)
Page
3.3.1 Fertilizer Effects on Growth, Nodulation and
Mycorrhizal Development in Buffalo-berry and
Silver-berry ......... .... 17
3.3.2 Effect of Container Volume and Inoculation on Growth
of Silver-berry 17
3.3.3 Growth of Silver-berry as Influenced by Soil Tempera-
ture and Symbiont Inoculation 18
3.3.4 Use of Soil, Nodule and Pure Culture Inocula for
Introducing N2-Fixing Frankia to Containerized
Silver-berry ............... 19
3.3.5. Effect of Inoculation Method and Inoculation Time
on Nodule and Mycorrhizal Development of Buffalo-
berry. 21
3.4 Field Trial to Test Growth Response of Inoculated
Silver-berry and Buffalo-berry 22
4. RESULTS. ................. 27
4.1 Mycorrhizal Status of Woody Shrubs ......... 27
4.2 Justification for Inoculation of Containerzied
Silver-berry and Buffalo-berry 27
4.2.1 The Dependency of Silver-berry and Buffalo-berry
on their Mycorrhizal and Na-Fixing Symbionts .... 27
4.2.2 Levels of VA Mycorrhizal Inoculum in Various Soils
in the Fort McMurray, Alberta Region 27
4.2.3 Growth Characteristics and Mycorrhizal Potential
of Undisturbed Bog Peat and Stockpiled Peat 29
4.2.4 Mycorrhizal Potential of Revegetated Dyke Peat ... 31
4.2.5 Effect of VA Mycorrhizal Inoculation on Plant
Performance of Slender Wheatgrass Grown in Stock-
piled Peat Under Fertilized and Unfertilized
Conditions ..... 31
4.2.6 Mycorrhizal and Nodule Status of Containerized
Shrubs Planted on the Oil Sands Tailings
Reconstruction Plots 31
4.2.7 Growth Characteristics and Symbiont Status of Woody
Shrubs Raised in Various Commercial Nurseries in
Alberta and British Columbia ... 34
4.2.8 Rates of Mycorrhi zation and Nodulation in Buffalo-
berry Grown in Woodland Soil and Amended Tailings
Sand in the Greenhouse 37
4.2.9 Rates of Mycorrhizal and Nodule Development in
Silver-berry Outplanted in an Undisturbed Woodland
and the Suncor Tar Island Dyke 40
4.3 Development of a Growing Regime for Greenhouse
Production of Mycorrhizal, Nodulated Silver-
berry and Buffalo-berry 42
4.3.1 Fertilizer Effects on Growth, Nodulation and
Mycorrhizal Development in Buffalo-berry and
Silver-berry 42
XXIV
TABLE OF CONTENTS (concluded)
Page
4.3c2 Effect of Container Volume and Inoculation on
Growth of Silver-berry 45
4.3.3 Growth of Silver-berry as Influenced by Soil
Temperature and Symbiont Inoculation ........ 45
4.3.4 Use of Soil, Nodule and Pure Culture Inocula for
Introducing Na-Fixing Frankia to Containerized
Silver-berry .................... 48
4.3.5 Effect of Inoculation Method and Time on Nodule and
Mycorrhizal Development of Buf falo-berry ...... 51
4.4 Field Trial to Test Growth Response of Inoculated
Silver-berry and Buffalo-berry ........... 51
4.4.1 Pre-Planting Symbiont Status of Inoculated and
Uninoculated Silver-berry and Buffalo-berry .... 51
4.4.2 Field Performance of Silver-berry After One and
Two Growing Seasons ................ 52
4.4.3 Field Performance of Buf falo-berry After One and
Two Growing Seasons ................ 59
4.4.4 Relationships Amongst Various Parameters Measured
on Inoculated and Uninoculated Silver-berry and
Buffalo-berry After One and Two Growing Seasons . . 63
5. DISCUSSION. ........... . 76
5.1 Mycorrhizal Status of Woody Shrubs. . 76
5.2 Justification for Inoculation of Containerized
Silver-Berry and Buffalo-Berry. .......... 76
5.2.1 The Dependency of Silver-berry and Buffalo-berry
on their Mycorrhizal and Na-fixing Symbionts. . . 76
5.2.2 Levels of VA Mycorrhizal Inoculum in Various Soils
in the Oil Sands Region and Effects of Stockpiling
on VAM Infectivity. ................ 77
5.2.3 Mycorrhizal and Nodule Status of Containerized Shrubs
Raised in Various Commercial Nurseries in Alberta and
British Columbia. ................. 79
5.2.4 Mycorrhization and Nodulation Rates of Buf falo-berry
and Silver-berry in the Greenhouse and the Field. , 80
5.2.5 Basis for Artificial Inoculation of Containerized
Buffalo-berry and Silver-berry for Outplanting on
Amended Oil Sands Tailings. . 81
5.3 Development of a Growing Regime for Greenhouse
Production of Mycorrhizal, Nodulated Silver-berry
and Buffalo-berry ................. 82
5.3.1 Fertilization Regimes ......... 82
5.3.2 Container Volume. ................. 83
5.3.3 Temperature ...... ........ 84
5.3.4 Frankia Inoculum Trials .............. 84
5.3.5 Growing Regimes for Greenhouse Production of Mycor-
rhizal, Nodulated Silver-berry and Buffalo-berry. . 85
5,4 Field Trial to Test Growth Response of Inoculated
Silver-berry and Buffalo-berry 85
6.0 REFERENCES CITED ..... 90
XXV
LIST OF TABLES
Page
1. Mycorrhizal status of selected woody shrubs
growing in the Fort McMurray and Kananaskis,
Alberta regions. .................. 28
2. Shoot and root production and symbiont development
in silverberry and buf falo-berry grown in recon-
structed soils with and without symbiont inoculum. . 29
3. Vesicular-arbuscular mycorrhizal (VAM) infection of
slender wheatgrass grown in the greenhouse in
various soils collected from the Fort McMurray,
Alberta region 30
4. Characteristics of slender wheatgrass grown in
peat from a muskeg bog and peat stockpiled for
eight months 32
5. Root and shoot production by slender wheatgrass
grown in the greenhouse in fertilized and unfer-
tilized dyke peat 33
6. Shoot and root production by slender wheatgrass grown
in stockpiled peat (50-100 cm) inoculated with Glomus
aqqreqatum and fertilized or left unfertilized ... 34
7. Mycorrhizal and nodule status of shrub species planted
on the RRTAC oil sand tailings reconstruction plots in
September, 1984 35
8. Size of container-grown and bareroot woody shrubs
obtained from four commercial nurseries in August,
1985 36
9. Vesicular-arbuscular mycorrhizal (VAM) status and
Frankia nodule development in woody shrubs sampled
from four commercial nurseries in 1985 38
10. Rates of shoot and root production, mycorrhizal
colonization and nodulation by buffalo-berry grown
in undisturbed woodland soil and peat/clay amended
tailings sand 39
11. Growth, nodulation and VA-mycorrhi zal development
in uninoculated silver-berry outplanted in the
boreal forest and Suncor Tar Island Dyke for 6
and 12 weeks 41
XX vi
c
LIST OF TABLES (continued)
Page
12. Fertilizer effects on growth, nodulation, and
mycorrhizal development in buffalo-berry grown in
woodland soil and peat/cl ay-amended tailings sand. . 43
13. Fertilizer effects on growth, nodulation, and
mycorrhizal development in silver-berry grown in
woodland soil and peat/cl ay-amended tailings sand. . 44
14. Effect of container volume and inoculation on shoot
and root production by si 1 ver-berry . 46
15. Growth of silver-berry as influenced by soil tempera-
ture and inoculation with mycorrhizal and Ns-fixing
symbionts 47
16. Effect of soil temperature and inoculation on mycor-
rhizal and nodule development in silver-berry grown
in a growth chamber for 13 weeks .......... 49
17. Use of soil, nodule, and pure culture inocula for
promoting growth and nodulation of container-grown
silver-berry .................... 50
18. Use of soil and soil slurry for promoting nodulation
and mycorrhizal development in container-grown
buffalo-berry. 52
19. Nodule and mycorrhizal development in container-grown
buffalo-berry inoculated with soil slurry at various
ages 53
20. Pre-planting mycorrhizal and nodule status of silver-
berry and buffalo-berry outplanted in the University
of Calgary soil reconstruction plots (with and
without surficial clay). .............. 54
21. Plant growth, nodulation, and vesicular-arbuscular
mycorrhizal development of inoculated and uninocu-
lated silver-berry outplanted for 1 year in the
University of Calgary soil reconstruction plots
at the Syncrude site ................ 55
XXV i i
LIST OF TABLES (concluded)
Page
22.
Plant growth, nodulation and vesicular-arbuscular
mycorrhizal development in inoculated and uninocu-
lated silver-berry outplanted for 2 years in the
University of Calgary soil reconstruction plots at
the Syncrude site. . .
56
23.
Plant growth, nodulation, and vesicular-arbuscular
mycorrhizal development of inoculated and uninocu-
lated buf falo-berry outplanted for 1 year in the
University of Calgary soil reconstruction plots at
the Syncrude site.
62
24.
Plant growth, nodulation and vesicular-arbuscular
mycorrhizal development in inoculated and uninocu-
lated buf falo-berry outplanted for 2 years in the
University of Calgary soil reconstruction plots at
the Syncrude site. ....
68
25.
Pearson product moment correlation coefficients for
various parameters measured on silver-berry grown
for 1 year in the University of Calgary soil
reconstruction plots at the Syncrude site. . . . . .
69
26.
Pearson product moment correlation coefficients for
various parameters measured on silver-berry grown
for 2 years in the University of Calgary soil
reconstruction plots at the Syncrude site
70
27.
Pearson product moment correlation coefficients for
various parameters measured on buffalo-berry grown
for 1 year in the University of Calgary soil
reconstruction plots at the Syncrude site
73
28.
Pearson product moment correlation coefficients for
various parameters measured on buf falo-berry grown
for 2 years in the University of Calgary soil
reconstruction plots at the Syncrude site
74
29.
Growing regime for greenhouse production of
mycorrhizal, nodulated silver-berry
86
30.
Growing regime for greenhouse production of
mycorrhizal, nodulated buf falo-berry ........
87
XXV i 1 i
c
LIST OF FIGURES
Page
Ic Map showing location of the study area ...... 7
2. Plot arrangement. ............ 24
3. Shoot heights of inoculated and uninoculated silver-
berry outplanted in reconstructed soil for two
growing seasons .................. 57
4. Shoot weights of inoculated and uninoculated silver-
berry outplanted in reconstructed soil for two
growing seasons .................. 58
5. Nodule development in inoculated and uninoculated
silver-berry outplanted in reconstructed soil for
two growing seasons ....... .... 60
6. Mycorrhizal development in inoculated and uninocu-
lated silver-berry outplanted in reconstructed soil
for two growing seasons .............. 61
7. Shoot heights of inoculated and uninoculated
buf falo-berry outplanted in reconstructed soil for
two growing seasons ................ 64
8. Shoot weights of inoculated and uninoculated
buffalo-berry outplanted in reconstructed soil for two
growing seasons ............ . 65
9. Nodule development in inoculated and uninoculated
buf falo-berry outplanted in reconstructed soil for
two growing seasons ................ 66
10. Mycorrhizal development in inoculated and uninocu-
lated buffalo-berry outplanted in reconstructed
soil for two growing seasons. ........... 67
11. Linear regression of shoot weights versus nodule
weights for one year-old silver-berry ....... 71
12. Linear regression of shoot weights versus nodule
weights for one year-old buffalo-berry. ...... 75
XXI X
APPENDIX TABLES
Page
1. Mycorrhizal infection of slender wheatgrass grown in
undisturbed muskeg and stockpiled peat (peat stock-
piled for 8 months) ...... .o ... 98
XXX
ACKNOWLEDGEMENTS
This research was funded by the Reclamation Research Technical
Advisory Committee of the Alberta Land Conservation and Reclamation
Council, using Heritage Savings Trust Fund monies. The cooperation of
Syncrude Canada Ltd. and Suncor Inc. for providing planting sites and
aid in plot establishment is gratefully acknowledged. In particular,
we would like to thank Tony Dai and Kim McCumber of Syncrude Canada
Ltd. for their help and advice during the field work. The support and
enthusiasm for this study demonstrated by Chris Powter and Jim Campbell
of the Research Management Division is very much appreciated. The
authors thank the following for providing nursery stock: T, Laidlaw
(Laidlaw Vegetation Consulting Ltd.), B. Hutchinson (Whitecourt
Mountain Seedling Nursery), G. Granger (Alberta Tree Nursery and Horti-
culture Centre), C. Jones (Reid, Collins Nurseries Ltd.), and B.
Novlesky (Syncrude Canada Ltd.). Silver-berry and buffalo-berry seeds
were provided by the Alberta Forest Service. In conclusion, this study
would not have been possible without the invaluable technical assis-
tance provided by L. Burton, P. Mazier, C. McBain and C. Anderson and
the statistical analyses provided by C. Griffiths. We are grateful to
Lynn Ewing and Della Patton for their help in typing and organizing the
report.
1
1 . INTRODUCTION
Actinorhizal plants are non-leguminous , N2-fixing plants
whose nodules are formed by the actinomycete, Frankia, rather than by
the bacterium, Rhizobium, as is the case for legumes. They are peren-
nial, woody trees or shrubs which often colonize nutrient-poor, margi-
nal or disturbed habitats such as sand dunes, wet bogs, dry sandy or
gravelly areas and mine wastes (Torrey, 1978). They have the ability
to fix up to 300 kg atmospheric nitrogen ha~^ year"^ and,
consequently, are seriously being considered as an alternative to
nitrogen fertilizer as a management tool for intensive forestry in
Canada (Fortin et al., 1984). Alder, in particular, has been shown to
significantly improve the nitrogen status of forest soils (Cot@ and
Camir@, 1985; Huss-Danell, 1986; Malcolm et al., 1985; Tarrant and
Trappe, 1971; Wheeler et al., 1986) and minespoils (Heilman and Ekuan,
1982; Tarrant and Trappe, 1971), but this has (Cot0 and Camir(9, 1985;
DeBell and Radwan, 1979; Hansen and Dawson, 1982) or has not (Heilman
and Ekuan, 1982; Malcolm et al., 1985) been manifested in improved
productivity of commercial tree species in mixed plantings. In addi-
tion to improving soil nitrogen levels, actinorhizal plants ameliorate
soil temperature and moisture conditions through the accumulation of
organic matter resulting from leaf and root litter deposition and
decomposition. The ability of actinorhizal shrubs to tolerate inhospi-
table conditions while improving soil fertility and organic matter
status has led to increased usage of these plants for land reclamation
and amenity planting purposes (Fessenden, 1979).
The actinorhizal plants which are native to Alberta include
green alder (Alnus crispa (Ait.) Pursh), river alder (A. tenuifolia
Nutt.), snow brush (Ceanothus velutinus Dougl. ex Hook.), silver-berry
(Elaeaqnus commutata Bernh. ex Rydb.), buffalo-berry (Shepherdia
canadensis (L.) Nutt.) and the yellow and white dryads (Dryas
drummondii Richards., D. octopetala ssp. hookeriana [Juz.] Hult.). Of
these, green alder, silver-berry and buffalo-berry are being tested as
potential candidates for the revegetation of oil sands tailings
resulting from the extraction of oil from the oil sand deposits located
in northeastern Alberta. This report is concerned exclusively with
silver-berry and buf falo-berry .
2
In addition to having the N2-fixing symbiont associated with
their roots, both silver-berry and buf falo-berry form mycorrhizae —
the mutual symbiosis between specific fungi, in this case vesicular-
arbuscular mycorrhizal (VAM) fungi, and the plant root. The fungus
improves the phosphorus nutrition of the plant by exploring a greater
volume of soil for the relatively immobile PO4 ion than the plant
root itself would be capable of doing, while the fungus benefits by
receiving carbohydrates from the plant. The potential importance of VA
mycorrhizae in enhancing the revegetation of minespoils has concen-
trated primarily on forage and crop species (Khan, 1981; Lambert and
Cole, 1980; Zak and Parkinson, 1982, 1983) with woody shrubs receiving
much less attention. However, due to the coarse-rooted nature of many
woody shrubs, it is possible that these species are more dependent on
the VA mycorrhizal symbiosis than fibrous-rooted species where soil-
root contact is high (Hayman, 1982). This would explain the signifi-
cant growth enhancement observed in many woody species inoculated with
a wide variety of VAM fungi (Furlan et al., 1983, Kormanik et al.,
1982; Plenchette et al., 1981; Pope et al., 1983) and the much improved
growth of ' VAM-inoculated rabbit brush and fourwing saltbush in coal
minespoil (Aldon, 1978; Lindsey et al., 1977).
Numerous studies, designed to clarify the interactions between
VAM fungi and Rhizobium in legumes, have shown that mycorrhizal infec-
tion can significantly stimulate nodulation, nitrogenase activity and
in some cases foliage N concentrations (Ames and Bethlenfal vay, 1987;
Azcon-Agui lar and Barea, 1981; Barea et al , , 1980; Barea and
Azcon-Agui lar, 1983; Carling et al., 1978; Ganry et al., 1982; Green et
al., 1983; Redente and Reeves, 1981; Smith and Daft, 1977; Smith et al,
1979). It has been demonstrated that both nodule initiation and N2-
fixation have a high P requirement which is satisfied by the mycor-
rhizae resulting in significantly greater plant productivity (Smith
et al., 1979). With the exception of Rose and Youngberg (1981), who
observed that the actinorhizal shrub, Ceanothus velutinus . exhibited
greater shoot and root weights, greater number and weight of nodules
and more nitrogenase activity if colonized by both the mycorrhizal
fungi and Frankia than if colonized by Frankia alone, research eluci-
dating the dependence of actinorhizal plants on both symbionts has been
3
lacking. Considering the potential importance of both the VAM fungi
and Frankia in the establishment, survival and growth of actinorhizal
shrubs on marginal and disturbed habitats such as mine tailings, it is
surprising that so little information is available on the role of these
symbionts in accelerating the revegetation process.
