The impacts of broadcast burning after clearcutting on the diversity of
ectomycorrhizal fungi associated with hybrid white spruce seedlings in central
British Columbia using morphological and molecular characterization techniques
by
Karen Mah
B.Sc., The University of Waterloo, 1994

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
BIOLOGY

© Karen Mah, 1999
THE UNIVERSITY OF NORTHERN BRITISH COLUMBIA
August, 1999

All rights reserved. This work may not be
reproduced in whole or in part, by photocopy or
other means, without the permission of the author.

UNIVERSITY OF NORTHERN
BRITISH COLUMBIA
LIBRARY
Prince George, BC

ABSTRACT
In British Columbia, broadcast burning following clearcutting has been used to meet forest management
objectives such as site preparation for restocking timber species. However, effects of broadcast burning
on ectomycorrhizae (ECM), which facilitate nutrient and energy cycling within forests, is poorly
understood. Difficulties include a complex soil environment and uncertainties in ECM identification. To
determine effects of broadcast burning following clearcutting on the diversity and abundance (percent
colonization) of ectomycorrhizal fungi, morphological and molecular (PCR-RFLP) methods were used to
assess naturally regenerating and outplanted hybrid white spruce seedlings growing in clearcut, clearcut
plus burned, and adjacent mature sites in the Interior of British Columbia. Morphological
characterization resulted in 24 fungal morphotypes. Significant treatment effects and seedling
differences occurred between naturally regenerating seedlings in clearcut and mature sites and between
naturally regenerating and planted seedlings in clearcut sites. The abundance of some morphotypes
differed on planted seedlings in clearcut compared with clearcut plus burned sites and for planted
seedlings in treated (clearcut and clearcut plus burned) compared with regenerating seedlings in
untreated (mature) sites. A Russulaceae type and Thelephora were the most abundant morphotypes on
regenerating seedlings in the mature and clearcut sites, respectively. Molecular characterization
showed no significant differences for treatment effects or seedling type. Amplification of the ITS region
for eight commonly occurring morphotypes revealed 12 genotypes (having a shared band pattern for
one or none of three restriction endonucleases) with 18 variants (having similar band patterns for two
restriction endonucleases). Cenococcum, Tuber, Hebeloma and Thelephora had only one genotype,
however, Amphinema, E-strain, MRA, and a Russulaceae type each exhibited two or three genotypes.
Morphology showed differences in occurrence and abundance of some ECM fungi following
clearcutting, and clearcutting plus burning, suggesting that disturbance may be altering the fungal
composition of hybrid white spruce seedlings on these sites towards ECM best able to adapt to changing
environmental conditions. Using both characterization techniques provided a comprehensive estimate
of diversity, specifically for total species richness when using morphology, and for increased
understanding of inter- and intra-specific variation with respect to molecular characterization of
ectomycorrhizal associations.

11

TABLE OF CONTENTS

Page

Abstract

ii

Table of contents

iii

List of tables

v

List of figures

vii

Acknowledgments

viii

Introduction

1

1. Literature review
1.1 Prescribed burning: objectives and current uses
1.2 Terminology and important definitions related to fire
1.3 Fire and the soil environment
1.4 Ectomycorrhizal fungi diversity and specificity
1.5 Functions of ectomycorrhizal fungi
1.6 Characterization of ectomycorrhizae
1.6.1 Morphological techniques
1.6.2 Molecular techniques
1.6.3 Comparison of characterization techniques
1.7 Calculating ectomycorrhizae diversity
1.8 Fire effects on mycorrhizae
1.8.1 Fire effects on soil organisms, soil properties and fungi
1.8.2 Effects of fire on mycorrhizal abundance and formation
1.8.3 Studies on mycorrhizal diversity and fire
1.9 Literature cited

5

6

7

8
8

9

10
10
11

12
14

17
18

2. Morphological characterization of ectomycorrhizae associated with hybrid white spruce seedlings in
the Aleza Lake Research Forest in the Central Interior of British Columbia
Abstract
23
2.1 Introduction
24
2.2 Materials and Methods
26
2.2.1 Site descriptions
2.2.2 Seedling sampling
29
30
2.2.3 Soil and seedling analysis
2.2.4 Ectomycorrhizae characterization
31
2.2.5 Statistical analysis of morphotype abundance and diversity indices
33
2.3 Results
2.3.1 Site and seedling characteristics
34
2.3.2 ECM morphotype occurrence, frequency of occurrence and abundance
35
2.3.3 ECM diversity
38
2.4 Discussion
2.4.1 ECM morphotype abundance
39
2.4.2 Treatment effects or seedling type differences on ectomycorrhizal diversity
46
51
2.5 Literature cited
3. Molecular characterization of mycorrhizae associated with hybrid white spruce seedlings in the
Aleza Lake Research Forest, Central Interior of British Columbia
Abstract
3.1 Introduction
3.2 Materials and Methods
3.2.1 Ectomycorrhizae sampling
3.2.2 DNA extraction
3.2.3 DNA amplification
3.2.4 Digestion , gel electrophoresis and photography
Il l

55
56
58
59
59
60

3.2.5 Analysis of molecular data
3.3 Results
3.3.1 Amplification and digestion success rates
3.3.2 Band patterns of selected ECM morphotypes
3.3.3 Molecular diversity for commonly occurring morphotypes
3.3.4 Treatment effects on ECM molecular diversity using the Phi , Shannon and Simpson
indices
3.4 Discussion
3.4.1 Genetic diversity between treatments and between seedling type
3.4.2 Genetic variation of hybrid white spruce ectomycorrhizae
3.4.3 Comparison between molecular and morphotyping characterization
3.4.4 Conclusions
3.5 Literature cited

61
63
65
69
70
72
73
76
80
81

Appendix A. Pre-harvest characteristics of treated sites in the Aleza Lake Research Forest (from
pre-harvest site prescription forms prepared by Northwood Pulp and Timber Limited for the B.C.
Ministry of Forests).

84

Appendix B. Macroscopic and microscopic characteristics of selected ectomycorrhizae found on
naturally regenerating and planted hybrid white spruce seedlings in mature forest, clearcut, and
cut plus burned sites in the Aleza Lake Research Forest, Central Interior of British Columbia.

85

Appendix C. Checklist for ectomycorrhizae morphological data.

86

Appendix D. Ectomycorrhizae descriptions of naturally regenerating and planted hybrid white spruce
seedlings growing in treated (clearcut, and cut plus burned) and untreated (mature) sites in the
Aleza Lake Research Forest, Central Interior of British Columbia.

87

Appendix E. An example of calculations for richness (Margalef}, evenness (Shannon) and composite
index (Shannon and Simpson) measures using ectomycorrhizae morphological abundance data
for seedlings growing in mature site 1 in the Aleza Lake Research Forest, Central Interior of
British Columbia.

91

Appendix F. Ectomycorrhizae morphotypes found on naturally regenerating (r) and planted (pi)
hybrid white spruce seedlings in treated (clearcut, and cut plus burned) and mature sites in the
Aleza Lake Research Forest. Table shows known or suspected genera or species from
comparisons made with the published literature.

92

Appendix G. Statistical summation (one-way ANOVA) for treatment (mature, clearcut and cut plus
burned) and seedling effect (naturally regenerating (n=16) and planted (n=28)) on mean
proportional abundance (±SE) for seven commonly occurring ectomycorrhizae associated with
hybrid white spruce growing in the Aleza Lake Research Forest, Central Interior of British
Columbia.

94

Appendix H. An example of a Phi index calculation for ectomycorrhizae molecular data (PCR-RFLP)
after PHYLIP analysis.

95

Appendix I. Sample phenogram of MRA

96

iv

LIST OF TABLES

Page

Table 1. Studies on ectomycorrhizae abundance following clearcutting and burning .

16

Table 2. Site descriptions and dates for clearcut (C), and cut plus broadcast burned (CB)
treatments in the Aleza Lake Research Forest.

29

Table 3. Sampling design for hybrid white spruce seedlings harvested from clearcut, cut plus
burned and mature sites in the Aleza Lake Research Forest.

30

Table 4. General site, seedling and soil characteristics (means ±SE) for mature, clearcut and cut
plus broadcast burned sites sampled in the Aleza Lake Research Forest.

35

Table 5. Morphotype occurrence on naturally regenerating and planted hybrid white spruce
seedlings in treated (clearcut, and cut plus burned) and untreated (mature forest) sites in
the Aleza Lake Research Forest, Central Interior of British Columbia.

36

Table 6. Mycorrhizae morphotype abundance (mean percent (±SE)) and frequency of occurrence
(%)for planted {pi) and naturally regenerating (r) hybrid white spruce seedlings in treated
(clearcut, and cut plus burned) and mature sites in the Aleza Lake Research Forest,
Central Interior of British Columbia.

37

Table 7. Ectomycorrhizae richness , evenness and diversity measures (Shannon and Simpson,
Shannon Evenness and Margalef) showing mean values (±SE). Indices were assessed
using one-way ANOVA to test for treatment effect (clearcut, cut plus burned, and
unburned, mature) and to test for seedling differences (naturally regenerating , (n=16)
versus planted (n=28) of hybrid white spruce seedlings growing in the Aleza Lake
Research Forest, Central Interior of British Columbia.