The value of actinorhizal shrubs for improving the fertility
and organic matter status of soils which are prone to significant
losses of N as a result of intensive forestry practices, has led
researchers in Quebec to develop a program for isolating, characteri-
zing and evaluating the effectiveness of Frankia strains from green
alder (Normand et al., 1984) with the final goal being large scale
inoculation of alder on a commercial basis (Perinet et al . , 1985).
Subsequent field trials with inoculated and uninoculated alders demon-
strated that, over the long term (3 years), inoculation with an effec-
tive strain of Frankia significantly improved growth of three species
of alder (Burgess et al., 1986). Inoculation with mycorrhizal fungi
was not addressed in these studies.
In Alberta the establishment of woody trees and shrubs on
disturbed sites is usually accomplished by outplanting containerized
seedlings which have been raised and hardened off in commercial or
provincial greenhouse operations. The use of containerized seedlings
offers a good opportunity for introducing N2-fixing and mycorrhizal
symbionts to the plants prior to being outplanted.
However, before embarking on a large-scale inoculation program
which will ultimately raise the cost of producing a seedling, a number
of factors should be considered. These include the degree of depen-
dency of a plant on its symbionts, the level of Frankia and mycorrhizal
inoculum in various soils into which the seedlings will be outplanted
and the effectivity of this inoculum, the nodule/mycorrhi zal status of
containerized seedlings leaving commercial greenhouses, and the rates
of nodulation and mycorrhizal colonization of seedlings once out-
planted. Prior consideration of these factors will determine whether
or not the time and effort required to develop an inoculation program
is worthwhile. Once the decision is made to enter into an inoculation
program, it becomes necessary to develop a growing regime for rearing
mycorrhizal, nodulated seedlings of an acceptable size. For this the
4
optimum nutrient conditions (fertilizer rates), soil pH, light condi-
tions, container volume, soil temperature, Frankia and mycorrhizal
inoculum source and method of inoculation should be evaluated.
Finally, it is essential that field trials be conducted to establish
unequivocally that inoculated seedlings will outperform uninoculated
seedlings over the long-term under field conditions. On the basis of
the foregoing discussion, research was conducted to fulfil the
following objectives.
1.1 OBJECTIVES
1. To determine definitively the mycorrhizal affinities of
various actinorhizal and other woody shrubs in the Fort
McMurray, Alberta region.
2. To establish a basis for promoting the symbiont inocula-
tion of two actinorhizal shrubs, buf falo-berry and
si 1 ver-berry. Factors which were investigated include:
i) the dependency of the shrubs on their mycorrhizal
and N2-fixing symbionts as expressed in plant
performance under inoculated and uninoculated
conditions .
ii) the Frankia and VAN! inoculum levels in the soil into
which the shrubs would be planted, i.e. peat stock-
piled on the Syncrude lease, soils reconstructed
from peat, mineral soil and oil sand tailings on the
tailings dykes and woody species trial plots on the
Syncrude lease.
iii) the mycorrhizal and nodule status of containerized
shrubs raised in commercial and provincial green-
houses.
iv) rates of mycorrhization and nodulation in undis-
turbed and reconstructed tailings dyke soils.
3. To develop a growing regime for the greenhouse production
of mycorrhizal, nodulated silver-berry and buffalo-berry.
5
There are basically two approaches to the development of
an inoculation program. The first, termed the "high tech"
approach, involves the isolation of species or strains of
VA mycorrhizal fungi and N2-fixing Frankia into pure
culture, propagating the isolates and then using pure
culture inoculum to inoculate containerized seedlings.
The second, the "low tech" approach, involves mixing field
soil with a high symbiont inoculum potential into the
planting mixture prior to filling and planting the
containers. Due to time constraints the second approach
was investigated in this study. This entailed testing
such factors as fertilization regimes, container volume,
soil temperatures , inoculum sources, and time and method
of inoculation.
4. To conduct a field trial on reconstructed soil on the
Syncrude site to critically evaluate the growth perfor-
mance of inoculated silver-berry and buf falo-berry as
compared with their uninoculated counterparts.
6
c
2. STUDY AREA
The study area was located in the Athabasca Oil Sands region
near Fort McMurray in northeastern Alberta (Figure 1). This region is
situated within the Mixedwood Section of the Boreal Forest Region
(Rowe, 1972) and has a gently undulating topography with sandy soils
dominating the upland areas and wet peatland occurring in the poorly
drained areas. The vegetation consists predominantly of white spruce
and aspen forest with jack pine-lichen woodlands occurring on the sandy
upland areas and black spruce/tamarack bogs in the poorly drained, low
lying areas. The climate is cool continental character! zed by rela-
tively short, cool summers and long cold winters. Mean annual tempera-
ture and precipitation in the Fort McMurray area are -0.2®C and
472 mm, respectively. Gray luvisolic soils are characteristic of the
aspen-white spruce mixedwood forests, while eluviated dystric brunisols
predominate in the jack pine-lichen woodlands. More detailed informa-
tion on the climate, vegetation and soils of the region can be obtained
from Strong and Leggat (1981) and Turchenek and Lindsay (1982).
Soils for greenhouse growth studies and VA mycorrhizal and
Frankia inoculum screening were sampled mainly from aspen-white spruce
mixed woodland in close vicinity to the Mildred Lake Research Facility
(Figure 1) and from reconstructed soil on the tailings dyke located on
the Syncrude Oil Sands Lease. Samples to determine the effect of
stockpiling muskeg peat on VA mycorrhizal inoculum were removed from an
eastern larch/black spruce/Labrador tea/moss peat bog and from the NT-2
peat stockpile, both located on the Syncrude Lease, The stockpile was
300 m wide and 3 m deep and was 8 months old when sampled. Rates of
nodulation and mycorrhi zation of silver-berry were determined on seed-
lings outplanted on plots established on the Suncor Tar Island Dyke
facing the Athabasca River on the Suncor Oil Sands Lease. The plots
were established in areas which had been revegetated with grass/legume
mixtures in 1971, 1974 and 1978.
The field trial to evaluate the growth response of inoculated
silver-berry and buf falo-berry was conducted on two plots established
adjacent to the RRTAC soil reconstruction-woody plant experimental area
on the Syncrude Oil Sands Lease. The experimental area was located on
a specially prepared pad of oil sand tailings which had been amended
7
Figure 1. Map showing location of the study area (excerpted from
Turchenek and Lindsay, 1982).
8
c
with various amounts of muskeg peat and surficial overburden clay.
Further details regarding the plots are given in reports prepared by
Hardy Associates (1983, 1984),
9
3. METHODS
3.1 MYCORRHIZAL STATUS OF WOODY SHRUBS
Although it is widely accepted that grasses and the majority
of herbaceous plants are mycorrhizal with vesicular-arbuscular (VA)
mycorrhizal fungi, the mycorrhizal affinities of woody shrubs,
including actinorhizal shrubs, are less well-known. In order to
establish the mycorrhizal condition of silver-berry, buf falo-berry and
various other shrubs growing in the wild, the following survey was
conducted.
Buffalo-berry and silver-berry plants were sampled from the
Vaartnou reclamation plots near the Mildred Lake campsite, from a
cutbank near the Suncor plant and from a roadcut near the University of
Calgary Research Station in the Kananaskis Valley. In addition,
saskatoon-berry and cinquefoil were sampled from a cutline near the
Mildred Lake campsite and the Vaartnou plots, respectively. Five
replicate plants were excavated at each sampling location, the roots
were washed, and only those roots which were attached to the stem of
the host species in question were assessed for mycorrhizal develop-
ment. The root systems were scanned under a dissecting microscope for
ecto-mycorrhizal development and subsamples subsequently cleared and
stained for the detection of VA mycorrhizae (Phillips and Hayman, 1970).
3.2 JUSTIFICATION FOR INOCULATION OF CONTAINERIZED SILVER-BERRY
AND BUFFALO-BERRY
Before undertaking a widescale inoculation program, a number
of factors regarding the necessity of such a program should be
considered. In this study the factors which were investigated include:
i) the dependency of the shrubs on their Na-fixing and
mycorrhizal symbionts, i.e. how do the shrubs perform in the presence
and absence of their symbionts and what benefits are conferred on the
host by the symbionts Woody species vary in their symbiont depen-
dency, and it could be argued that if they are not highly dependent
(based on stimulation of root and shoot production in the presence of
the symbionts) there is less of a need to inoculate them prior to
outplanting.
10
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ii) symbiont inoculum levels in the soil into which the shrubs
will be planted. The symbiont inoculum potential is determined
primarily by the degree of disturbance of a soil and by the host plant
species present in a particular site. For example, massive soil
upheaval characteristic of most mining operations can reduce symbiont
inoculum potential substantially, while the dominance of non-host plant
species, i.e. those which are neither Na-fixing or VA mycorrhizal,
can also lead to a reduction in symbiont inoculum. Shrubs would be
expected to benefit most from pre-planting inoculation when planted
into soil lacking symbiont inoculum.
iii) VA mycorrhizal and Na-fixing nodule status of
containerized shrubs prior to outplanting. While being raised in the
greenhouse, containerized plants may become colonized by their
symbionts via inoculum in the planting mixture, the water or the
atmosphere. If this is the case, artificial inoculation may be
unnecessary.
iv) rates of mycorrhizal colonization and nodulation. Rapid
colonization by the symbionts after outplanting will ensure that the
host plants will derive maximum benefit from the mycorrhizal/N2-f ixing
relationship. If colonization is slow, however, inoculation may be
necessary to accelerate plant establishment and growth. This would
apply, particularly, in northern regions where the short growing season
could reduce symbiont colonization rates to such a degree that the
plant would not begin to benefit from its symbionts until the end of
the growing season. The various experiments which were designed to
elucidate the preceding factors follow.
3.2.1 The Dependency of Silver-berry and Buffalo-berry on their
Mycorrhizal and N2-Fixing Symbionts
Oil sand tailings and peat from the Syncrude NT2 stockpile
were mixed based on 11 and 5.5 cm depth equivalents for the sand and
peat, respectively. A 3.5 cm depth equivalent of forest soil collected
from beneath buf falo-berry shrubs growing in an aspen stand near
Mildred Lake and containing a high symbiont inoculum potential was
added to half of the oil sands/peat mixture. This represented the
inoculated treatment. The uninoculated treatment was identical to the
11
inoculated treatment with the exception that the buf falo-berry inoculum
was autoclaved to eradicate the symbionts. Sections of sewer pipe,
20 cm deep and 7.7 cm diameter, the bottoms of which were covered with
a layer of polyester batting and a piece of fiberglass screening, were
filled with soil mixtures from each treatment.
Buffalo-berry seeds were scarified in concentrated H2SO4
for 30 min (King, 1980), rinsed in cold running water overnight and
germinated on moist filter paper. Silver-berry seeds were leached in
cold, running water for 4 days and germinated on moist filter paper
(King et al . , 1983). One germinant of each species was planted in each
of 10 replicate cores (total = 40 cores) and the cores placed in the
greenhouse in December in a random arrangement. Light intensity on
clear days was 500 yEm ^sec ^ (198 W m 26 klx), 156 yEm ^sec ^
(67 W m”^, 9 klx) on cloudy days and day length was extended to 20
hours with a minimum of 74 yEm ^sec ^ (20 W m 3.5 klx). Temperatures
were generally between 18 and 25°C, but occasionally fell to 5®C at
night, and were 30®C during the day. Plants were watered twice
weekly without any additions of nutrients. All seedlings were
harvested after 12 weeks.
Shoots were removed, dried at 80°C and weighed. Roots were
separated from the soil, washed and the nodules counted and weighed.
Vesicular-arbuscular mycorrhizal assessments were determined on sub-
samples by the method of Zak and Parkinson (1982). Remaining roots
were dried at 80®C and weighed.
3.2.2 Levels of Mycorrhizal Inoculum in Outplanting Soil
During the course of the last decade numerous soils collected
from the Fort McMurray region have been assayed for their VA-mycor-
rhizal inoculum potential. Assays consisted of a baiting technique in
which slender wheatgrass germinants were planted in soil or peat which
had been well-mixed and packed into 65 or 150 cc Leach Cone-tainers .
The plants were grown in the greenhouse and were not fertilized. After
8 to 12 weeks, the roots were separated from the soil, cleared, stained
and assessed for mycorrhizal development using the methods given in
3.2.1. The various soils which were assayed are listed in Table 3 of
the Results section.
12
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3.2.3 Growth Characteristics and Mvcorrhizal Potential of Undis-
turbed Bog Peat and Stockpiled Peat
Before mining the oil sands, the muskeg peat, which often
overlies the oil-bearing sand, is drained, stripped and stockpiled for
subsequent revegetation of the tailings sand dykes. Little is known of
the VA mycorrhizal status of undisturbed peat and the impact of stock-
piling on mycorrhizal propagules. The plants being used to revegetate
the dykes (e.g. grasses, legumes) and those being considered for recla-
mation purposes (e.g. woody shrubs such as saskatoon-berry, buffalo-
berry, si 1 ver-berry, wild rose etc.) are VA mycorrhizal and may be
heavily dependent on their mycorrhizal associates for both growth and
survival. Therefore, it was considered essential that the mycorrhizal
potential of both undisturbed and stockpiled peat be investigated.
Twenty-five peat samples were randomly removed from each of
two depths (0-15 cm; 50-100 cm) in an undisturbed peat bog and a peat
stockpile on the Syncrude site near Fort McMurray. The vegetation on
the bog was predominantly eastern larch, swamp birch, Labrador tea and
feather and sphagnum mosses while the peat stockpile, which was 8
months old, was largely unvegetated. Samples were removed every 10 m
along a 250 m transect on each site.
Each sample was thoroughly mixed, packed into a 65 cc Leach
Cone-tainer and planted with a slender wheatgrass germinant. Plants
were grown in the greenhouse under the light conditions described in
3.1.1 and received no fertilizer. After 12 weeks, shoot weights, root
weights, total root length and % VAM infection were determined using
the methods described in 3.2.1.
3.2.4 Mvcorrhizal Potential of Revegetated Dyke Peat
The University of Calgary experimental plot on the Syncrude
dyke was reclaimed with peat from a 6 year old stockpile which had been
revegetated with a grass mixture. Therefore, it was decided to sample
this peat to determine if storage time and presence of a VA mycorrhizal
host had changed the mycorrhizal inoculum potential.
Five peat samples (0-20 cm deep) were randomly removed from
the northern boundary of the experimental plot, mixed and packed into
20, 65 cc Leach Cone-tainers , 4 per sample. Each Cone-tainer was
13
planted with a pre-germinated slender wheatgrass seedling. The seed-
lings were then divided into two treatments — ten seedlings received
Plant Prod Soilless Feed at a rate of 100 mg L ^ 15-15-18 twice per
week and sequestrene-Fe at 56 ppm twice per week while the remaining 10
seedlings received only deionized water. Plants were grown in the
greenhouse under a 20h daylength, with a minimum of 3.5 klx light
intensity. The parameters measured after 9 weeks included shoot and
root weights, root length and % VAM colonization using the methods
described in 3.2.1 .
3.2.5 Effect of VA Mycorrhizal Inoculation on Plant Performance of
Slender Wheatgrass Grown in Stockpiled Peat under Fertilized
and Unfertilized Conditions
Preliminary examination of plants grown in peat stockpiled for
8 months revealed that there was a paucity of VAM inoculum in this
peat. Therefore, an experiment was conducted to determine if the
addition of VA inoculum would improve the growth of slender wheatgrass
under fertilized or unfertilized conditions.
Peat from the 50-100 cm depth of the 8 month old NT-2 stock-
pile was bulked and separated into two batches. One batch was mixed
50/50 (v/v) with root/sand inoculum from a Glomus aggregatum (a common
VA fungus having a wide distribution in Alberta) pot culture while the
other batch was mixed 50/50 (v/v) with autoclaved root/sand inoculum to
serve as a control. Twenty Leach Cone-tainers were packed from each
batch and one pre-germinated slender wheatgrass seedling was planted in
each container. Seedlings were grown in the greenhouse under the
conditions outlined in 3.2.4. Five seedlings from each treatment
(inoculated, fertilized; uninoculated, fertilized; inoculated, unfer-
tilized; uninoculated, unfertilized) were sampled when the plants were
4 and 10 weeks old. Shoot and root weights and VA mycorrhizal develop-
ment were assessed as described previously.
3.2.6 Mycorrhizal and Nodule Status of Containerized Shrubs Planted
on the Oil Sands Tailings Reconstruction Plots
If shrub species which are highly dependent on their symbionts
for growth and survival are used for reclamation purposes, both the
14
symbiont inoculum potential in the reconstructed soil and the symbiont
status of the shrubs prior to outplanting should be determined.
Consequently, it was decided that the mycorrhizal and nodulation status
of the shrubs outplanted on the RRTAC oil sands tailings reconstruction
plots (pad plots) in the fall of 1984 should be assessed.
Wild rose, pin cherry, saskatoon-berry, Canada buffalo-berry
and silver-berry, grown in either the Syncrude or Laidlaw nurseries,
were sampled in August. Twenty-five plants of each species were
randomly selected to determine plant weights and mycorrhizal and nodule
status. All plants had been grown in 150 cc Spencer-Lemai re Hillson
book containers. Roots were washed free of soil, examined for nodules
and then cleared, stained and examined for VA mycorrhizae (3.2.1).
Shoot and root weights were determined after drying at 80C.