39

Table 8. Sorenson similarity coefficients calculated for ectomycorrhizae of naturally regenerating (r)
and planted (pi) hybrid white spruce seedlings from unburned mature, clearcut, and cut
burned sites in the Aleza Lake Research Forest, Central Interior of British Columbia.

39

Table 9. Summary of DNA amplification (PCR) of mycorrhizal root tips from naturally regenerating
and planted hybrid wh ite spruce seedlings growing in the Aleza Lake Research Forest,
Central Interior of British Columbia.

64

Table 10. RFLP band patterns of four ascomycete morphotypes amplified (PCR) from naturally
regenerating and planted hybrid white spruce seedlings in the Aleza Lake Research Forest,
Central Interior of British Columbia.

66

Table 11. RFLP band patterns of four basidiomycete morphotypes amplified (PCR) from naturally
regenerating and planted hybrid white spruce seedlings in the Aleza Lake Research Forest,
Central Interior of British Columbia.

67

Table 12. Comparison of RFLP band patterns of lightly colonized but unknown ECM with known
morphological types amplified (PCR) from naturally regenerating and planted hybrid white
spruce seedlings in the Aleza Lake Research Forest, Central Interior of British Columbia.

68

Table 13. Molecular genotypes and variant occurrence and diversity (Phi index) for commonly
occurring ECM morphotypes found on naturally regenerating and planted hybrid white
spruce seedlings growing in treated (clearcut, and cut plus burned) and untreated (mature)
sites in the Aleza Lake Research Forest, Central Interior of British Columbia.

69

v

Table 14. Number of molecular genotypes and variants for ECM found on naturally regenerating and
planted hybrid white spruce seedlings growing in treated (clearcut, and cut plus burned) and
untreated (mature) sites in the Aleza Lake Research Forest, Central Interior of British
Columbia.
70
Table 15. Statistical summation for treatment effect and seedling type on molecular diversity
assessed using Phi, Shannon and Simpson index values (mean±SE) for ECM associated
with naturally regenerating and planted hybrid white spruce seedlings growing in treated
(clearcut, and cut plus burned) and untreated (mature) sites in the Aleza Lake Research
Forest, Central Interior of British Columbia.

VI

72

LIST OF FIGURES

Page

Figure 1. Location of sites in the Aleza Lake Research Forest, Prince George Forest District (insert
from Prince George Forest District recreation map, BC MOF, FRBC, Feb. 1997. Scale
approximately 1:400 000).

27

Figure 2. A portion of the forest floor in the mature site located in the Aleza Lake Research Forest,
Central Interior of British Columbia (June 1997).

27

Figure 3. a) cut plus broadcast burned site and b) clearcut site located in the Aleza Lake Research
Forest, Central Interior of British Columbia (June 1997).

28

Figure 4. Root systems of a) naturally regenerating and b) planted hybrid white spruce seedlings
harvested from mature forest and cut plus burned sites, respectively, in the Aleza Lake
Research Forest, Central Interior of British Columbia.

32

vii

ACKNOWLEDGMENTS
The author would like to thank the following for their help:
Forest Renewal BC, for financial support;
UNBC, for providing travel grants and laboratory facilities ;
Dr. Hugues Massicotte, my supervisor, for countless hours of editing and discussion and constant good
cheer;
Committee members, Drs. Keith Egger and Chris Opio, for their expertise on molecular and fire issues,
respectively ;
Dr. Dan Durall, the external examiner, for reviewing my thesis;
BC Ministry of Forests, Dr. Paul Sanborn and Sharon Dow who provided maps and data on the study
sites;
Ron Jansen, from Northwood Pulp and Paper Limited , for providing site treatment information;
Linda Tackaberry, for training and support in morphological characterization of regenerating seedlings
and endless hours of editing and discussion ;
Tamara Bereck and Brent Young , for processing tips of regenerating seedlings for molecular analysis;
Quentin Baldwin , for training and helpful suggestions dealing with molecular and computer glitches;
Dr. Lito Arocena, Kevin Driscoll and Christine Breed for help with soil analysis;
Dr. Bruno Zumbo, for fundamental and vital statistical aid ;
Dr. Mike Walters, for statistical advice and input for the thesis proposal ;
Dr. David Dick, for advice on chemical and thesis matters;
Keith Williams and Aniko Varga, for suggestions (helpful and otherwise) about theses and life in general;
Secretaries Judy Armstrong, Paula Poirier and Yvonne Smith on the third floor of the administration
building for help with the little details;
And to all those working in lab 403 for listening to my "progress" on the thesis.

vii i

Introduction
In British Columbia, broadcast burning (a form of prescribed burning) has been commonly used as a
method of site preparation following clearcutting to help create a favourable environment for seedling
establishment (Hawkes et at. 1990). To determine the efficacy of this treatment, it is necessary to
examine the fire effects on the soil environment because the soil is a major determinant of site productivity
(Agee 1993, Wells eta/. 1979). For example, plant growth and productivity potential are affected by soil
moisture-holding capacity, nutrient status and porosity (Hungerford eta/. 1991 ). Numerous studies have
been conducted on soil physical and chemical properties following clearcutting and burning. Burning
effects are often confounded with those of clearcutting but in general, as the fire severity increases,
negative impacts on the soil also occur, such as decreased soil porosity, increased soil erosion, and
increased nutrient volatilization (Agee 1993, Wells eta/. 1979).

The effects of broadcast burning on soil organisms have not been well documented. Of particular interest
are fire effects on the overall diversity and abundance (percent colonization) of ectomycorrhizal fungi that
live in symbiotic association with conifers, particularly commercial forest tree species. In forming
ectomycorrhizae (ECM}, these fungi contribute to nutrient and water uptake by roots and to protection
against root pathogens (Harley and Smith 1983}, providing a key link in nutrient and energy cycling within
forest ecosystems (Dighton and Mason 1985). Some previous studies have reported a decrease in ECM
abundance following fire and harvesting (Wright and Tarrant 1958; Harvey eta/. 1980; Perry eta/. 1982;
Schoenberger and Perry 1982; Parke eta/. 1984). However, other studies reported an increase (Pilz and
Perry 1984; Brainerd and Perry 1987; Richter and Bruhn 1993) or no decrease (Visser 1995) in ECM
abundance following these disturbances. Obstacles in determining responses of ECM formation to fire
include the complexity of the soil environment, differences in response to fire intensity and severity as well
as difficulty in identifying fungal symbionts.

Few studies have been conducted concerning the effects of fire on ECM diversity and these had limited or
no descriptions of fungal types, making comparisons with current studies difficult. Recent efforts have
been made to describe ECM using more detailed morphological characterization (Simard eta/. 1997a;
Horton eta/. 1998; Visser eta/. 1998) as well as molecular (the polymerase chain reaction-restriction

fragment length polymorphism, PCR-RFLP) methods (Kernaghan eta/. 1997; Horton and Bruns 1998,
Jonsson eta/. 1999). Some weaknesses can be attributed to these two methods, including the inability of
some morphotypes to be identified to the species level and the fact that some tips fail to amplify for
molecular analysis. Traditional diversity indices (such as Shannon or Simpson) have been used in
morphological analysis of ECM communities, however species uncertainty can be problematic because all
species in a sample must be known (Magurran 1988). To assess diversity using molecular data, the Phi
index has been derived by Egger (Baldwin 1999, M.Sc. Thesis). Using the Phi index, PCR-RFLP band
patterns from ECM root tips are matched with every other tip in the sample and their distances
(representative of their relatedness) are used instead of species richness and abundance data, in
calculating molecular mycorrhizal diversity (Egger, pers. comm. 1999). By using a combination of
morphological and molecular approaches, a more detailed assessment of mycorrhizal diversity and a
better understanding of responses to burning effects can hopefully be obtained .

The main objective of the present study was to determine, using both morphological and molecular
characterization (PCR-RFLP) methods, the effect of broadcast burning following clearcutting on the
diversity of ECM on planted and naturally regenerating hybrid white spruce (Picea engelmannii (Parry ex
Engelm.) x g/auca (Moench) Voss). Mature, clearcut, and clearcut plus burned (cut plus burned) sites in
the sub-boreal spruce (SBS) biogeoclimatic zone of central British Columbia were examined. In addition,
the study was to explore differences in ECM diversity between planted and regenerating seedlings and to
compare molecular results with previous morphological assessments. One of the main species used in
reforestation in the Central Interior of British Columbia is hybrid wh ite spruce, however, few studies have
examined the ECM diversity of these seedlings planted on broadcast burned sites following harvesting.