3.2.7 Growth Characteristics and Symbiont Status of Woody Shrubs
Raised in Various Commercial Nurseries in Alberta and British
Columbia
As mentioned previously, containerized shrubs can become
mycorrhizal or nodulated during the growing and hardening-off phases in
the greenhouse and shadehouse via inoculum in the planting mixture,
water or atmosphere. If seedlings become heavily mycorrhizal or nodu-
lated prior to outplanting, artificial inoculation may not be neces-
sary. Since little is known of the symbiont status of shrub species
grown in commercial greenhouses, a survey of various nursery-grown
shrubs was conducted.
Nine species of woody shrubs were sampled from the Whitecourt,
Laidlaw, Oliver and Syncrude nurseries in Alberta and the Reid-CoHins
nursery in British Columbia in August, 1985. Names of the shrubs, crop
year, container size and number of seedlings assayed are detailed in
Table 8 of the Results. Shoot heights were measured for some of the
species, while shoot and root weights were determined for all plants
after drying at 80C. The N2-fixing shrubs were assessed for nodule
numbers and weights. Vesicular-arbuscular mycorrhizal status of all
the shrubs was determined by clearing, staining and examining under a
dissecting microscope, a 10% subsample of the total wet weight of each
root system.
15
3.2.8 Rates of Mvcorrhization and Nodulation in Buffalo-berry Grown
in Woodland Soil and Amended Tailings Sand in the Greenhouse
The rate of mycorrhizal and nodule development from indigenous
soil inoculum may determine to a large degree the benefits derived by
the plant from its symbionts during the first growing season after out-
planting. This applies particularly to the oil sands region where the
short growing season and potentially low symbiont inoculum levels in
the reconstructed soil on the tailings dykes may result in such slow
colonization rates that the plants do not benefit from their symbionts
until the end of the growing season. Therefore the rates of infection
may determine whether or not pre-planting inoculation is necessary.
With this in mind a study was conducted to determine the rates of
mycorrhizal and nodule development of buffalo-berry planted in undis-
turbed woodland soil and amended tailings sand.
Five soil samples were removed from the forest floor of a
mixed woodland (poplar, spruce, buf falo-berry, alder) near the Mildred
Lake campsite. The roots were coarsely chopped, the samples were
bulked and the soil/root mixture packed into 25, 150 cc Leach
Cone-tainers . The procedure was repeated with 0-15 cm deep recon-
structed soil (tailings sand, 3% peat, 12% clay) removed from the
University of Calgary soil reconstruction plots adjacent to the RRTAC
soil reconstruction site. Buf falo-berry seed was stratified and germi-
nated as described previously (3.2.1) and one germinant planted in each
container. Plants were grown in the greenhouse under the conditions
outlined in 3.2.1 and were watered with deionized water when required.
At 2, 4, 8, 12, and 20 weeks after planting, 5 seedlings were destruc-
tively sampled and shoot and root weights, % mycorrhizal colonization
and nodule weights measured using the methods described in 3.2.1.
3.2.9 Rates of Mycorrhizal and Nodule Development in Silver-berry
Outplanted in an Undisturbed Woodland and the Suncor Tar
Island Dyke
This study was conducted to gain some insight into the rates
of symbiont colonization under field conditions.
Silver-berry seed was stratified and germinated using the
technique described in 3.2.1. Germinants were planted in autoclaved
16
peat/vermiculite (50/50, v/v) in 150 cc containers. Plants were grown
in the greenhouse for 2 months using the light conditions described in
3.2.1. Seedlings were fertilized with 200 mg 28-14-14 twice weekly and
flushed with deionized water between fertilizer applications. Prior to
outplanting, seedlings were hardened off outdoors for 2 weeks without
any fertilization.
Three 10m x 10m plots were established on the Suncor Tar
Island Dyke in areas which had been revegetated with grass/legume
mixtures in 1971, 1974 and 1978. Fertilization of the plots had been
discontinued in 1979. Seedlings were planted in June, 1985. In each
plot, 50 seedlings were planted at Im intervals in 5 rows. The rows
were 2m apart, 10 seedlings per row. The procedure was repeated in one
additional plot established in an undisturbed mixed woodland (poplar,
pine, spruce, wild rose, grasses etc.) located near the Mildred Lake
campsite.
At 6 and 12 weeks after planting, 10 seedlings were randomly
sampled from each plot, 2 seedlings per row. Shoot and root weights,
root growth out of the planting plug, nodule weights and mycorrhizal
development were measured for each plant using the methods described in
3.2.1. Survival was measured at the 6 week sample time.
3.3 DEVELOPMENT OF A GROWING REGIME FOR GREENHOUSE PRODUCTION OF
MYCORRHIZAL, NODULATED SILVER-BERRY AND BUFFALO-BERRY
If, based on the factors discussed in 3.2, actinorhizal seed-
lings would benefit significantly from being colonized by their
symbionts prior to being outplanted, it may become necessary to develop
a program for inoculating containerized plants in the greenhouse or the
shadehouse. There are many factors which are important in achieving
successful inoculation; those investigated in this research program
included fertilizer regimes, growing temperatures, container volume,
inoculum source, and time and method of inoculation. Details of the
various experiments follow.
17
3.3.1 Fertilizer Effects on Growth. Nodulation and Mvcorrhizal
Development in Buffalo-berry and Silver-berry
Ten random forest floor samples were removed from a mixed
woodland (aspen poplar, white spruce, wild rose, buffalo-berry,
grasses, herbs) located near the Mildred Lake campsite. An additional
10 samples were removed from plot 2 (tailings sand amended with 11 cm
(3% organic C) muskeg peat and 2.9 cm (12% clay) surficial overburden
clay) in the University of Calgary reclamation site (pad plot study).
Each sample was subsampled and the subsamples for each soil type
bulked. Forty containers were packed for each soil type and planted
with either silver-berry or buf falo-berry which had been scarified and
germinated as described previously. Five replicates of each species in
each soil type were subjected to one of the following four fertilizer
regimes: no fertilizer, 100 mg L 200 mg L ^ and 400 mg L ^
28-14-14 Plant Prod Soilless Feed. Fertilizer applications were made
twice weekly and plants were flushed with deionized water between
applications to remove excess fertilizer salts. Plants were raised in
the greenhouse in conditions similar to those described in 3.2.4.
After 20 weeks shoot and root weights after drying at 80®C,
nodule wet weight and % root length colonized by mycorrhizal fungi were
determined for each seedling using the methods described previously.
3.3.2 Effect of Container Volume and Inoculation on Growth of
Silver-berry
. Silver-berry seed was germinated as described in 3.2.1.
Germinants were planted in 65 cc and 150 cc Cone-tainers (Ray Leach,
Canby, OR) filled with 50/50 (v/v) autoclaved (sterilized) peat/
vermiculite growing medium treated with autoclaved (control treatment)
or unautoclaved VAM inoculum (inoculated treatment). The inoculum was
collected from beneath buffalo-berry shrubs in a mixed woodland near
the Mildred Lake campsite and was added to the planting mixture at a
rate of 10% by volume. The seedlings were grown in the greenhouse
(daylength extended to 20h with 6ro-1ux lights; minimum light intensity
3.5 klx) and received 200 mg L~^ 15-15-18 Plant Prod Soilless Feed
once weekly during the first 4 weeks of growth and twice weekly for the
remaining 16 weeks. Excess fertilizer was flushed out with deionized
18
c
water between fertilizer applications. There were 10 replicates for
each of the uninoculated and inoculated treatments in each container
size category.
Half of the seedlings in each treatment (i.e. 5 replicates)
were harvested at 12 weeks and the remainder at 20 weeks. Shoot
weight, root weight, nodule and mycorrhizal status were determined at
each sample time. Mycorrhizal status was assessed by the methods of
Phillips and Hayman (1970) and Zak and Parkinson (1982).
3.3.3 Growth of Silver-berry as Influenced by Soil Temperature and
Symbiont Inoculation
Silver-berry was stratified and germinated as described
previously (3.2.1). Leach Cone-tainers (150cc) were filled with soil
mixtures belonging to each of the following four treatments:
i) uninoculated control - autoclaved peat/vermicul ite
(50/50, v/v) amended with autoclaved soil (20% by volume)
from a Mildred Lake mixed woodland.
ii) woodland soil inoculum - as above with the exception that
the woodland soil was not autoclaved,
iii) silver-berry soil inoculum - as above with the exception
that soil inoculum originated from beneath silver-berry
planted by Vaartnou in a reclamation plot near the
Mildred Lake camp.
iv) VAM pot culture inoculum - as above but using Glomus
aqqreqatum inoculum. Glomus aqqreqatum, a VAM fungus
indigenous to Alberta, had been maintained in pure
culture in the greenhouse on silver-berry planted in
autoclaved peat/vermicul ite. The inoculum consisted of
pot culture soil and chopped silver-berry roots con-
taining the fungus.
Half the seedlings were grown in a growth chamber programmed
to maintain a constant temperature of 26®C in the root zone while the
other half were placed in a chamber programmed to maintain a 16®C root
zone. The experimental temperatures were based on soil temperatures
measured in the University of Calgary greenhouse. These generally fell
in the range of 20 to 30®C with extremes at 15® and 40®C. Day
19
length was set at 18 h and the air temperature was 24®C day/26®C night
in the 26®C chamber and 11®C day/16®C night in the 16®C chamber.
Light intensity 25 cm from the lights was 180 yE m~^ sec~^
(170 w m~^, 30 klx) in the 26®C chamber and 135 yE m~^ sec~^
(170 w m~^, 21 klx) in the 16®C chamber. Fertilizer (28-14-14
Plant Prod Soilless Feed) was applied twice weekly at a rate of
100 mg beginning 2 weeks after planting. Excess fertilizer was
leached out with deionized water between fertilizer applications.
There were 10 replicates/inoculum treatment/temperature.
Seedlings were harvested when 13 weeks old. Shoot height,
root collar diameter, and shoot weights after drying at 80 C, were
measured. Nodule number and weight were determined for the whole root
system while root length colonized by VAM fungi was estimated from a
10% subsample of the total root weight. Roots were cleared and stained
according to Phillips and Hayman (1970) and mycorrhizal infection quan-
tified by the methods of Zak and Parkinson (1982). Roots not used for
VAM quantification were dried at 80®C and used to estimate total root
weights .
3.3.4 Use of Soil, Nodule and Pure Culture Inocula for Introducinq
Na-Fixinq Frankia to Containerized Silver-berry
There are numerous methods for introducing Frankia into
containerized silver-berry seedlings in the greenhouse; however, the
methods vary in their practicality and the efficiency with which the
inoculum becomes established. This study was conducted to determine
the most effective method for inoculating actinorhizal shrubs on a
relatively large-scale basis.
Silver-berry seed was stratified and germinated as discussed
previously. The planting mixture consisted of autoclaved peat/
vermiculite (50/50, v/v) inoculated as follows:
i) No inoculum - control
ii) Frankia pure culture - Frankia inoculum specific for
silver-berry was purchased from Rhizotec Laboratories Inc.
in Quebec. Fifty milliliters of the inoculum were diluted
in 150 ml deionized water and applied to 15, 7 week old
silver-berry seedlings at a rate of 10-15 ml/plant.
20
iii) Frankia pure culture - Frankia culture SCN 10a was kindly
provided by Dr. M. Lalonde, Faculty of Forestry, Laval
University, Quebec. The culture was originally isolated
from Shepherdia. Frankia was cultured in Qmod media
(Quispel, 1960 modified by Carpenter and Robertson, 1983)
for 3.5 months and then inoculated with a pipette into the
root region of each of 15, 7 week old silver-berry at a
rate of 10 ml of Frankia Qmod culture/seedling.
iv) Soil slurry A - soil from the root region of a heavily
nodulated silver-berry seedling outplanted on the
University of Calgary reclamation plot on the Syncrude site
for 1 year was well-mixed and a 25 g subsample placed in
160 ml deionized water. The soil/water slurry was stirred
for 2 min and inoculated with a pipette into the root
region of 15, 7 week old silver-berry seedlings at a rate
of 10 ml/container.
v) Soil slurry B - Twenty-five grams of forest floor soil from
beneath buffalo-berry in a mixed poplar-spruce woodland
located near the Mildred Lake camp was mixed into 170 ml
deionized water and blended for 2 min at 10,000 rpm. The
soil slurry was then injected into each seedling as
described in iv).
vi) Nodule inoculum A - Fresh nodules were picked from the
roots of one year old silver-berry shrubs which had been
planted in the University of Calgary reclamation plots
(RRTAC pad plot) on the Syncrude site. Approximately 6.4 g
wet nodules were washed in deionized water on a 1 mm mesh
sieve, crushed to a thick paste in a mortar, and suspended
in 150 ml deionized water. Ten milliliters of nodule/water
slurry were then inoculated into 7 week old silver-berry
seedlings as described in iv).
vii) Nodule inoculum B - Nodules were collected from silverberry
shrubs planted by Vaartnou in a reclamation site situated
near the Mildred Lake camp. Many of the nodules were found
associated with roots permeating rotten wood buried in the
sandy soil. Approximately 6.3 g wet weight nodules were
21
washed, crushed and suspended in deionized water as
described in vi). The nodule slurry was inoculated into
containerized silver-berry following the method described
in i v) .
viii) Nodule inoculum C - Fresh nodules were collected from the
same source as for vi). Approximately 6 g of wet nodules
were rinsed 3 times in deionized water and then mashed in a
mortar until they formed a thick paste. The nodule paste
was then suspended in polyvinyl pyrrolidine-phosphate
buffer solution (a treatment which reduces oxidation of
phenols which appear to inhibit the growth of Frankia,
Loomis and Battaille, 1966), shaken for 1 min and
centrifuged for 10 min at 5000 rpm at 20°C. The
supernatent was decanted and the procedure repeated 3
times. The resultant nodule pellets were resuspended in
170 ml deionized water and injected into containerized
silver-berry seedlings as described above.
The seedlings were grown in the greenhouse under the
conditions detailed in 3.3.2. Two weeks after planting the seedlings
began receiving 15-15-18 Plant Prod Soilless Feed fertilizer at a rate
of 100 mg twice weekly. Excess fertilizer was flushed out with
deionized water between fertilizer applications. The seedlings were
grown for 18 weeks after which height, root collar diameter, shoot
weight, root weight, nodule number and nodule wet weight were deter-
mined for each replicate using the methods described previously.
3.3.5. Effect of Inoculation Method and Inoculation Time on Nodule
and Mvcorrhizal Development of 8uf falo-berry
This study was performed to determine if inoculum soil mixed
into the planting mixture was a more effective means of introducing
inoculum and promoting symbiont development than applying the inoculum
as a soil slurry. Also, the timing of inoculum application was tested
by inoculating seedlings of various ages.
8uf falo-berry seed was scarified and germinated as described
previously with the exception that seed was treated with sulphuric acid
22
c
for 40 rather than 30 minutes. The germinants were planted according
to the following treatments:
i) 10 seedlings were planted in sterilized peat/vermiculite
ii) 10 seedlings were planted in peat/vermiculite which had
been amended with mixed forest (Mildred Lake camp vici-
nity) floor soil at a rate of 10% by volume,
iii) 10 seedlings were planted in peat/vermiculite and treated
with a mixed forest soil/water slurry. Approximately
18 g of forest soil (same as that used in ii) was mixed
with 120 ml deionized water, blended at 10,000 rpm for
2 min and applied to each seedling at a rate of 10 ml/
container.
iv) 40 seedlings were planted in peat/vermiculite and 10
seedlings were treated as described above (iii) when they
were 2, 3, 4 and 5 weeks old.
Seedlings were grown in a growth chamber programmed for an
18 hour day/6 hour night. Light intensity was measured at
420 yE m~^ (54 klx or 330 W m~^). After 10 weeks the seedlings were
transferred to the greenhouse where daylength was extended to 20 hours
and light intensities were in the vicinity of 207 yE m~^ (39.6 klx,
255 W m~^). Fertilizer was applied at a rate of 200 mg L~^ 28-14-14
Plant Prod Soilless Feed once weekly for the first 7 weeks and was
increased to 400 mg L ^ twice weekly thereafter. The seedlings were
harvested after 17 weeks and assessed for shoot height, branching,
shoot weight, root weight, nodule weight and mycorrhizal root length as
per the methods discussed previously.
3.4 FIELD TRIAL TO TEST GROWTH RESPONSE OF INOCULATED SILVER-BERRY
AND BUFFALO-BERRY
In order to determine if the development of an inoculation
program is worthwhile, a field trial to assess the growth response of
inoculated and uninoculated plants should be conducted. If inoculation
confers few benefits on the plant, in terms of growth and survival,
inoculation of containerized shrubs prior to outplanting may be
unnecessary. Therefore, a study was conducted to determine the effect
of inoculating silver-berry and buffalo-berry with soil containing both
23
VA mycorrhizal and Frankia inoculum on plant growth and symbiont
development under field conditions. The seedlings were outplanted on
two reconstructed soils, one amended with peat and one amended with
peat and clay.
The two University of Calgary plots were located on the east
side of the RRTAC soil reconstruction-woody plant experimental area on
a specially prepared pad of oil sand tailings from the Syncrude Canada
Ltd. extraction plant (Figure 2). The two soil treatments, mixed to a
depth of 20 cm, were the application of (1) 11 cm (3% organic C) of
muskeg peat (P-1) and (2) 11 cm of muskeg peat plus 2.9 cm (12% clay)
of surficial overburden clay (P-2), The plots were 12 x 44 m with
buffer strips on all sides leaving 10 x 40 m for planting. The plots
were constructed by Hardy Associates in June 1984. The muskeg peat was
from the Syncrude NT-2 stockpile and the clay from the 0-pit located
close to mixed aspen woodland. The clay was from a depth of
approximately 1 to 3 m and consisted of 39% clay, 29% silt and 32% sand
(Hardy Associates, 1983). Both plots were fertilized with 0-45-0 rock
phosphate at a rate of 112 kg ha ^ and all amendments thoroughly
mixed into the top 20 cm of sand with a Madge Rotoclear machine.
Further details on plot construction are given in reports by Hardy
Associates ,
Each 10 X 40 m plot was staked to delineate 40 rows with 20
planting positions per row. This resulted in 1 m spacing between rows
and 0.5 m spacing within rows. Provisions were made to accommodate a
total of 10 treatments per plot, each treatment with 4 randomly
assigned rows (i.e. 20 plants/row x 4 rows = 80 replicates). The plant
species used in the trial were si 1 ver-berry, buffalo-berry, green alder
and jack pine. Only silver-berry and buffalo-berry will be discussed
here.
Silver-berry and buf falo-berry seed were scarified and germi-
nated as outlined previously. The germinants were then planted (65 cc
containers) in sterilized peat/vermiculite (50/50, v/v) containing
either autoclaved (control treatment) or unautoclaved (inoculated
treatment) symbiont inoculum at a rate of 20% by volume. The source of
the inoculum was the soil mixture used in the shrub dependency study
(3.1.1). The Frankia and mycorrhizal fungi in this soil were
24
25
propagated in pot culture in the greenhouse by growing silver-berry in
a mixture of inoculum soil and peat/vermiculite for 2 months. The pot
culture soil was then used to set up the inoculated and uninoculated
treatments .
The seedlings were grown in the greenhouse with daylength
extended to 20 h and a minimum of 3.5 klx light intensity. They were
fertilized at a rate of 100 mg L ^ of 15-15-18 Plant Prod fertilizer
from weeks 4 to 7 and at a rate of 200 mg L ^ during weeks 8 and 9.
In week 10 fertilization was reduced to 100 mg and the silver-
berry was supplemented with 100 mg L ^ of NH4NO3. This regime
continued until week 13 when buffalo-berry was also supplemented with
100 mg of NH4NO3. Both silver-berry and buffalo-berry
required additional N to stimulate growth and counteract chlorosis. In
weeks 15 and 16, fertilizers were applied only once per week. Fertili-
zation was stopped in week 17 and the seedlings were hardened off out-
doors for two weeks prior to outplanting.
Preplanting mycorrhizal and nodulation assessments were made
by randomly selecting 10 plants from each species-inoculation treat-
ment, removing the shoots, washing the planting mixture from the roots
and determining shoot weight, root weight, VA mycorrhizal status and
nodule status. The methods used have been presented previously.
Survival was measured in the field one year after outplan-
ting. In each of the inoculated and uninoculated treatments in each of
the two plots, 10 randomly chosen seedlings/species were excavated and
transported to the laboratory. Excavation consisted of digging an
approximately 25 cm square to a depth of 20 cm around each plant,
shaking the excess sand from the roots and placing the plant in a
plastic bag. The shoots were clipped at the root/shoot interface and
shoot height and weight (after drying at 80®C) were measured. The
root systems were washed free of soil, nodules were counted, separated
from the roots and weighed. Five of the 10 root systems in each treat-
ment were selected randomly and subsampled for VA mycorrhizal assess-
ments. The size of the subsamples varied with the size of the root
system and ranged from 15% of the total wet weight for large root
systems to 50% of the total for small root systems. Only relatively
young roots (2 mm diameter or less) were sampled since older, thicker
26
roots usually lacked a cortex - the site of VA mycorrhizal infection.
The roots were cleared (8 min) and stained (7 min) following the
methods of Phillips and Hayman (1970) and mycorrhizal infection quanti-
fied as outlined by Zak and Parkinson (1982). The remaining roots were
dried at 80®C and dry weights determined.
Silver-berry foliage was analyzed for total N and P. All 10
replicates for each inoculated and uninoculated treatment in each plot
were ground in a Wiley rotary mill to pass a 40 mesh (425 ym)
screen. The samples were digested with concentrated sulphuric acid and
30% hydrogen peroxide in a Technicon BO-block digester. Acid digests
were filtered through a Whatman No. 1 filter and stored. Both total N
(as ammonium N) and P (as orthophosphate P) were determined colorime-
trically on a Technicon Autoanalyzer II system using the ammonium
molybdate/ascorbic acid chemistry for PO4-P and the Berthelot
Reaction for NH4-N.
Sampling was repeated two years after outplanting. Due to
poor survival (particularly in the inoculated treatment) during the
first winter after outplanting, the number of surviving plants in each
treatment in each plot was often less than 10. To increase replica-
tion, shrubs from both plots were pooled resulting in 15 replicate
silver-berry and 7 replicate buffalo-berry plants in each of the inocu-
lated and uninoculated treatments. It was felt that pooling the
silver-berry from both plots was justified since plot treatment effects
on shrub growth and nodule development were insignificant for this
species after the first year. Although buffalo-berry exhibited plot
treatment effects on growth and nodule development after the first
growing season, pooling of second year plants was necessary to improve
replication so statistical analysis could be performed.
Two-year old plants were excavated and shoot heights, shoot
weights, root weights, nodule weights and mycorrhizal colonization
assessed as described for the one-year old plants. In addition, root
collar diameters and frequency of branching was measured at this sample
time.
27
4. RESULTS
4.1 MYCORRHIZAL STATUS OF WOODY SHRUBS
Buffalo-berry, si 1 ver-berry, saskatoon-berry and cinquefoil
were all found to be strictly vesicular-arbuscular mycorrhizal (VAM)
regardless of sampling location (Table 1). Mycorrhizal infection of
young, active roots (dia. <2 mm) ranged up to 60% for both buffalo-
berry and silver-berry and was slightly higher for saskatoon-berry and
lower for cinquefoil .
4.2 JUSTIFICATION FOR INOCULATION OF CONTAINERIZED SILVER-BERRY
AND BUFFALO-BERRY
4.2.1 The Dependency of Silver-berry and Buf falo-berrv on their
Mycorrhizal and N2-Fixinq Symbionts
Shoot weights were 4 and 9 times greater for inoculated
silver-berry and buffalo-berry, respectively, than for their uninocu-
lated counterparts , while the roots of the inoculated shrubs weighed 3
and 4 times more, respectively, than the roots of the uninoculated
shrubs (Table 2). In the inoculated treatments, nodule development was
more pronounced on the buffalo-berry than the si 1 ver-berry, but VAM
colonization was approximately the same for the two shrub species (i.e.
64 and 70%). Low levels of nodulation and mycorrhization were detected
in the uninoculated treatments suggesting contamination of the planting
mixture occurred during the course of the experiment.
4.2.2 Levels of VA Mycorrhizal Inoculum in Various Soils in the Fort
McMurray^ Alberta Region
Although slender wheatgrass was used to assay various soils
for VAM inoculum potential, the results should be applicable to
buf falo-berry and silver-berry since both the grass and the shrubs are
VA mycorrhizal and evidence to date suggests that VAM fungi are
generally not host specific. Because grasses grow faster and, due to
the fibrosity of their roots, tend to exploit a greater volume of soil
than many shrub species do, they offer a more rapid and efficient means
for surveying soils for VAM inoculum.
28
c
Table 1. Mycorrhizal status of selected woody shrubs growing in the
Fort McMurray and Kananaskis, Alberta regions. VAM =
vesicular-arbuscular mycorrhizae.
Shrub
species
Sampling
Location
and Date
Adjacent
Woodland^
Mycorrhizal Infection
Status density (%)
Buf falo-berry
Vaartnou plot,
Mildred L. camp
(June, n = 5)
Jack pine/
lichen
VAM
Most roots dead
Infection low,
5-10%
Buf falo-berry
Cutbank near
Suncor plant
(Oct. , n = 5)
Mixed/
aspen
VAM
20-40
Buf falo-berry
Kananaskis ,
roadcut
(Aug., n = 5)
Mixed/
aspen
VAM
20-60
Si 1 ver-berry
Vaartnou plot,
Mildred L. camp
(June, n = 5)
Jack pine/
lichen
VAM
20-50
Si 1 ver-berry
Kananaskis
(Aug., n = 5)
Mixed/
aspen
VAM
40-60
Saskatoon-berry
Outline near
Mildred L. camp
(Oct. , n = 5)
Jack pine/
lichen
VAM
60-80
Cinquef oi 1
Vaartnou plot
(June, n = 5)
Jack pine/
lichen
VAM
20-30
i Woodland located in close vicinity to sampling location.
29
Table 2. Shoot and root production and symbiont development in silver-
berry and buf falo-berry grown in reconstructed soils with and
without symbiont inoculum. Data are means ± SD.^
Shrub Inoculum
Shoot Weight
(mg dwt)
Root Weight
(mg dwt)
Nodule Weight
(mg wet wt)
VAM Coloni-
zation (%)
Silver-berry +
422 ± 21*5
139 ± 23b
6 + 3*5
70 ± 16*5
-
108 ± 27a
45 ± 12a
1 ± ia
1 ± 2a
Buffalo-berry ^
244 + 5lb
72 ± 25b
44 ± 20
64 ± 6*5
-
27 ± ga
1+
00
0)
0
2 ± 3a
1 Data analyzed by a two sample T-test. Values in each column followed
by the same letter for either silver-berry or buffalo-berry are not
significantly different (p = 0.05).
Vesicular arbuscular mycorrhizal inoculum potential was negli-
gible (0-5% VAM infection) in the majority of peat samples (Table 3).
Peat, stockpiled for 8 to 12 months on the Syncrude site, lacked VAM
inoculum, but increased levels of inoculum were evident in stockpiled
peat which had been revegetated with a grass/legume (VAM hosts) mixture
for 6 years. Mixed woodland soil exhibited the highest VAM inoculum
potential with coarse textured soil from an aspen/shrub/grass woodland
being the most infective (64% mycorrhizal infection) of all the soils
tested.
4.2.3 Growth Characteri sties and Mycorrhizal Potential of
Undisturbed Bog Peat and Stockpiled Peat
Again, slender wheatgrass, rather than a shrub species, was
used in this VAM assay.
30
c
Table 3. Vesicular-arbuscular mycorrhizal (VAM) infection of slender
wheatgrass grown in the greenhouse in various soils collected
from the Fort McMurray, Alberta region. Infection expressed
as % total root length infected.
Soil Description
Age of Plant
When Sampled (wk)
% VAM
Infection
Aspen woodland mineral
8
13.0
Fine textured soil from beneath
undisturbed mixed woodland
12
37.0
Coarse textured soil from beneath
undisturbed mixed woodland
12
64.0
Carex/Sphagnum peat mixture from
Syncrude and Suncor leases
9
1 .6
Undisturbed peat, Canstar lease
12
0.6
Undisturbed peat, 0-15 cm depth.
Syncrude lease
12
14.5
Undisturbed peat, 50-100 cm depth,
Syncrude lease
12
4.8
NT 2 (Syncrude) stockpile peat
stockpiled for:
8 mo. , 0- 1 5 cm depth
12
0
8 mo. , 50-100 cm depth
12
3.1
1 2 mo. , 0- 1 5 cm depth
8
0.2
Peat stockpiled for 6 years on
Syncrude site (East Muskeg)
9
13.7
31
Shoot weights, root weights, shoot/root (S/R) ratios, total
root lengths and % mycorrhizal infection were very similar in the
undisturbed and stockpiled peat (Table 4). There was very little
effect of sampling depth on the majority of parameters tested, with the
exception of shoot weights, which were greater in the 0 - 15 cm deep
undisturbed and stockpiled peat than in the 50 - 100 cm deep peat.
Percent VAM colonization was highest in the undisturbed surface peat,
but not significantly so since sample variation was high (Appendix
Table 1). Mycorrhizal inoculum levels were generally very low with the
infection consisting mainly of hyphae and arbuscules (Appendix Table 1).
4.2.4 Mycorrhizal Potential of Revegetated Dyke Peat
Slender wheatgrass shoot and root weights and root lengths
were slightly lower in 6 year-old stockpiled peat than 8 month-old
stockpiled peat, while the reverse was true for VA mycorrhizal inoculum
potential (Tables 4, 5). Fertilization of the 6 year-old stockpiled
peat significantly improved plant productivity while not significantly
altering VAM inoculum potential (Table 5).
4.2.5 Effect of VA Mycorrhizal Inoculation on Plant Performance of
Slender Wheatgrass Grown in Stockpiled Peat Under Fertilized
and Unfertilized Conditions
Inoculation of slender wheatgrass with the VAM fungus. Glomus
aqqreqatum, significantly improved shoot and root production but only
if the plants received no fertilizer and only when the plants were 10
weeks old (Table 6). As expected, fertilization greatly improved plant
growth but counteracted the potentially beneficial effects of the VAM
fungus, resulting in very few differences between inoculated and unino-
culated treatments at the two sample times.
4.2.6 Mycorrhizal and Nodule Status of Containerized Shrubs Planted
on the Oil Sands Tailings Reconstruction Plots
Shoot and root weights of the containerized VAM shrub species
planted on the RRTAC oil sands tailings reconstruction plots (i.e. pad
plots) are given in Table 7. Buf falo-berry and silver-berry shoots
appeared to be underweight and the symbiont status of all the shrubs
32
Table 4. Characteristics of slender wheatgrass grown in peat from a
muskeg bog and peat stockpiled for eight months
Sampling Depth (cm)
Plant Parameter
Peat Source
0-15
50 - 100
Shoot weight
Undisturbed
94.3b
54.4a
(mg dwt planf^)
Stockpile
73.9b
59.7a
Root weight
Undisturbed
127.9b
96.2a
(mg dwt plant“^)
Stockpile
97.2a
100.1a
Shoot/root ratio
Undisturbed
0.74 (.23)
0.57 (.09)
Stockpile
0.77 (.15)
0.60 (.13)
Total root length
Undisturbed
545.0a
578.6a
(m L-^)
Stockpile
563.5a
623. ia
Percent VAM infection
Undisturbed
14.5a
4.sa
Stockpile "
oa
3.1a
^ Shoot and root weight data analyzed by a Kruskal-Wallis test. Root
length and VAM infection analyzed by two-way ANOVA. Values in each
data set followed by the same letter are not significantly different
(p = 0.05). Values in brackets are standard deviations.
33
Table 5. Root and shoot production by slender wheatgrass grown in the
greenhouse in fertilized and unfertilized dyke peat. Peat
had been stockpiled for 6 years prior to spreading on the
dyke. 1
Treatment
Measurement
Unfertilized
Ferti lized
Shoot weight
(mg plant
55^
144^
Root weight
(mg plant
81 "
139'^
Shoot/Root
0.68
1 .04
Root length
(m soil)
326®
508*’
Mycorrhizae (%)
14®
16®
^ Data analyzed by Hotelling's test. Means in each row not
followed by the same letter differ significantly (p = 0.05).
was poor. With the exception of one pincherry plant, which exhibited a
small patch of mycorrhizal infection caused by the "fine endophyte",
all the shrubs were nonmycorrhizal . None of buf falo-berry and 16% of
the silver-berry were nodulated. The fungal root pathogen, Thielaviopsis .
was occassional ly observed in the silver-berry roots while Olpidium,
another fungal parasite, occurred frequently in the roots of all the
shrub species.
34
Table 6. Shoot and root production by slender wheatgrass grown in
stockpiled peat (50-100 cm deep) inoculated with Glomus
aqqreqatum and fertilized or left unferti lized. ^
4 week old plants
10 week
old plants
Measurement
Fertilized
Unfertilized
Fertilized
Unfertilized
Shoot weight
Inoculated 22^
132*^
22'^
-1
(mg dwt plant)
be
Uninoculated 14
7^
113''
12ab
Root weight
Inoculated 22*^
10^
194''
30*^
-1
(mg dwt plant)
Uninoculated 18^
10^
146'
11"
^ Data for each parameter analyzed by three-way ANOVA (MSE = .0715
and .1077 for shoots and roots respectively). A three-way interaction
was observed for shoot data, hence Scheffe multiple contrasts were
applied to individual treatment means. No three-way interaction was
observed for root data, hence Scheffe multiple contrasts were applied
to two-way means. Values within each data set followed by the same
letter(s) do not differ significantly (p = 0.05). Data required
LN (Y + 1) transformation.
4.2.7 Growth Characteristics and Symbiont Status of Woody Shrubs
Raised in Various Commercial Nurseries in Alberta and British
Columbia
Shoot heights, shoot weights, root weights and S/R ratios for
all species surveyed are summarized in Table 8. Shoot weights for each
plant species varied greatly amongst nurseries mainly due to
differences in crop year, growing regimes (i.e. fertilization rates,
light conditions) and possibly container size. Silver-berry from the
Laidlaw Nursery were of an acceptable size (0.98 g) while those from
35
■a <o
o f—
z Q.
>>
s:
o •
Q
O I/)
2 +1
QC
oeL ^
m
0) CM
^ II
+J c
O 1/1
cr
-O «3
O) 0>
-M e
c
CO 0)
r— S_
Q. CO
in (O
0> +->
♦r- (O
u a
<u
a.
in
'=r
00
3 01
i. r-
sz
in »
&-
<4_ (U
O JO
E
j/1 0)
3 -H
•»-> Q.
(O <U
+-> 00
in
c.
O) *r-
3 (/I
“O -H
O O
C f—
Q.
o.
<
fO o
(O o
M 3
&-
J= +->
i- in
i. c
O O
u o
>1 <u
s:
<u
Ql
00
oj L.
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oi x:
1/1 r— a
O -r- i.
od 3 Q_
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i.
t-
(U
JO
C (U
i_ >
<U I—
CD -r-
00
One small patch of VAM infection was observed in the root system of one pincherry plant. The mycorrhizal
fungus was identified as the “fine endophyte." In general, roots were in good condition, although extensive
infection by Thielaviopsi s . a root pathogen was observed in two of the silver-berry plants. Olpidi urn, another
root parasite, was observed regularly in the roots of all species.