Previous studies have reported decreased abundance of ECM tips with an increase in disturbance (from
undisturbed to clearcut, to cut plus burned sites) (Wright and Tarrant 1958; Harvey eta/. 1980; Perry eta/.
1982; Schoenberger and Perry 1982; Parke eta/. 1984 ). Due to the more extreme environments created
by site disturbance and possible decreases in available fungal inoculum, this trend might be expected. As
well , the fungal community composition between treatments may differ as those species best able to
adapt to particular cond itions of clearcut and cut plus burned sites may be favoured . Initial site changes
2

caused by clearcutting can include increased temperature extremes due to loss of shading from
vegetation , destruction of the organic layer as well as disturbance of the mineral layer, and decreased soil
porosity and water infiltration due to soil compaction. Following the removal of boles, crowns and forest
floor, Amaranthus eta/. (1996) reported that at moderate to severe soil compaction levels, decreased
ECM abundance on outplanted Douglas-fir (Pseudotsuga menziesii var. g/auca [Beissn .] Franco) and
western white pine (Pinus montico/a Dougl. ex D. Don) resulted . Disturbance of the organic and mineral
layers is important, especially for fine roots. In a wh ite spruce (Picea g/auca [Moench] Voss) -subalpine fir
(Abies /asiocarpa [Hook.] Nutt.) stand, Kimmins and Hawkes (1978) found that approximately 70% of
overstory and understory fine-root biomass was in the LFH and Ae horizons, at an average depth of 8 em.
The initial flush of nutrients from the finer slash (Kimmins 1997) may be quickly lost when no roots or
organisms are present to use them.

Depending on the fire severity and intensity, subsequent burning following clearcutting could destroy
additional sources of fungal inoculum such as roots, fungal spores and sclerotia or might discourage the
presence of remaining animal vectors. However, broadcast burns of light to moderate severity may in fact
create an environment of increased nutrient availability and reduced competition, for those organisms
remaining on , or colon izing the site. Furthermore, a few years after disturbance, soil conditions should
improve, due to the regrowth of vegetation , increased shading , the formation of an organic soil layer and
the return of animal vectors. Although perhaps not as great as might be seen immediately after
disturbance, differences in ECM abundance and diversity might still be expected to persist.

ECM abundance and diversity might also be expected to be higher where there are many niches that
specialized mycorrhizal fungi can colon ize, such as in the mature forest. A steady supply of nutrients and
water, regulation of temperature extremes and abundance of fungal inoculum sources and animal vectors
should maintain a diverse and competitive community of ECM . Thus, there might be notable differences
in fungal community compos ition between regenerating seedlings in the treated and untreated sites.
Naturally regenerating seedlings in mature sites could provide a reference for the possible inoculum
(native fungi) existing on adjacent sites following disturbance.

3

Differences may exist between regenerating and planted seedlings in the same site. Seedlings originating
from greenhouse and nursery stock may initially possess fungal inoculum when outplanted two years
later. However, new roots are gradually replaced or colon ized by in situ fung i. Bledsoe and Tennyson
(1982) reported that previously inoculated Douglas-fir seedlings, outplanted on dry, burned over sites in
eastern Washington were colonized by native fungi within five months, although some fungi from the
original inoculum were still present. Inoculated ectomycorrhizal fungi on outplanted black spruce (Picea
mariana [Mill.] B.S.P.) and jack pine (Pinus banksiana Lamb.) nursery seedlings, declined sharply after

two growing seasons and indigenous fungi were noted 11 weeks after outplanting (Browning and Whitney
1992). Another difference between planted and regenerating seedlings relates to the size of seedlings
and their root systems. The larger root systems of the planted seedlings, grown under optimal conditions
in the greenhouse and nursery for the first two years, might confer a greater chance of colonization by
different and more ectomycorrhizal fungi when outplanted onto treated sites.

4

1. Literature Review
1.1 PRESCRIBED BURNING : OBJECTIVES AND CURRENT USES
Prescribed burning is the knowledgeable application of fire to a specific land area to accomplish
predetermined forest management or other land use objectives (Merrill and Alexander 1987). In contrast,
a wildfire is an unplanned or unwanted natural or human-caused fire (Merrill and Alexander 1987).
Historically, wildfires have been the most important regenerative agent in coniferous forests (Ahlgren and
Ahlgren 1960; DeByle 1976) but only recently have forest managers acknowledged this. After decades of
fire suppression , forest managers are now attempting to utilize methods that mimic natural disturbance
regimes, to meet goals of sustaining biological diversity and forest productivity (Delong and Tanner
1996). In theory, fire should be reintroduced in the form of prescribed burns to those forest ecosystems
that require periodic fire to maintain the character, diversity and vigour of the intrinsic plant and animal
communities (Poulin et at. 1994). In British Columbia, such ecosystems include interior and northern
forests in the sub-boreal spruce (SBS) biogeoclimatic zone (Vasbinder eta/. 1996).