36
Table 8
Size of
container
-grown
and bareoot woody shrubs
obtained
from four
commerical nurseries in
August,
1985.
Data are
means ± SD
Container
No. of
Shoot Height Shoot Weight Root Weight
S/R
Plant species
Nursery
Crop
size (cc)
seedlings
(cm)
(g)
(g)
( range)
Pincherry
Reid-Collins
Unknown
SL 47(?)
8
NM
1.58 ± 0.43
2.26 ± 0.63
.7 ( .5 - 1.5)
Whitecourt
Heeled in ( *84)
NA
2
60, 46
17.64, 8.16
NA
NA
Laidlaw
1984
SL 150
10
38 ± 8
1 .64 1 0.5
1.76 ± 0.48
.9 (0.6 - 1 .4)
Laidlaw
1985
SL 150
10
46 ± 10
1 .91 1 0.85
1.36 1 0.55
1 .4 (0.9 - 1 .8)
Saskatoon
Reid Collins
Unknown
SL 47(?)
10
NM
1.70 ± 0.56
1 .97 ± 0.95
0.9 (0.6 -1.5)
Whitecourt
1984
Styro-20
10
35 ± 4
4.46 ± 1.19
2.01 ± 0.58
2.3 (2.0 - 3.4)
Whitecourt
1985
Styro-4
10
21 ± 4
1 .84 1 0.53
NM
NM
Laidlaw
1985
SL 150
10
17 ± 4
1 .42 ± 0.41
0.83 ± 0.37
2.0 (1.0 - 3.7)
Oliver
1983
SL 150
10
30 ± 11
2.36 1 1 .41
1 .48 ± 0.92
1.7 (1.0 - 2.3)
Oliver
3 - 5 y(?)
Bare root
10
35 ± 5
NA^
NA*
NA*
Dogwood
Oliver
1985
SL 150
10
18 ± 3
0.46 1 0.13
0.18 ± 0.06
2.5 (2.0 - 3.6)
Willow
Reid-Collins
1985(?)
SL 150
10
NM
NA*
0.18 ± 0.08
NM
Cinquefoil
Whitecourt
1984
Styro-8
10
39 ± 7
2.62 1 1.61
0.68 ± 0.56
4.6 (2.5 - 7.6)
Silver-berry
Reid-Collins
?
SL 47(?)
10
NM
0.39 1 0.06
0.18 1 0.07
2.8 (1.1 - 3.3)
Laidlaw
1985
SL 150
10
23 ± 6
0.98 1 0.48
0.39 ± 0.24
2.8 (1.3 - 6.1)
Oliver
1983
SL 150
10
19 13
0.77 1 0.29
0.30 ± 0.12
2.7 (1.8 - 3.2)
Buffalo-berry
Reid Collins
?
SL 47(?)
10
NM
1.11 ± 0.53
1 .97 1 0.77
0.6 (0.4 - 0.9)
Syncrude^
1981
7
10
NM
0.59 1 0.16
0.58 ± 0.19
1 .0
1982
7
10
NM
0.57 ± 0.23
0.57 ± 0.17
1 .0
1983
7
10
NM
0.11 1 0.04
0.12 1 0.03
0.9
Silver
Oliver
1985
SL-150
10
12 ± 1
0.18 1 0.04
0.05 1 0.02
3.7 (2.6 - 6.3)
buffalo-berry
Oliver
3 - 5 yr(?)
8a re root
10
36 ± 4
NA*
NA*
NA*
Syncrude*
1983
7
10
NM
0.09 1 0.04
0.07 ± 0.03
1.3
Russian olive
Oliver
3 - 5 yr (?)
Bareroot
10
32 1 4.3
NA*
NA*
NA*
^ Seedlings pruned
* Cutting included in shoot weight determination
* 1981 crop sampled in June 1984; 1982 and 1983 crops sampled in October, 1983
* Sampled in June 1984
NM = not measured; NA = not applicable; SL = Spencer Lemaire; Styro = Styroblocic
37
the Reid-Col lins Nursery were unusually small (0.39 g) in comparison.
The 1981 and 1982 buffalo-berry from the Syncrude Nursery also tended
to be underweight and silver buf falo-berry from the same nursery were
particularly stunted. The shoot weights of the actinorhizal shrubs
were generally less than those of the non-actinorhi zal shrubs of
equivalent age. Shoot/root ratios were highly variable, ranging from
0.6 (Reid-Col lins buf falo-berry) to 4.6 (Whitecourt cinquefoil).
Seedlings sampled in the same year that they were planted were
usually non-mycorrhizal (Table 9). Seedlings which were one year old
or more and had probably overwintered in the shadehouse or outdoors
were quite often mycorrhizal (Reid-Col 1 ins pincherry, saskatoon,
buf falo-berry, Whitecourt saskatoon, Oliver saskatoon) but not always
so (Laidlaw pincherry, Oliver si 1 ver-berry. Syncrude buf falo-berry) .
The bareroot stock was heavily mycorrhizal. The “fine endophyte",
characterized by its narrow hyphae (2-3 ym dia) and finely branched
arbuscules, was frequently observed on the roots of saskatoon (White-
court) and pincherry (Reid-Collins, Laidlaw '85 pincherry). Many of
the seedlings were infected with the fungal root parasite, Olpidium,
regardless of nursery or seedling age. Thielaviopsis . a pathogenic
fungus which causes black root rot of tobacco and many vegetables,
occurred in 60% of the silver-berry from the Laidlaw nursery although
this was not evident from the condition of the shoot.
There was no evidence of nodulation on the actinorhizal shrubs
unless the shrubs were older than one year or were bareroot stock.
4.2.8 Rates of Mycorrhization and Nodulation in Buffalo-berry Grown
in Woodland Soil and Amended Tailings Sand in the Greenhouse
Shoot and root weights increased with time in both soil treat-
ments and, by the end of the study, were significantly greater in the
woodland soil than in the tailings sand (Table 10). Seedlings raised
in woodland soil became mycorrhizal much more rapidly (25% infection at
2 weeks) than seedlings grown in amended tailings sand (no infection
until 8 weeks after planting). Percent mycorrhizal infection increased
significantly with time in the woodland soil but not in the tailings
sand where it remained relatively stable. A significantly greater
amount of VAM infection was attained in the woodland soil (87%) than in
38
Table 9. Vesicular-arbuscular mycorrhizal (VAM) status and Frankia nodule
development in woody shrubs samples from four commercial nurseries
in 1985,
Plant species
Nursery
Crop
No. of % Seedlings
Seedlings Mycorrhizal
Surveyed
* VAM""
(range)
X Seedlings
Nodulated
Nodule
No. Plant->-
Nodule Weight
(g wet plant"^)
Pincherry
Reid-Collins
Unknown
8
63
<1 - 20
NA
NA
NA
Whitecourt
Heeled in (1984)
2
100
33
NA
NA
NA
Laidlaw
1984
8
0
0
NA
NA
NA
Laidlaw
1985
10
30
<1-2
NA
NA
NA
Saskatoon
Reid-Col 1 ins
Unknown
10
50
<2 - 73
NA
NA
NA
Whitecourt
1984
10
100
<1 - 58
NA
NA
NA
Laidlaw
1985
10
0
0
NA
NA
NA
Oliver
1983
10
40
<1 - 66
NA
NA
NA
Oliver
3-5 y (?)
10
100
40 - 60
NA
NA
NA
Dogwood
Oliver
1985
10
0
0
NA
NA
NA
Willow
Reid-Collins
1985 (?)
10
0
0
NA
NA
NA
Cinquefoi 1
Whitecourt
1984
10
0
0
NA
NA
NA
Silver-berry
Reid-Collins
Unknown
25
0
0
0
0
0
Laidlaw
1985
10
0
0
0
0
0
Oliver
1983
10
0
0
10
4
NM
Buffalo-berry
Reid-Collins
Unknown
25
20
<1 - 33
88
NM
0.27 ± 0.24
Syncrude*
1981
10
0
0
60
1 - 3
0.019 - 0.098
1982
10
0
0
20
NM
NM
1983
10
0
0
0
0
0
Silver
Oliver
1985
10
0
0
0
0
0
Buffalo-berry
3 - 5 y (?)
Bare root
100
50 - 80
100
22 ± 13
0.47 ± 0.26
Syncrude*
1983
5
0
0
0
0
0
Russian olive
Oliver
3 - 5 y (?)
Bareroot
100
40 - 70
100
6 ± 3
0.12 ± 0.10
^ % VAM refers to % of fine (<5 mm diameter) roots infected with VAM
* 1981 crop sampled in June, 1984; 1982 and 1983 crops sampled in October, 1983
s sampled in June 1984
NA = not applicable; NM = not measured
Table 10. Rates of shoot and root production, mycorrhizal colonization and nodulation by buffalo-berry
grown in undisturbed woodland soil and peat/clay amended tailings sand. Data are means
39
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40
the tailings sand (33%). The mycorrhizal data were highly variable
suggesting a high degree of variation in VAM inoculum potential amongst
the replicate soil samples. Nodules were first visible at 8 weeks when
100% of the woodland soil buffalo-berry had formed nodules compared
with 40% for seedlings planted in the tailings sand. Seedlings, older
than 8 weeks, were all nodulated with nodules produced on woodland
seedlings weighing more than those produced on tailings seedlings,
Nodulation increased with seedling age.
4.2.9 Rates of Mycorrhizal and Nodule Development in Silver-berry
Qutplanted in an Undisturbed Woodland and the Suncor Tar
Island Dyke
Shoot weights did not increase over the growing season in any
of the planting locations; in fact, there tended to be a loss in weight
between weeks 6 and 12 due to leaf abscission (Table 11). Root growth
was also negligible, as was evident from the lack of root growth out of
the planting plug in all treatments except the dyke plot revegetated in
1978. Seedling survival over the first 6 weeks after outplanting was
higher in the undisturbed mixed woodland (100%) than in the dyke plots,
particularly in the plot revegetated in 1974 where only 36% of the
seedlings were alive after 6 weeks. Vegetation in the 1974 plot was
dominated by sweet clover.
Nodulation was minimal after 6 weeks although 2 plants in the
1978 dyke plot possessed small nodules. After 12 weeks all the seed-
lings in the undisturbed woodland plot had become nodulated, but, with
the exception of the 1978 dyke plot where 4 plants became nodulated,
none of the seedlings planted on the dyke formed nodules. Nodule
number was highest on plants from the woodland plot. Almost all seed-
lings, regardless of planting location, became mycorrhizal within
6 weeks of being outplanted. Percent mycorrhizal infection, however,
was patchy, possibly a result of poor root growth out of the planting
plug.
41
Table 11, Growth, nodulation and VA-mycorrhi zal development in uninoculated
silver-berry out-planted in the boreal forest and Suncor Tar
Island Dyke for 6 and 12 weeks. Dyke locations revegetated in
1971, 1974, 1978. Data are means ± SD^.
Measurement
Age
(wk)
Preplant
Planting
locations
Undisturbed
Dyke 1971
Dyke 1974
Dyke 1978
Shoot weight
6
216 ± 60^
229 ± 84^
195 ± 58®
205 ± 50®
230 ± 80®
(mg dry plant~^)
12
216 ± 68^
153 ± 34^*^
111 ±42®
158 ± 47®^
176 ± 63®^
Root weight
6
119 ± 34*^
80 ± 27^*^
87 ± 24®^
73 ± 24®
94 ± 37®
(mg dry plant”^)
12
119 ± 34^
145 ± 23®
83 ± 34®
119 ± 66®
127 ± 67®
Survival/50
6
NA
50
38
18
43
Root growth
6
NA
0
0
0
0
out of plug
12
0
0
0
4/10
Plants with
6
0
0/10
0/10
0/10
2/10
nodules
12
0
10/10
0/10
0/ 7
4/10
Nodules/plant
6
0
0
0
0
1
(wet wt plant"^)
12
0
4-21 (.006-
.028) 0
0
1-8
Plants with
6
0
10/10
9/10
10/10
8/10
VAM
12
0
10/10
9/10
7/ 7
10/10
% VAM
6
0
<1-58
<1-71
5-31
5-29
12
0
<1-20
5-70
<2-30
1-20
Shoot and root weight data analyzed by one-way ANOVA and differences detected by Scheffe
multiple contrasts for pairwise comparisons. Values in each row followed by the same
letter(s) are not significantly different (p = 0.05).
42
4.3 DEVELOPMENT OF A GROWING REGIME FOR GREENHOUSE PRODUCTION OF
MYCORRHIZAL, NODULATED SILVER-BERRY AND BUFFALO-BERRY
4.3.1 Fertilizer Effects on Growths Nodulation and Mvcorrhizal
Development in Buffalo-berry and Silver-berry
Shoot production by buffalo-berry was greater in the woodland
soil than in the tailings sand but only in the 0 and 100 mg L ^
fertilizer regimes (Table 12). At the higher fertilizer regimes (200
and 400 mg L shoot production in the two soils was similar.
There was an increasing trend in shoot weights with increasing fertili-
zation for plants in the tailings sand but not for plants in the wood-
land soil where no significant differences in shoot weights were
detected amongst the three fertilizer rates tested. Shoot response of
silver-berry was similar to that of buffalo-berry (Table 13), with the
exception that the 400 mg L ^ fertilizer regime greatly stimulated
shoot production, particularly in the tailings sand treatment.
The patterns of root production at the various fertilizer
regimes were very similar for the two shrub species (Tables 12, 13).
Overall, root weights were lower in the tailings sand than in the
woodland soil. Fertilization at 200 mg L~^ significantly increased
buf falo-berry root production, while silver-berry root weights were 1.7
and 2.0 times greater at the 400 mg L~^ rate than at the 0 and
100 mg L”^ rates, respectively. Silver-berry plants were generally
heavier than buf falo-berry plants.
At the 0 and 100 mg L~^ fertilizer regimes, almost all the
buffalo-berry and silver-berry seedlings in both test soils developed
nodules. At 200 mg L ^ fertilizer (56 mg N), nodule formation based on
nodule weight, appeared to be inhibited but not significantly so. However,
application of 400 mg L ^ fertilizer or 112 mg N significantly reduced
nodule production rate on both species in both soil types. Although
nodule formation was reduced at the high fertilizer regime, all the
silver-berry and the majority of buffalo-berry in the woodland soil
became nodulated.
Mycorrhi zation appeared to be less sensitive to fertilization
than nodulation. The majority of seedlings at all fertilizer treat-
ments in both soils developed mycorrhizae, but the extent of
43
Table 12. Fertilizer effects on growth, nodulation, and mycorrhizal
development in buffalo-berry grown in woodland soil and
peat/clay-amended tailings sand. Age = 20 weeks. ^
Measurement
Soil
Fertilizer (mq/L 28-14-14)
0 100 200 400
Row Means
Shoot weight
Woodland
377*’*=
473*’='^
497^°
552^°
NA
(mg)
Tailings
144^
269®*’
519°^
664^
NA
Root weight
Woodland
239
234
404
388
316''
(mg)
Tailings
51
178
273
288
198®
Column means 145^
206^
339°
338°
% Seedlings
Woodland
100
100
80
100
with nodules
Tailings
100
80
20
40
Nodule weight
Woodland
60
34
11
7
00
C\J
(mg wet)
Tailings
24
22
6
3
14®
Column means 42°
28^
8.5"°
5"
% Seedlings
Woodland
100
100
100
100
with
Tailings
100
80
100
80
mycorrhizae
% Mycorrhizal
Woodland
94
90
82
40
infection
Tailings
46
56
73
23
50®
Column means 70°
73°
78°
32"
^ Data analyzed by two-way ANOVA. Differences amongst means were
detected by Scheffe multiple contrasts for pairwise comparisons
applied to individual means where a significant interaction occurred
or to row or column means if no interaction was detected in the ANOVA.
Values in each data set followed by the same letter are not signi-
ficantly different. (MSE for shoot weight, root weight, nodule weight,
and % mycorrhizal infection are 12040, 7944.9, 270.21, and 515.28,
respectively. )
44
Table 13. Fertilizer effects on growth, nodulation, and mycorrhizal
development in silver-berry grown in woodland soil and
peat/cl ay-amended tailings sand. Age = 20 weeks. ^
Measurement
Soil
Fertilizer (mq/L 28-14
0 TOO 200
-14)
400
Row Means
Shoot weight
Woodland
515
637
586
622
590^
(mg)
Tailings
286
399
453
863
500®
Column means 401^
518^
520^
Root weight
Woodland
337
334
444
507
406*^
(mg)
Tai lings
257
174
230
523
296®
Column means 297^
254^
337^*^
515^
% Seedlings
Woodland
100
100
100
100
with nodules
Tailings
100
100
50
40
Nodule weight
Woodland
91
58
30
15
49®
(mg wet
Tailings
42
76
64
12
49®
plant ^)
Column means 67^
67^
47^'
14^
% Seedlings
Woodland
100
100
100
100
with
Tai lings
100
100
50
80
mycorrhi zae
% Mycorrhizal
Woodland
94
90
93
85
91®
infection
Tailings
37
52
29
17
34‘>
Column means 66^
7l"
61^
51^
^ Data analyzed by two-way ANOVA. Shoot weight data required a LO
transformation (MSE = .02788) while root weight data required a SQRT
transformation (MSE = 11.319). MSE for nodule weight and mycorrhizal
infection = 1273.9 and 298.71, respectively. No significant inter-
action was detected, hence Scheffe multiple contrasts for pairwise
comparisons were applied to row and column means. Values in either the
row or column means for each measurement followed by the same letter
are not significantly different (p = 0.05).