From a forest management standpoint, prescribed burning has been used to meet several objectives.
These include reduction of the fire hazard, site preparation for replanting and seedling establishment,
reduction of brush competition, facilitation of stand tending, site sanitization of disease organisms or insect
pests, and natural ecosystem management as mentioned above (Hawkes et at. 1990; Weber and Taylor
1992; Mutch 1994; Feller 1996). Ninety percent of the prescribed burning in Canada has occurred in
British Columbia, from the period of 1984 to 1992 (Feller 1996), and fire has been used as an economical

and efficient tool to meet some of the above objectives (DeByle 1976; Weber and Taylor 1992). Since
1992, however, a decline in the amount of land burned in Canada for silvicultural purposes has occurred
(Feller 1996). The main reasons for this decline are the logistical difficulties and economic costs in
controlling fires that involve liability issues, shortage of qualified personnel and smoke concerns (Arno
1996). Meeting criteria of biodiversity hinders the use of fire in preference for other silvicultural treatments
such as partial or selective cutting , combined with the increased availability of machines for mechanical
site preparation (Feller 1996; Dow, pers. comm. 1997). Despite this downward trend, prescribed fire still
remains an important tool in vegetation management (Feller 1996).

5

Normally, a burn prescription uses guides such as the Canadian Forest Fire Weather Index and Canadian
Forest Fire Behavior Prediction system (Merrill and Alexander 1987) and is laid out once a site has been
assessed . Three options are available to burn slash , or logging debris, namely broadcast, windrow (piling
of slash into long rows) or pile burning. Broadcast burning is lower in intensity and its effects are
homogeneous throughout the site as slash is distributed evenly on the ground, compared to windrow and
pile burning where soil effects are more localized and severe (DeByle 1976; Hawkes eta/. 1990).
Broadcast burning imitates a natural fire better that the other two methods because there is less mineral
soil disturbance, however, these fires are more difficult to control than slash pile burns (DeByle 1976).

1.2 TERMINOLOGY AND IMPORTANT DEFINITIONS RELATED TO FIRE
Critical concepts describing prescribed burns are the fire type, fire intensity, and fire severity. There are
three basic types of fire in forests, categorized by the vertical strata where the burn occurs: the ground,
surface and crown . In slash burns, only ground and surface fires occur. Ground fires, though of low
intensity, are the most destructive as they smoulder slowly through packed organic matter and can kill
roots in the forest floor. Surface fires exhibit flaming and burn rapidly , scorching bark and needles, killing
seedlings and saplings, and opening serotinous cones. Each fire type releases different heat intensities
and spreads at different rates , resulting in variable amounts of fuel consumed and levels of heating aboveand belowground (Barbour eta/. 1987; Hungerford eta/. 1991 ; Kimmins 1997).

Fire intensity is defined as the rate of heat release per unit of ground surface area (kW/m) and is
proportional to flame height and rate of spread (Wells eta/. 1979). The rate of spread is the speed at
which the leading edge of the fire travels downwind; duration refers to the time over which energy release
occurs at any particular location (Kimmins 1997). The combined effects of fire intensity and duration are
expressed by the term fire severity. This can be a qualitative assessment of litter, duff and soil
appearance (or disappearance) after burning (Wells eta/. 1979) or a quantitative measurement of the
reduction in forest floor th ickness (Merrill and Alexander 1987; Haeussler 1991 ; Feller 1996). Most fires in
the SBS zone have been characterized as medium to high intensity surface and crown fires (Parminter
1992).

6

Fire severity is primarily influenced by four factors: fuel properties (compaction, composition and moisture
content); weather conditions before and during fire (temperature, wind and precipitation); site conditions
(topography and soil texture) ; and finally type of prescribed burn method used (Haeussler 1991 ). The
timing of a fire is important in many respects . In particular, one must consider changes in plant phenology
occurring throughout the year. For example, a fire in the early spring may not be as detrimental as one
occurring in the summer because plants can resprout resulting in very little impact on the vegetation in the
following year (Haeussler 1991 ; Kimmins 1997). Dormant plants (from late summer to early spring) are
better protected against fire due to the presence of high levels of belowground carbohydrates and
protected buds (Haeussler 1991 ).

1.3 FIRE AND THE SOIL ENVIRONMENT
Soil organisms play a major role in soil formation and nutrient cycling (Borchers and Perry 1990), yet soil
biological responses to fire are some of the least studied aspects of the soil environment (Agee 1993).
This may be due in part to the complexity of the soil environment, especially in the rhizosphere, the root
surface and surrounding area where intense soil biological activity occurs (Borchers and Perry 1990).
Here, nitrogen fixers, mycorrhizal fungi and root pathogens exist, interact, and use and/or produce
carbon-rich root exudates, secretions of enzymes, chelators, growth hormones and antibiotics (Harley and
Smith 1983). Extraction, identification and the study of small soil organisms such as bacteria and fungi
often involves methods that are time-consuming, have a high degree of uncertainty, and that require
constant revisions of taxonomy.