45
mycorrhizal colonization was significantly less in the tailings sand
than in the woodland soil. Percent mycorrhizal infection in the
buffalo-berry was significantly reduced at the high fertilizer rate,
but mycorrhizal formation in the silver-berry was not inhibited at any
of the fertilizer rates.
Based on these data, it appears that the fertilizer concentra-
tion required to produce a mycorrhizal, nodulated silver-berry or
buffalo-berry should not exceed 56 mg N L"^.
4.3.2 Effect of Container Volume and Inoculation on Growth of
Silver-berry
Both 12 and 20 week-old seedlings were significantly larger
and heavier when grown in 150 cc containers than when grown in 65 cc
containers (Table 14). Shoot and root weights of inoculated plants
were significantly greater than those of the uninoculated plants, but
this was the case only when plants were grown in the 150 cc
containers. Plants grown in the 65 cc containers demonstrated no
significant response to inoculation.
Almost all the seedlings became nodulated, including those in
the uninoculated treatments, suggesting that there was some cross-
contamination of the Frankia symbiont between treatments. All the
plants in the inoculated treatments became mycorrhizal, but percent
infection was low (20-25%) and variable. None of the uninoculated
plants became mycorrhizal and container size did not seem to influence
mycorrhizal development.
4.3.3 Growth of Silver-berry as Influenced by Soil Temperature and
Symbiont Inoculation
Shoot heights, shoot weights and root weights were, with the
exception of roots in the Glomus aggregatum treatment, significantly
greater at 26®C than at 16®C (Table 15). Root collar diameters were
also greater at 26°C but only for seedlings inoculated with woodland and
silver-berry soil. The largest, heaviest plants with the heaviest root
systems were obtained in the 26®C, woodland soil-inoculated treatment
(shoot ht = 24 cm, shoot wt = 966 mg, root wt = 424 mg). Inoculation with
silver-berry soil also stimulated shoot production but not to the same
Table 14. Effect of container volume and inoculation on shoot and root production by silver-berry.
Plants received 200 mg 15-15-18 fertilizer twice weekly.
46
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Data analyzed by two-way ANOVA. Data for 12 week shoot weights required a LO transformation,
MSB's for 12 week shoot and root data and 20 week shoot and root data are .0059, 948,15, 4287,
and 2264, respectively. Significant interactions were detected in each age group, consequently
Scheffe multiple contrasts were applied to individual means. Values in each column for each age
class followed by the same letter are not significantly different (p < 0.05).
Table 15. Growth of silver-berry as influenced by soil temperature and inoculation with
mycorrhizal and Na-fixing symbionts. Seedlings were grown in a growth chamber for
13 weeks. ^
47
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Data analyzed by a two-way ANOVA. Shoot height, shoot weight, and root weight data
required a LO transformation. MSE's for shoot height, root collar diameter, shoot weight,
and root weight are .00477, .06803, .01097, and .01377, respectively. Differences detec-
ted by Scheffe multiple contrasts for pairwise comparisons. Values for each measurement
followed by the same letter(s) are not significantly different (p = 0.05).
48
degree as inoculation with woodland soil. Inoculation with Glomus
aqqreqatum pot culture soil resulted in seedlings which were signifi-
cantly smaller than the uninoculated controls. Shoot/root ratios
ranged from 1.6 to 2.8 with the biggest, healthiest seedlings attaining
a S/R of 2.5.
Almost all the seedlings in the soil-inoculated treatments
became nodulated, regardless of temperature (Table 16). Some of the
seedlings in the uninoculated and Glomus-inoculated treatments at 26®C
also became nodulated, presumably due to contamination from the soil
treatments, but the weight of nodules produced was minimal. Nodule
weight produced per plant in the soil-inoculated treatments was 5 to 11
times greater at 26®C than at 16®C. Inoculation with woodland soil
resulted in more nodule production per plant than inoculation with
si 1 ver-berry soi 1 .
All of the inoculated seedlings, at both temperatures,
developed mycorrhizae. However, the % mycorrhizal root length in the
soil-inoculated seedlings was significantly greater at 26®C than 16°C.
There was no significant effect of temperature on % mycorrhizal root
length for seedlings inoculated with G. aqqreqatum.
In this experiment, the largest seedlings with the best mycor-
rhizal and nodule development occurred in the 26°C, woodland soil-
inoculated treatment.
4.3.4 Use of Soil, Nodule and Pure Culture Inocula for Introducing
N2-Fixinq Frankia to Containerized Silver-berry
After 18 weeks growth, the tallest (24-27 cm), heaviest
(1.1 - 1.3 g shoot wt) silver-berry seedlings with the biggest
(0.44 - 0.65 g) and most heavily nodulated (170 - 200 mg/plant) root
systems were produced in the treatments inoculated with soil from
beneath wild buf falo-berry, crushed silver-berry nodules or crushed
silver-berry nodules treated with polyvinyl pyrrolidine (Table 17).
Plants inoculated with Frankia ordered from Rhizotec successfully
formed nodules but this was not reflected in shoot heights and shoot
and root weights which were not significantly different from the unino-
culated controls. No nodulation occurred in plants inoculated with
Frankia which had been isolated from buffalo-berry. Treatment of
Table 16, Effect of soil temperature and inoculation on mycorrhizal and nodule
development in silver-berry grown in a growth chamber for 13 weeks. ^
49
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50
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Data analyzed by one-way ANOVA. Shoot and root weight data required SQRT and LO transformations
respectively. MSE's for height, root collar diameter, shoot weight, root weight, nodule number and
nodule weight are 5.96, .1012, 9.864, .02004, 26.54, and 2404, respectively. Differences detected
by Scheff@ multiple contrasts for pairwise comparisons. Values in each column followed by the same
letter(s) are not significantly different, S/R ratios are means ± SD.
51
nodules with polyvinyl pyrrolidine significantly improved nodule forma-
tion and growth. Nodules were also formed on plants inoculated with
soil from beneath silver-berry planted in the RRTAC pad plot, but lack
of stimulation in shoot and root growth suggests the nodules were
ineffective at the time of sampling.
4.3.5 Effect of Inoculation Method and Time on Nodule and Mycor-
rhizal Development of Buffalo-berry
Although inoculation with soil or soil slurry did not signifi-
cantly affect shoot height or branching, shoot and root weights were
significantly greater in the soil mixture treatment than in the soil
slurry or uninoculated treatments (Table 18). Plants in both the soil
mixture and soil slurry treatments developed nodules but nodule
weights/plant were greatest on seedlings in the soil mixture. Only
plants inoculated by mixing woodland soil into the planting medium
became mycorrhizal.
The age (2, 3, 4, 5 weeks) of the seedlings when soil slurry
inoculum was injected into the containers did not have a significant
impact on shoot height, branching, shoot weight, root weight and nodule
development (Table 19). None of the seedlings inoculated with soil
slurry became mycorrhizal, regardless of seedling age.
Based on these data, it appears that mixing soil with a high
symbiont inoculum into the planting medium prior to planting is the
most effective means for inoculating containerized seedlings.
4.4 FIELD TRIAL TO TEST GROWTH RESPONSE OF INOCULATED SILVER-BERRY
AND BUFFALO-BERRY
4.4.1 Pre-Planting Symbiont Status of Inoculated and Uninoculated
Silver-berry and Buffalo-berry
The mycorrhizal and nodulation status of the silver-berry and
buffalo-berry grown in the University of Calgary greenhouse and out-
planted on the University of Calgary reclamation plots adjacent to the
RRTAC soil reconstruction-woody plant experimental area on the Syncrude
lease are presented in Table 20. Both silver-berry and buf falo-berry
were small and underweight compared with the seedlings surveyed from
Table 18. Use of soil and soil slurry for promoting nodulation and
mycorrhizal development in container-grown buffalo-berry, ^
Measurement
Inoculum Source
None
Woodland Soil
Mixture
Woodland Soil
Slurry
Shoot height (cm)
17.9®
22.9®
20.7^
Branches seedling ^
6.1®
9.3®
5.4^
Shoot weight (g)
1.14®
2.06*’
1.21^
Root weight (g)
0.43®
o.ss'’
0.34^
S/R ratio
2. 7+0. 4
3.411.00
3.9±0.8
Nodule weight (g wet plant^
) 0
0.25+0.09
0,095±0,07
Mycorrhizal root length (%)
0
25+20
0
^ Data analyzed by one-way
ANOVA and
differences detected
by Scheffe
multiple contrasts for
pairwise
comparisons. Shoot
height data
required a LO transformation. MSE's are .0064, 14.99, ,1248, and
.0273 for shoot height, branches, shoot weight, and root weight data,
respectively. Values in each row followed by the same letter(s) are
not significantly different (p =0.05). S/R, nodule weight, and mycor-
rhizal values are means ± SO.
many of the commercial greenhouses (Table 8). Although 100% and 90% of
the inoculated shrubs became mycorrhizal and nodulated, respectively,
this was not evident in the shoot and root weights which were almost
identical in the inoculated and uninoculated treatments. None of the
uninoculated plants developed mycorrhizae, but nodules did develop on
20% of the silver-berry in the uninoculated treatments, presumably a
result of contamination from the inoculated treatments.
4.4.2 Field Performance of Silver-berry After One and Two Growing
Seasons
Percent survival of the uninoculated silver-berry after the
first winter in the field was substantially better than was the case
for the inoculated shrubs (Table 21). More seedlings survived in
Plot 1 than in Plot 2.
53
Table 19. Nodule and mycorrhizal development in container-grown buffalo-
berry inoculated with soil slurry at various ages.^
Seedling Age When
Inoculated
(Weeks)
Measurement
2
3
4
5
Shoot height (cm)
20.7®
21.7®
20.2®
21 .6®
Branches seedling ^
5.4®
4.7®
4.2®
6.3®
Shoot weight (g)
1.21®
1.04®
1.14®
1.19®
Root weight (g)
0.34®
0.32®
0.36®
0.37®
S/R ratio
3.910.8
3.310.4
3.611 .4
3.410.1
-1
Nodule weight (g wet plant )
.095®
.076®
.087®
.072®
Mycorrhizal root length (%)
0
0
0
0
^ Data analyzed by one-way ANOVA and differences detected by Scheffe
multiple contrasts for pairwise comparisons. Values in each row fol-
lowed by the same letter(s) are not significantly different (p = 0.05).
S/R ratios are means ± SO.
With the exception of mycorrhizal root length, which was sig-
nificantly greater in plants from Plot 2 than plants from Plot 1, there
were no significant differences in plant performance and symbiont deve-
lopment between the two plot treatments.
After one growing season, shoots of the inoculated shrubs were
1.4 - 2.3 times taller than those of the uninoculated shrubs, while shoot
weights were 3-7 times greater in the inoculated treatment than in the
uninoculated treatment. The difference in shoot production by inoculated
and uninoculated shrubs was extended into the second growing season when
inoculated silver-berry were 1.6 times taller and 3.4 times heavier than
their uninoculated counterparts (Tables 21, 22; Figures 3, 4). Although
the root weights are probably a gross underestimate because of the
difficulty in excavating entire root systems, they, nevertheless, were
significantly greater in the inoculated treatment after both the
Table 20. Pre-planting mycorrhizal and nodule status of silver-berry and buffalo-berry outplanted
in the University of Calgary soil reconstruction plots (with and without surficial
clay). Data are means (n=10) ± SO.
54
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55
Table 21. Plant growth, nodulation, and vesicular-arbuscular mycor-
rhizal development of inoculated and uninoculated silver-
berry outplanted for 1 year in the University of Calgary soil
reconstruction plots at the Syncrude site. Data are means
(n=10) ± SD.i
Measurement
Inoculated
(V-)
Plot 1
(tailings +
peat)
Plot 2
(tailings,
peat + clay)
Row
X
Survival(%)
-H
40
16
-
67
47
Shoot height (cm)
+
23 ± 7
20 ± 8
21 .s'’
-
10 ± 6
14 ± 4
12.03
Column
X
16.5a
17.03
Shoot weight (g dry
5.35 ± 3.96
3.26 ± 2.54
4.31^
planf^)
_
-
0.70 ± 0.83
1 .01 ± 0.67
0.86
Column
X
3.03^
2.143
Root weight (g dry
+
2.46 ± 1 .77
1 .49 ± 0.96
1 .98'’
plant ^)
-
0.45 ± 0.45
0.64 ± 0.46
0.55a
Column
X
1 .46^
1 .073
Nodules (no.
+
77 ± 79
55 ± 44
os')
planf^)
-
13 ± 6
20 ± 9
16.5a
Column
X
453
37.53
Nodules (g wet
+
1.59 ± 1.33
0.89 ± 0.70
1.24'’
planf^)
_
-
0.11 ± 0.08
0.30 ± 0.25
0.21a
Column
X
0.85^
0.603
Mycorrhizal root
+
56 ± 15
58 ± 14
57'’
length (%) (n=5)
-
2 ± 2
22 ± 24
12a
Column
X
29a
40b
Shoot N (%)
+
2.69 ± 0.21
2.63 ± 0.46
2.66'’
_
-
2.56 ± 0.37
2.12 ± 0.54
2.34a
Column
X
2.63^
2.383
Shoot P (%)
+
0.15 ± 0.02
0.14 ± 0.04
0.145a
_
-
0.13 ± 0.03
0.12 ± 0.06
0.125a
Column
X
0.143
0.133
^ Data analyzed by two-way ANOVA and differences detected by Scheffe
multiple contrasts for pairwise comparisons. Shoot weight, root
weight, nodule number and nodule weight data were L0(x + l)
transformed. MSE's are 42.45, 0.0463, 0.0238, 0.1220, 0.0174, 242.37,
0.1701 and 0.0014 for each measurement in sequential order. For each
measurement row means or column means followed by the same letter are
not significantly different (p = 0.05).
56
Table 22. Plant growth, nodulation and vesicular-arbuscular mycorrhizal
development in inoculated and uninoculated silver-berry
outplanted for 2 years in the University of Calgary soil
reconstruction plots at the Syncrude site. Data are means
(n = 15) ± SO.i
Treatment
Measurement Uninoculated Inoculated
Shoot height (cm)
34.5 ± 163
56
±
14b
Shoot weight
(g dry planf^)
10.3 ± 10.83
35.4
+
21 .7b
Root weight
(g dry planf^)
2.0 ± 1.43
5.8
+
3.5b
Root collar diameter (mm)
5.9 ± 243
10.8
+
33b
Branches
(number plant ~^)
15 + 163
39
+
21b
Nodule weight
(g wet planf^)
0.7 + 0.63
2.1
+
1.3b
Mycorrhizal roots (%)
43 ± 293
67
+
14b
^ Data analyzed by a two sample T test. Shoot weight, root weight
and nodule weight data required LO transformations . Values in each
row followed by the same letter are not significantly different
(p = 0.05).
100
57
C\l
cr
<
Ld
(N
I
o ^
CL Cd
^ <
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_i
CL
oooooooooo
OTOOr^COlD'^fOCMT-
(no) 1H0I3H lOOHS
Figure 3 Shoot heights (± SO) of inoculated and uninoculated silver-berry outplanted in
reconstructed soil for two growing seasons.
58
CNJ
Dr:
<
LU
(o) IHOGM iOOHS
Figure 4 Shoot weights (± SD) of inoculated and uninoculated silver-berry outplanted in
reconstructed soil for two growing seasons.
59
first and second growing seasons. Branching was also significantly
better in the inoculated shrubs than in the uninoculated shrubs.
The significant growth response of the inoculated shrubs
aboveground was reflected in the symbiont status on the roots below-
ground. The inoculated shrubs had significantly more nodules per plant
and weighed an average of 3 (Plot 2) to 14 (Plot 1) times more than
those which developed on the uninoculated shrubs from indigenous soil
inoculum (Table 21, Figure 5). Even after two growing seasons the
weight of nodules produced by the inoculated plants was 3 times greater
than that produced by the uninoculated shrubs. The length of root
occupied by mycorrhizal fungi was also significantly higher in the
inoculated shrubs than in the uninoculated shrubs after both growing
seasons (Tables 21, 22; Figure 6). After the first growing season
mycorrhizal development was significantly better in the tailings sand
amended with peat and clay (Plot 2) than in the sand amended only with
peat (Plot 1) suggesting that the type of emendation influenced mycor-
rhizal inoculum potential .
After one growing season, the nitrogen concentrations in the
silver-berry foliage were significantly higher for the inoculated
shrubs than the uninoculated shrubs; however, no differences were
detected in foliage P between the inoculated and uninoculated treat-
ments (Table 21 ) .
4.4.3 Field Performance of Buf falo-berrv After One and Two Growing
Seasons
As was the case for the si 1 ver-berry, survival of buffalo
berry after the first winter was significantly less for the inoculated
shrubs than the uninoculated shrubs with a greater majority of the
seedlings surviving in Plot 1 than Plot 2 (Table 23).
The pattern of response of surviving buf falo-berry to inocula-
tion was also very similar to that of silver-berry with inoculated
buffalo-berry being taller (1.5 times) and having heavier shoots
(3.6 - 4.5 times), roots (2.1 - 2.6 times), and nodules (1.6 - 1.8
times) and more mycorrhizal colonization (2.0 - 2.7 times) than
60
c
(iNvid/o) s3inaoN
Figure 5 Nodule development in inoculated and uninoculated silver-berry outplanted in
reconstructed soil for two growing seasons. Data are means ± SO.
100
61
(N
0:1
<
UJ
CSj
h-
O
_i
□_
I —
o
_J
Q_
CK
<
l_U
oooooooooo
ocot^coin^t-ioiNT-
SiOOd IVZIHddOOAH ^
Figure 6 Mycorrhizal development in inoculated and uninoculated silver-berry outplanted
in reconstructed soil for two growing seasons. Data are means ± SO.
62
Table 23. Plant growth, nodulation, and vesicular-arbuscular mycor-
rhizal development of inoculated and uninoculated buffalo-
berry outplanted for 1 year in the University of Calgary soil
reconstruction plots at the Syncrude site. Data are means
(n=10) ± SO.i
Measurement
Inoculated
(+/-)
Plot 1
(tailings +
peat)
Plot 2
(tai lings ,
peat -1- clay)
Row
X
Survival(%)
+
48
40
-
75
53
Shoot height (cm)
10 ± 6
14 ± 6
12.0‘>
-
6.5 ± 2
10 ± 3
8.3a
Column
X
8.25^
12.0b
Shoot weight
0.67 ± 0.72
1 ,04 ± 0.91
0.86*5
(g dry planf^)
—
-
0.15 ± 0,07
0.29 ± 0.21
0.22a
Column
X
0.41^
0.67b
Root weight
+
0.30 ± 0.21
0.47 ± 0.30
0.39*5
(g dry plant"^)
_
-
0.14 ± 0.06
0.18 ± 0,07
0.16®
Column
X
0.223
0.33b
Nodules
22 ± 21
34 ± 32
28.03
(no. plant~^)
-
12 ± 8
21 ± 13
16.53
Column
X
173
27.53
Nodules
+
0.26 ± 0.26
0.41 ± 0.36
0.34*5
(g wet plant"^)
-
0.03 ± 0.03
0.08 ± 0.07
0.063
Column
X
0.153
0.25b
Mycorrhizal root
+
64 ± 21
57 ± 28
61*5
length (%) (n=5)
_
-
24 ± 17
28 ± 7
263
Column
X
443
433
^ Data analyzed by two-way ANOVA and differences detected by Scheffe
multiple contrasts for pairwise comparisons. Shoot height, shoot
weight, root weight, nodule number and nodule weight data were LN
transformed. MSE's are 0.171, 0.657, 0,331, 0.933, 0.885 and 383.5
for each measurement in sequential order. Values for row or column
means for each measurement followed by the same letter are not
significantly different (p = 0.05).
63
uninoculated buf falo-berry after one growing season (Table 23,
Figures 7 to 10). This pattern was carried over into the second
growing season when inoculated shrubs were still significantly taller,
heavier and more heavily nodulated and mycorrhizal than the
uninoculated shrubs (Table 24, Figures 7 to 10).
In contrast to the si 1 ver-berry, where very few significant
differences were detected between plot treatments, many of the measure-
ments made on buffalo-berry were significantly affected by the type of
amendment applied to the tailings sand. After one growing season,
shoot heights and weights, root weights and nodule weights per plant
were significantly greater for seedlings planted in the tailings sand
amended with peat and clay (Plot 2) than for seedlings planted in
tailings sand amended with peat only (Plot 1) (Table 23). Mycorrhizal
infection was not affected.
4.4.4 Relationships Amongst Various Parameters Measured on
Inoculated and Uninoculated Silver-berry and Buffalo-berry
After One and Two Growing Seasons
Pearson product moment correlation coefficients were calcu-
lated to determine if any strong relationships existed between plant
performance and the mycorrhizal/nodulation status of the roots. After
one growing season there were high correlations between silver-berry
shoot weights and nodule numbers (coefficient = 0.947) and shoot
weights and nodule weights (coefficient = 0.912) (Table 25). Shoot
productivity appeared to be more closely related to nodule status than
mycorrhizal status (coeff icient = 0.628) . Also, percent mycorrhizal
root length was not closely correlated with nodule number and weight.
Neither shoot productivity nor symbiont development exhibited strong
correlations with foliage nutrient (N and P) status. Correlation
coefficients calculated for silver-berry data collected after the
second growing season followed the same pattern as that observed for
the first year data with strong correlations still present between
nodule weights and shoot and root weights, but not between % mycor-
rhizal roots and shoot and root weights (Table 26).
A linear regression of the silver-berry shoot weights vs
nodule weights for the first year data is presented in Figure 11, and
64
(FNO) iHOGH lOOHS
Figure 7 Shoot heights (± SD) of inoculated and uninoculated buffalo-berry outplanted
in reconstructed soil for two growing seasons.
65
OOOCD'^CNOOOCDtJ-CNIO
CM •«- -r- 1- t- r-
C\|
(H
<
LJ
cr
<
UJ
(O) 1H0I3M lOOHS
Figure 8 Shoot weights (± SO) of inoculated and uninoculated buffalo-berry outplanted
in reconstructed soil for two growing seasons.
□ UNINOCULATED
W INOCULATED
66
' " I
ro
— — , —
CM ^ O
(iNvid/o) s3inaoN
0 •
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s_
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0^
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1 c
O to
I— Oi
H3 E
u- o>
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ja (O
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C C
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100
67
oooooooooo
oioDh'Coin'^rocM^
CM
cr
<
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q:
<
LU
SiOOd nVZIHddOOAn %
Figure 10 Mycorrhizal development in inoculated and uninoculated buffalo-berry outplanted
in reconstructed soil for two growing seasons. Data are means ± SD.
68
Table 24. Plant growth, nodulation and vesicular-arbuscular mycorrhizal
development in inoculated and uninoculated buffalo-berry
outplanted for 2 years in the University of Calgary soil
reconstruction plots at the Syncrude site. Data are means
(n = 7) ± SO.i
Treatment
Measurement Uninoculated Inoculated
Shoot height (cm)
Shoot weight
(g dry plant"^)
Root weight
(g dry planf^)
Root collar diameter (mm)
Branches
(number plant ~^)
Nodule weight
(g wet planf^)
Mycorrhizal roots (%)
13.4 ± 6.73
1.8 ± 2.2^
0.6 ± 0.5a
3.2 ± 0.9a
10 ± 9.9a
0.3 ± 0.5a
46 ± 33a
25.5 ± 9.9b
7.5 + 8.0b
2.0 ± 1.5b
5.7 + 2.0b
23 ± 263
1 .0 ± 1 .13
83 + 9.5b
1 Data analyzed by a two sample T-test. Shoot weight, root weight
and nodule weight data were LO transformed. Values in each row
followed by the same letter are not significantly different (p = 0.05).
Table 25. Pearson product moment correlation coefficients for various parameters measured on
silver-berry grown for 1 year in the University of Calgary soil reconstruction plots
at the Syncrude site.
69
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Table 26. Pearson product moment correlation coefficients for various parameters measured on
silver-berry grown for 2 years in the University of Calgary soil reconstruction plots
at the Syncrude site.
70
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71
Figure 11 Linear regression of shoot weights versus nodule weights for
one year-old silverberry.
72
illustrates the very close relationship (r^ = 0.89) between nodu-
lation and shoot performance. This relationship was continued into the
second growing season when the regression equation was LN SHOOT WEIGHT
(MG) = 1.92 + 1.11 (LN NODULE WEIGHT (MG)) and the r^ was 0.91.
Correlation coefficients for the buffalo-berry parameters
followed a very similar pattern to those calculated for the
silver-berry data. Relationships between shoot weight or root weight
and nodule weight were high (0.82 and 0.72 for shoot and root vs
nodules, respectively) after the first growing season (Table 27) and
very high during the second growing season (0.96 and 0.95 for shoot and
root vs nodule weights, respectively) (Table 28). As was the case for
the si 1 ver-berry, the correlation between % mycorrhizal root length and
shoot weight was low (0.58 and 0.31 for years 1 and 2, respectively) as
was the correlation between % mycorrhizal root length and nodule
weights (0.57 and 0.26 for years 1 and 2, respectively). The close
relationship between nodulation and shoot production of buffalo-berry
during the first growing season is exemplified in the linear regression
presented in Figure 12. The relationship during the second growing
season was even stronger with the regression equation being LN SHOOT
WEIGHT (HG) = 1.97 + 0.996 (LN NODULE WEIGHT (MG)) and the being
0.93.
Table 27. Pearson product moment correlation coefficients for various parameters
measured on buf falo-berry grown for 1 year in the University of Calgary soil
reconstruction plots at the Syncrude site.
73
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Table 28. Pearson product moment correlation coefficients for various parameters measured on
buf falo-berry grown for 2 years in the University of Calgary soil reconstruction plots
at the Syncrude site.
74
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BUFFALO-BERRY
Shoot weight = 2.531 + 0.718 (Nodule weight )
= 0.88
. I t \ I \ ^ \ ^ L
123456789 10
LN NODULE WEIGHT (MG)
Linear regression of shoot weights versus nodule weights
for one year-old buffalo-berry.
76
5. DISCUSSION
5.1 MYCORRHIZAL STATUS OF WOODY SHRUBS
Members of the Rosaceae are considered to be strictly
vesicular-arbuscular mycorrhizal (Trappe, 1981); thus it was to be
expected that both cinquefoil and saskatoon-berry would bear
VAM-colonized roots. Silver-berry and buf falo-berry were also found to
be exclusively VA mycorrhizal contrary to observations made by Rose
(1980) that buf falo-berry was both VA and ectomycorrhi zal . All of the
plants sampled were mycorrhizal emphasizing the fact that, in the wild,
the mycorrhizal condition is the norm. The high level of VAM coloni-
zation in some of the plants is indicative of a potentially high
dependence by woody shrubs on the VAM symbiont.
5.2 JUSTIFICATION FOR INOCULATION OF CONTAINERIZED SILVER-BERRY
AND BUFFALO-BERRY
5.2.1 The Dependency of Silver-berry and Buf falo-berry on their
Mycorrhizal and N2-fixing Symbionts
Mycorrhizal dependency is expressed as the dry weight of a
mycorrhizal plant as a percentage of the dry weight of a nonmycorrhi zal
plant at a given level of soil fertility (Menge et al., 1982). Under
the low nutrient regimes used in this experiment (plants were not
fertilized and previous experiments revealed that N and P contents of
muskeg peat were so low that plants responded strongly to the addition
of NPK fertilizer) the mycorrhizal dependencies of silver-berry and
buffalo-berry were 368% and 727%, respectively.
Soil collected from beneath buf falo-berry shrubs was used as
inoculum in this study; consequently, the relative contributions of the
N2-fixing Frankia and the VA mycorrhizal fungus in stimulating plant
growth could not be ascertained. However, based on the numerous studies
conducted on the Rhi zobi um-VAM-1 equme symbiosis, it appears that the
VAM relationship is necessary to satisfy the high P demand of the nodu-
lation and N2-fixation processes (Barea and Azcon-Agui lar, 1983).
The high dependence of buffalo-berry and silver-berry on their
symbionts suggests that early colonization by Frankia and the VAM fungi
77
is essential to ensure plant success in both natural and disturbed soil
systems. In regards to the revegetation of oil sands tailings, it is
apparent that the growth performance of buf falo-berry and silver-berry
would be significantly better if the shrubs were mycorrhizal and nodu-
lated prior to outplanting or if they were outplanted into soil with a
high inoculum potential.
5,2.2 Levels of VA Mycorrhizal Inoculum in Various Soils in the Oil
Sands Region and Effects of Stockpiling on VAM Infectivitv
The VA mycorrhizal inoculum potential in a soil is dependent
to a large extent on the mycorrhizal host plants in a specific site and
on the degree of disturbance of the soil. Sites dominated by
ectomycorrhizal conifers or ericaceous species (ericoid mycorrhizae)
would be expected to lack VAM inoculum due to the lack of VA hosts.
For example, Kovacic et al. (1984) found that VAM spores and
mycorrhizal colonization were extremely low in a Ponderosa pine forest
in Colorado compared with a similar forest which had been killed and
colonized by VA herbs and grasses. Severe soil disturbance, such as
that which occurs during mining, has been shown to significantly reduce
VAM inoculum potential (Allen and Allen, 1980; Moorman and Reeves,
1979; Mott and Zuberer, 1987; Zak and Parkinson, 1982). Loss of VAM
inoculum potential or reduced rates of mycorrhizal colonization as a
result of topsoil storage during surface mining has also been reported
(Gould and Liberta, 1981; Miller et al, 1984; Rives et al, 1980; Visser
et al, 1984; Warner, 1983). Lack of VAM inoculum in a minespoil may
necessitate emendation with soil having a high inoculum potential in
order to ensure establishment and growth of outplanted seedlings.
Both undisturbed and stockpiled muskeg on the Syncrude lease
contained negligible quantities of VAM inoculum. This is not surpri-
sing since the vegetation in undisturbed muskeg is often dominated by
non-VA hosts such as tamarack, black spruce and ericaceous plants.
Both tamarack and black spruce are hosts for ectomycorrhizae while the
Ericales form arbutoid or ericoid-type mycorrhizae. The fungi involved
in the formation of ectomycorrhizae and arbutoid or ericoid-type mycor-
rhizae are taxonomical ly very different from those which form VA mycor-
rhizae.
78
c
Although many of the plant species in the undisturbed muskeg
were non-VA hosts, VA inoculum did occur in areas where VA hosts
(grasses) were present. However, compared with mixed woodland soil
where 64% mycorrhizal colonization was attained after 12 weeks growth,
the infectivity potential of the peat, even when originally occupied by
a VA host, was poor. Interestingly, VA inoculum, albeit low, was also
present in the 50-100 cm deep undisturbed peat which was well below the
rooting zone for most species. In a survey of VA inoculum in a peat
deposit formed under a white spruce stand, Danielson, Zak and Parkinson
(1984) also reported viable VA inoculum down to a depth of 100 cm.
No impact of stockpiling on VAM infectivity was recorded due
to the lack of inoculum in the peat prior to stockpiling. Mycorrhizal
infectivity was low in the stockpiled peat, but increased by 10 to 12%
when the stockpile was vegetated with grass for 6 years indicating a
very slow rate of increase in inoculum levels, even in the presence of
VAM hosts.
The importance of the mycorrhizal symbiosis in stimulating
plant growth in the peat stockpiled for 8 months was evidenced in
greater shoot and root production by slender wheatgrass when a VAM
fungus was artificially introduced to the peat. Some of the P defi-
ciency symptoms (deep green to purple leaves) noted for plants grown in
the uninoculated peat appeared to be partially alleviated by VAM infec-
tion. Although mycorrhizal infection stimulated plant production,
10 week old plants were still extremely small. Low rates of fertiliza-
tion (15 mg N L~^) substantially improved shoot production, but in
the presence of fertilizer, VAM colonization did not have a significant
effect on shoot weights. Numerous studies have demonstrated that,
under conditions where nutrients are not limiting to non-mycorrhi zal
plants, growth stimulation due to mycorrhizal inoculation is lost
(Abbott and Robson, 1984). This becomes particularly evident when
comparing phosphate response curves (i.e. phosphorus applied versus
dry weight of plant produced) for mycorrhizal and non-mycorrhizal
plants (Abbott and Robson, 1984).
It appears, therefore, that undisturbed muskeg peat and stock-
piled peat used in the reclamation of the Syncrude tailings dykes have
neglible quantities of VAM inoculum while soil in mixed aspen white
79
spruce woodlands exhibit high infectivity. The lack of VAM inoculum in
stockpiled peat and the potential benefits derived by the plant if
inoculum is introduced into the growing medium suggests that, when
revegetating tailings sand with highly symbiont-dependent shrubs such
as buf falo-berry and si 1 ver-berry, attempts should be made to ensure
that containerized seedlings are inoculated with their symbionts prior
to outplanting. Alternatively, the tailings could be amended with
inoculum-rich soil, possibly mixed woodland soil. The former approach
would be more economically feasible that the second.
5.2.3 Mvcorrhizal and Nodule Status of Containerized Shrubs Raised
in Various Commercial Nurseries in Alberta and British Columbia
The practice of raising seedlings in containers or nursery
beds offers an opportunity to manage both mycorrhizal and N2-fixing
symbionts by allowing artificial inoculation with selected symbionts
prior to outplanting. Mycorrhizal inoculation of sterilized nursery
soil has been demonstrated to significantly improve the growth of a
range of woody plant species including fruit trees (particularly
citrus), timber trees and ornamentals (Powell, 1984). As a result of
this, mycorrhizal inoculation of nursery-grown citrus is now a common
practice in the U.S. (Powell, 1984). The growth response of various
actinorhizal plants, especially alder, to Frankia inoculation has
resulted in large scale inoculation of these plants in Quebec (Perinet
et al., 1985).
While in the greenhouse, containerized seedlings can become
colonized by either the VA-mycorrhi zal fungi or N2-fixing bacteria
residing in the planting mixture (unless it is sterilized), the atmos-
phere or the water. However, a survey of woody shrubs grown in Alberta
and B.C. nurseries revealed that containerized seedlings seldom become
mycorrhizal or nodulated during the first year of growth. If the seed-
lings are older than one year and have spent some time in the shade-
house or outdoors they may become mycorrhizal or nodulated but not
necessarily so. The slow rates of mycorrhi zation and nodulation in the
nurseries may be a result of a combination of factors including high
fertilizer regimes (which inhibit symbiont development), a lack of
symbiont inoculum and inefficient dispersal of inoculum from adjacent
80
inoculum sources. Regardless of the reasons for poor symbiont develop-
ment in the nurseries, it can be concluded that containerized shrubs
outplanted when less than one year old (which is the situation for most
greenhouse operations) will be symbiont-free and, therefore, completely
dependent on the inoculum present in the reconstructed soil in which
they are planted. The low inoculum potential of reconstructed soil on
the tailings sands dykes combined with the high dependency of woody
plants on their symbionts suggests that woody species used in the
revegetation of the oil sands, and possibly other disturbed areas,
would benefit greatly from artificial inoculation.
Silver-berry, buf falo-berry and silver buffalo-berry surveyed
in this study were often underweight and chlorotic - a condition which
may have been partially due to the poor development of N2-fixing
nodules.