Perhaps the most important soil organisms affecting the survival of seedlings are the fungi that live in
symbiotic association with living plant roots, forming mycorrhizae (Harley and Smith 1983). These
symbiotic fungi represent about 10% of all recognized soil fungal species (Molina eta/. 1992).
Furthermore, an estimated 90% of all terrestrial plant species belong to families that are commonly
mycorrhizal (Trappe 1987; Molina eta/. 1992).

7

1.4 ECTOMYCORRHIZAL FUNGI DIVERSITY AND SPECIFICITY

Mycorrhizal fungi involved in mutualistic symbioses belong in the phyla Asco-, Basidia- and Zygomycotina
and are grouped into seven currently recognized groups: vesicular-arbuscular, ecto- (ECM), ectendo-,
arbutoid , monotropoid , ericoid, and orchid mycorrhizae (Harley and Smith 1983). ECM are symbiotic
associations between fung i and angiosperm or gymnosperm hosts, many of which are important timber
species worldwide and include species such as pine (Pinus} , spruce (Picea) , Douglas-fir (Pseudotsuga)
and Eucalyptus. An excess of 5000 ectomycorrhizal fungi associate with likely more than 2000 plant host
species (Kendrick 1992}, providing for a multitude of combinations. Different hosts may possess a few or
many mycorrhizal fungal partners and fungi may be host specific, to intermediate, to broad host ranging,
capable of forming functionally compatible mycorrhizae on few to several members of diverse families.
Douglas-fir and pines for example, have possibly 2000 associated fungal species worldwide, based on
sporocarp-host associations (Trappe 1977). Currently, the data suggest that most ectomycorrhizal fungi
are intermediate to broad host ranging (Molina eta/. 1992). An example of a broad host ranging fungus is
Thelephora , sometimes found on greenhouse-grown seedlings and capable of forming on members of
many plant genera (lngleby eta/. 1990). However, several hundred species of fungi can be genusspecific such as Sui/Ius granulatus, which only associates with Pinus species (Molina eta/. 1992).

1.5 FUNCTIONS OF ECTOMYCORRHIZAL FUNGI
Increased absorption and access to minerals, increased nutrient and water uptake by plant roots,
protection against root pathogens and the ability to act as large reservoirs of plant derived carbon are
several documented functions of ectomycorrhizal fung i (Harley and Smith 1983). Different fungi vary in
the degree to which they can perform these functions (Perry and Rose 1983) but the details of their
specific functional contributions largely remain unknown .

The persistence and distribution of ectomycorrhizae in the absence of living hosts is not well documented
(Harvey eta/. 1980; Amaranth us 1991 ). There is potential for woody shrubs colonizing a planted site to
provide a source of inoculum for host tree species. For example, Arctostaphylos uva-ursi, may provide
ectomycorrhizal fungal inoculum for pine and spruce in clearcuts in B.C. (Molina eta/. 1992). The
suggestion of fungal linkages between plants of the same or different species implies a greater role by

8

mycorrhizae in seedling regeneration than is currently acknowledged. Newman (1988) proposed some
important possibilities due to fungal linkages such as benefits to seedlings or to nutrient deficient plants
that can link into a "hyphal network" and receive photosynthates from other hosts or from direct nutrient
transfers from dying roots to living roots without going through the soil (where nutrients may be lost).
Simard eta/. (1997b), using reciprocal isotope labeling, showed bidirectional carbon transfer between
birch (Betula papyrifera Marsh.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seedlings
growing in the cedar-hemlock biogeoclimatic zone, and reported a net carbon gain by Douglas-fir.
Transfer between hosts was facilitated by hyphal linkages but net carbon transfer must be demonstrated.

1.6 CHARACTERIZATION OF ECTOMYCORRHIZAE
1.6.1 Morphological techniques
Morphologically, ECM are different from other mycorrhizae in that the fungus forms a sheath or mantle
that surrounds the plant root and a Hartig net develops between the root epidermal and cortical cells
(Harley and Smith 1983). In angiosperms, the Hartig net intercellular penetration is barred by the
exodermis, whereas in gymnosperms (which do not possess an exodermis) penetration occurs up to the
endodermis (Kendrick 1992). ECM morphotypes are characterized by using light microscopy to
distinguish features such as colour, mantle structure, external hyphae, and presence and structure of
rhizomorphs (Agerer 1987-98; lngleby eta/. 1990; Agerer 1991 ; Goodman eta/. 1996). Only a few ECM
have been unambiguously identified to genus and species level using this characterization method
(Mehmann eta/. 1995) and the uncertainty may be partly due to the fact that morphology may vary
according to changes in the root or soil environment, or to changes in host partner (Egger 1995).