5.2.4 Mvcorrhization and Nodulation Rates of Buffalo-berry and
Silver-berry in the Greenhouse and the Field
The rapidity with which an uninoculated actinorhizal shrub
seedling becomes mycorrhizal and nodulated after outplanting will
determine to a large degree the benefits it will gain from the symbio-
sis during the first growing season. A short growing season and low
symbiont inoculum potential are two factors which could reduce rates of
infection to such an extent that seedlings would not benefit from their
symbionts until the second growing season, if they survive the winter.
Artificial inoculation would ensure that a seedling derived maximum
benefit from its symbionts immediately after outplanting.
Buffalo-berry raised in amended tailings sand exhibited signi-
ficantly slower rates of mycorrhi zation and a lower degree of mycor-
rhizal colonization than did seedlings grown in mixed woodland soil,
presumably a result of lower inoculum levels in the tailings sand.
Under ideal conditions in the greenhouse, plants in the amended
tailings sand did not become mycorrhizal or obviously nodulated until
eight weeks after planting. Since rates of colonization would be
expected to be much slower in the field than in the greenhouse and
since the growing season in the oil sands region is short, it is
doubtful that containerized shrubs would gain much from the symbiosis
81
during the first growing season after outplanting in reconstructed soil
unless they were artificially inoculated.
The relatively rapid mycorrhi zation of silver-berry seedlings
outplanted on the Suncor dyke in June can be explained by the predomi-
nance of VAM hosts which were no doubt instrumental in raising the
level of VAM inoculum in the reconstructed soil. However, compared with
seedlings outplanted in a mixed woodland, seedlings on the dyke
exhibited very poor nodulation over the growing season. This may have
been due to a lack of Frankia inoculum in the reconstructed soil or due
to very poor root growth out of the planting plug. The lack of both
shoot and root growth during the term of the study is difficult to
explain, and should, perhaps be investigated further. The high rate of
mortality of seedlings outplanted in the Suncor plot revegetated in
1974 may have been a result of intense competition by sweet clover.
Results from the field trial supported those obtained in the greenhouse
and form a strong basis for considering artificial inoculation of
containerized seedlings.
5.2.5 Basis for Artificial Inoculation of Containerized Buffalo-
berry and Silver-berry for Outplanting on Amended Oil Sands
Tailings
It appears that artificial inoculation of containerized
buffalo-berry and silver-berry is justified for the following reasons:
1. Silver-berry and buf falo-berry are heavily dependent on
their symbionts as evidenced by the significant growth
response when they are inoculated with Frankia and VAM
fungi .
2. Mycorrhizal inoculum is lacking in the reconstructed soil
causing rates of nodulation and mycorrhi zation to be so
slow that containerized seedlings would benefit tremen-
dously if armed with their symbionts when outplanted.
3. Containerized seedlings seldom become mycorrhizal or
nodulated while in the nursery, and are, therefore,
virtually symbiont-free if outplanted within a year of
being seeded.
82
5.3 DEVELOPMENT OF A GROWING REGIME FOR GREENHOUSE PRODUCTION OF
MYCORRHIZAL, NODULATED SILVER-BERRY AND BUFFALO-BERRY
5.3.1 Fertilization Regimes
Many factors, including the type of growing medium, water/
aeration conditions, pH, light intensity and photoperiod, temperature,
container size and pesticide or herbicide applications can signifi-
cantly influence the infectivity of symbiont inoculum (Menge, 1984).
However, the factor which appears to have the greatest influence is the
fertilization regime. It is now widely accepted that high available P
levels in the soil can severely inhibit mycorrhizal infection due to an
increase in the P content of the host tissue (Cooper, 1984). High
concentrations of N fertilizer can also reduce mycorrhizal formation
particularly if the N is in the ammonium form (Cooper, 1984; Menge,
1984). Consequently, in order to produce mycorrhizal, nodulated seed-
lings of suitable size and quality for outplanting, it is necessary to
develop fertilization regimes which will maximize both plant production
and mycorrhizal development.
Fertilizer studies on silver-berry and buffalo-berry grown in
woodland soil and reconstructed soil, revealed that for both species of
shrub, fertilizer applications in excess of 200 mg L 28-14-14 (i.e.
56 mg N, 12 mg P, 23 mg K) severely reduced mycorrhizal and nodule
development. Plant response to fertilization was greater in the recon-
structed soil than in the woodland soil, presumably because the recon-
structed soil was more nutrient -poor and lacked the symbiont inoculum
required to compensate for the low N and P levels. Nodulation and
mycorrhization were significantly less in the reconstructed soil than
in the woodland soil due to lower inoculum levels in the reconstructed
soi 1 .
High N concentrations in the soil inhibit nodulation (Bond
et al., 1954; MacConnell and Bond, 1957) whereas high P concentrations
in the plant inhibit mycorrhization (Cooper, 1984). Mycorrhization did
not appear to be as sensitive to the concentrations of NHa-N and P
used in this study as was nodulation. At the higher fertilizer regime
the dependence of the shrubs on their symbionts was lost due to greater
availability of nutrients in the soil solution.
83
Under the conditions set forth in this study, fertilization at
a rate of 200 mg L ^ 28-14-14 for 20 weeks produced a seedling whose
shoot weight was very similar to that produced at the 400 mg rate, but
whose root system exhibited wel 1 -developed mycorrhizae and nodules.
5.3.2 Container Volume
As expected, plant performance in the 150 cc containers was
much superior to that in the 65 cc containers. That inoculated
silver-berry grown in 150 cc containers exhibited a symbiont growth
response whereas inoculated seedlings in 65 cc containers did not, is
probably related to root density and the volume of soil available for
exploitation by the symbionts. Baath and Hayman (1984) observed that
the mycorrhizal growth response of onions was highly dependent on
container size and plant density in each container. As the soil volume
was reduced there was a concomitant decrease in mycorrhizal growth
response. Danielson, Griffiths and Parkinson (1984) suggested that the
high root density which may develop in small containers could reduce
the effectiveness of the mycorrhizae, since the roots themselves would
efficiently exploit the soil for nutrients with little or no dependence
on the fungal mycelium. In fact, in situations such as this and where
P availability is not limiting to growth, a growth depression may occur
in the presence of the mycorrhizal fungi as the host and the fungus
compete for plant-produced C (Buwalda and Goh, 1982). Therefore, it
appears that container size and the degree to which a particular plant
species can exploit the available soil volume are important factors to
consider when producing mycorrhizal, nodulated seedlings for commercial
purposes.
It is interesting to note that after 20 weeks growth almost
all the seedlings were nodulated including those planted in the auto-
claved planting medium. The nodulation of silverberry in the uninocu-
lated (autoclaved) treatment suggests that Frankia is readily dispersed
and may have been introduced by insects, particularly dipteran larvae,
which were observed in the soil during the dismantling of the
experiment.
84
c
5.3.3 Temperature
Temperature can have significant effects on symbiont develop-
ment and alter plant growth response to the symbiosis. Maximum mycor-
rhizal colonization appears to occur at the point of optimum plant
growth which for onion, soybean and cotton falls in a temperature range
of 21 to 30®C (Furlan and Fortin, 1973; Pugh et al., 1981; Schenck and
Smith, 1982; Smith and Roncadori , 1986). Below 20®C, mycorrhizal deve-
lopment and plant response appears to be suppressed. Similarly, infection
and development by Frankia has been observed to be delayed at tempera-
tures below 20°C with nitrogen fixation being totally inhibited at
15®C (Reddell et al., 1985). The optimum temperature for nodulation
and growth of Casuarina, an actinorhizal plant found in warm temperate to
tropical climates, falls in the range of 25°C to 30®C (Reddell
et al., 1985).
These observations are in agreement with those obtained in
this study where seedlings inoculated with woodland soil exhibited
better mycorrhization and nodulation and a greater growth response at
26®C than at 16°C. In the Glomus aggregatum inoculated treatment,
however, shoot weights were greater at 26°C than at 16®C but mycorrhi-
zation was not affected by temperature. This aggressive fungus caused a
growth depression presumably due to an excessive drain of host
photosynthate by the symbiont (Cooper, 1984).
5.3.4 Frankia Inoculum Trials
The preferred method for inoculating actinorhizal shrubs with
Frankia has been the application of a liguid suspension of Frankia pure
culture using either spraying or injecting technigues (Burgess et al.,
1986; Fortin et al.,1983; Stowers and Smith, 1985; Vogel and Dawson,
1985). Perinet et al. (1985) compared crushed nodule and pure culture
inoculum on alder seedlings and found that the use of nodule homoge-
nates resulted in variable nodulation which was not reproducible. This
was not the case in this study where inoculation of silver-berry with
wild buffalo-berry soil, crushed nodules or polyvinyl pyrrolidine-
treated nodules resulted in the biggest seedlings with the most heavily
nodulated root systems. Seedlings inoculated with Frankia pure culture
obtained from Rhizotec Labs in Quebec became heavily nodulated but this
85
was not manifested in improved plant growth. A slower rate of nodula-
tion and a delay in the N2-fixation process in this treatment may
explain the lack of a growth response. It is possible a positive
growth response would have occurred if the experiment had been
extended. No nodule formation was evident on silver-berry inoculated
with a strain of Frankia isolated from buf falo-berry . This suggests
that the inoculum did not survive the inoculation treatment or that
buf falo-berry Frankia may not be compatible with si 1 ver-berry .
Treatment of nodules with polyvinly pyrrolidine to prevent oxidation of
phenols greatly improved the effectivity of the Frankia and is strongly
recommended if inoculating with crushed nodule homogenate.
The most practical source of Frankia inoculum appears to be
forest floor soil removed from beneath wild buffalo-berry. This inocu-
lum was much more effective if mixed into the planting mixture than if
applied as a slurry, presumably as a result of better distribution of
the inoculum. The time at which the seedlings were inoculated did not
appear to be important in determining the rate and degree of nodulation
suggesting that inoculum can be introduced either before or shortly
after planting, whichever is most convenient. The lack of mycorrhizal
development in the slurry treatments is difficult to explain, but may
have been due to a reduction in mycorrhizal infectivity caused by
vigorous stirring (10,000 rpm) during slurry preparation.
5.3.5 Growing Regimes for Greenhouse Production of Mycorrhizal,
Nodulated Silver-berry and Buffalo-berry
Based on the foregoing results and discussion, growing regimes
for the greenhouse production of mycorrhizal, nodulated silver-berry
and buf falo-berry were formulated. These are presented in Tables 29 and
30. It should be kept in mind that the final heights and weights of
the seedlings are dependent to a large degree on the use of highly
infective and effective symbiont inoculum.
5.4 FIELD TRIAL TO TEST GROWTH RESPONSE OF INOCULATED SILVER-BERRY
AND BUFFALO-BERRY
At the time of outplanting, the size of the silver-berry
shrubs compared favorably with those outplanted on the RRTAC plots
86
c
Table 29. Growing regime for greenhouse production of mycorrhizal,
nodulated si 1 ver-berry .
Planting Time:
March, April
Inoculum:
Silver-berry field or pot culture soil with
high inoculant load; crushed nodules for
N2-fixinq Frankia
Inoculum Quantity/Container:
10-15% Inoculum soil/planting mixture (V/V);
inoculum mixed into planting mixture
Planting Mixture:
1/1 (V/V) Peat/Vermiculite
Container Volume:
150 cc
Grower Fertilizer:
200 mg 28-14-14 L~^ applied twice weekly or
56 mg N, 28 mg P2O5, 28 mg K2O L"^
applied twice weekly
Temperature:
25-30OC
Growing Time:
12-14 weeks
Product:
24-26 cm tall, mycorrhizal, nodulated seedling
with 1 - 1.2 g shoot weight
87
Table 30. Growing regime for greenhouse production of mycorrhizal,
nodulated buffalo-berry.
Planting Time:
March, April
Inoculum:
Buf falo-berry field or pot culture soil with
high symbiont inoculum levels; crushed nodules
for Frankia
Inoculum/Container:
10-15% inoculum soil/planting mixture (V/V);
inoculum mixed into planting mixture
Planting Mixture:
1/1 (V/V) Peat/Vermiculite
Container Volume:
150 cc
Grower Fertilizer:
200-400 mg 28-14-14 or 56-112 mg N,
28-56 mg P2O5, 28-56 mg K2O L”'' applied
twice weekly
Temperature:
25-30OC
Growing Time:
16-18 weeks
Product:
20-22 cm tall, mycorrhizal, nodulated seedling
with 1.2 - 2 g shoot weight
88
(0o68 g plant ^). However, the buffalo-berry seedlings were small and
underweight (0.23 g plant compared with those outplanted on the
RRTAC plots (0.77 g plant ^). The poor growth exhibited by buffalo-
berry, particularly during the early phases of growth, is believed to
have been the result of inadequate N fertilization. It is postulated
that N2-fixing shrubs such buffalo-berry may be heavily dependent on
the Na-fixing Frankia, and therefore, require either rapid infection
by this symbiont or high soil N levels to compensate for the lack of
the symbiont. Supplementing the 15-15-18 fertilizer with NH4NO3
improved the growth of the buf falo-berry seedlings tremendously.
Both uninoculated and inoculated seedlings were of a similar
size thereby justifying treatment comparisons. All inoculated seed-
lings developed mycorrhizae and nodules but infection was patchy
possibly due to variation in the fertilizer regime.
High mortality during the first winter can be explained by
insufficient hardening off and freezing weather conditions within days
after outplanting. Mortality was higher for the inoculated than unino-
culated seedlings, also presumably due to improper hardening off. Some
studies have shown that mycorrhizal infection can reduce stomatal
resistance, thereby increasing transpi ration rate (Allen et al., 1981;
Allen and Boosalis, 1983; Levy and Krikun, 1980) although recent inves-
tigations by Graham et al. (1987) failed to find any effect of mycor-
rhizal infection on the water relations of Citrus . It is possible,
however, that, due to improper hardening off, the inoculated silver-
berry and buffalo-berry were not in the same physiological condition as
their uninoculated counterparts when outplanted. Greater stomatal
conductivity and higher rates of transpiration may have increased the
susceptibility of the inoculated seedlings to frost damage. It may be
that mycorrhizal seedlings require a longer period of hardening off
than non-mycorrhizal seedlings do. The relationship between symbiont
infection and susceptibility to winter kill should be investigated in
more detail .
The much superior growth performance of inoculated seedlings
compared with uninoculated seedlings over two growing seasons provides
unequivocal proof that pre-inoculation with mycorrhizal and N2-fixing
symbionts can, in the case of buffalo-berry and si 1 ver-berry , result in
more rapid revegetion of oil sands tailings. These findings support
89
those of Burgess et al. (1986) where Frankia-inoculated alders signifi-
cantly outperformed uninoculated alders over a three year period.
It is unknown why both inoculated and uninoculated buffalo-
berry were more productive in the peat and clay-amended tailings sand
than in the tailings sand amended with peat only. Differences in site
characteristics and soil chemical/physical properties are possible
explanations .
It is difficult to determine if the symbionts introduced with
the seedlings persisted and continued to colonize after outplanting;
however, the much greater nodule production and mycorrhizal development
in the inoculated treatments suggests that this was the case. The
indigenous soil inoculum successfully infected the uninoculated seed-
lings, but it is postulated that symbiont inoculum potential and rates
of colonization were so low that infection could not approach that in
the pre-inoculated seedlings over the two year period.
The relative contributions of the mycorrhizal fungi and the
Frankia to plant growth could not be discerned in this study. However,
nodule number/weights exhibited a much stronger correlation with shoot
weights than mycorrhizal root lengths inferring that, under the
conditions of this study, shoot production was more dependent on nodule
status than mycorrhizal condition. The very close relationship between
shoot weights and nodule weights is emphasized in the regressions
presented in Figures 7 and 8.
Vesicular-arbuscular mycorrhizae can enhance nodulation and
N2-fixation by satisfying the high P demand required for these
processes (Barea and Azcon-Agui lar, 1983). Consequently, their contri-
bution may be a subtle one and should not be underestimated. However,
the relatively poor correlation between nodule status and mycorrhizal
development in both silver-berry and buffalo-berry indicates that,
under field conditions, other factors besides mycorrhizal status may
strongly influence nodulation. Whatever the mechanisms behind the
superior growth performance of the inoculated shrubs, this study has
demonstrated that actinorhizal seedlings can benefit greatly over the
long term from artificial introduction of their symbionts prior to out-
planting. Therefore, when using actinorhizal shrubs for reclamation,
and possibly for amenity and forestry purposes also, symbiont inocula-
tion of seedlings is strongly recommended.
90
c
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Appendix Table 1. Mycorrhizal infection of slender wheatgrass grown in
undisturbed muskeg and stockpiled peat (peat stockpiled for 8
months) . ^
Sampling Depth (cm)
Root Parameter
Peat Source
0-15
50-100
Total root length
Undisturbed
545.0®
578.6®
H
_J
E
Stockpile
563.5®
623.1®
Mycorrhizal root length
Undisturbed
61 .5
21 .9
(m L”")
Stockpile
0
23.8
with arbuscules
Undisturbed
19.7 ± 35.5
5.3 t
7.0
Stockpile
0
3.4 +
5.1
with vesicles
Undisturbed
4.4 + 8.3
2.3 t
5.1
Stockpile
0
2.1 +
3.0
with hyphae
Undisturbed
37.4 ± 55.3
14.3 ±
22.8
Stockpile
0
18.3 ±
26.8
Percent infection
Undisturbed
14.5®
4.8®
Stockpile
0®
3.1®
1 Where possible, data were analyzed by two-way ANOVA (MSE = 322.82
and 117.05 for total root length and percent infection respectively).
Values in each data set followed by the same letter do not differ
significantly (p = 0.5). Standard deviations are included for those
data which could not be analyzed.
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