Another approach to ECM identification is by linking reproductive structures or sporocarps (epigeous or
hypogeous) to mycelia connected to underground root systems. ECM morphotypes in different areas of
the world have been described and identified by this method (Agerer 1987-98; lngleby eta/. 1990; Agerer
1991 ; Agerer eta/. 1996-98). ECM can also be identified by pure culture synthesis from sporocarps and
re-inoculating seedlings to describe ECM . However a minority of fungal species have been successfully
grown in culture (Danielson 1984).

9

1.6.2 Molecular techniques
Recent advances in molecular biology should help to provide a measure of ECM identification that is
independent of environmental variation (Egger 1995). Restriction fragment length polymorphism (RFLP)
analysis is used in combination with the polymerase chain reaction (PCR, Mullis and Faloona 1987), a
process that amplifies target DNA sequences (from root tips, sporocarps or cultures) by the use of fungalspecific primers (Egger 1995; Mehmann eta/. 1995; Gardes and Bruns 1996a). The internal transcribed
spacer (ITS) region of the ribosomal unit of DNA (rONA) has been chosen for amplification because it is
variable enough to identify most fungi to the species or species group level, although it may not be
variable enough to distinguish between closely related species (Gardes and Bruns 1996a). The amplified
DNA is then digested by restriction enzymes and the fragments (target sequences) are run on a gel,
where they migrate at different speeds and separate during electrophoresis, due to their varying lengths
(Egger 1992, Gardes and Bruns 1996a). The resulting bands, visible by staining the gel, can be
compared to known DNA patterns for identification or stored for reference if no match is found. The
success in identifying (or matching) a given gel will depend on the database used for comparison (Gardes
and Bruns 1996a).

1.6.3 Comparison of characterization techniques
Recent studies indicate that the methods outlined above vary in the precision of fungal identification.
Mehmann eta/. (1995) conducted a study on ectomycorrhizal diversity in a 40-year-old pure spruce
(Picea abies) stand in Switzerland, and reported 23, 18 and seven types respectively, using molecular
(ITS, PCR-RFLP method) , morphological (using a dissecting microscope) and sporocarp techniques for
identification. Sporocarp identification was the least reliable for assessing belowground diversity as
fruiting varied with environmental conditions such as temperature, humidity and precipitation (Mehmann et
a/. 1995). Morphotyping of soil cores was more reliable , however, molecular characterization revealed
that one morphotype could represent one species, or that one morphotype could represent more than one
species, or that several morphotypes could represent one species (Mehmann eta/. 1995). Gardes and
Bruns (1996b) examined ECM diversity of a 40-year-old bishop pine (Pinus muricata) stand in California
and reported 10 sporocarps and 20 RFLP types over a four-year period . Due to the inherent challenges
of characterization approaches, a combination of morphological and molecular techniques may provide
10

more information about mycorrhizae than if using only one method. Environmental differences in
morphology may reflect differences in functional diversity whereas molecular techniques may provide
understanding of inter- and intra-specific diversity.

1.7 CALCULATING ECTOMYCORRHIZAE DIVERSITY
Simple measures of diversity are richness (the number of species), and evenness (the distribution of
species abundance). Richness measures can simply be counts of species or an average number of
species per sampling unit to take unequal sample size into account, such as the Margalef index: DM9 = (S1) /In n, where S is the number of species and n is the number of individuals (Magurran 1988). Evenness
measures the distribution of species abundance (Magurran 1988). Heterogeneity indices take both
richness and evenness into account and two types are the information statistics indices and dominance
measures, represented by the Shannon and Simpson index respectively (Magurran 1988). In some
studies, morphological data has been analysed using the Shannon and Simpson composite diversity
indices to measure species diversity for each seedling (Brainerd and Perry 1987; Simard 1997a). The
Shannon index is calculated asH= -L P; In (P;), where P; =the proportional abundance of the ith species;
Simpson index is computed as C = 1- L P;2 (Magurran 1988; Brewer 1994). H is sensitive to rare species
(affected more by species richness) , while Cis heavily weighted to the most abundant (dominant) species
(Magurran 1988). As the value of the indices increase, so does diversity. These indices are nonparametric, making no assumptions of normal data distribution however all species in the sample must be
accounted for or known (Magurran 1988), which is rarely the case in soil microbial studies. A measure of
evenness for the Shannon index can be calculated using the ratio of observed diversity to the maximum
diversity: E = H I In S.

The Sorenson coefficient of similarity measures beta diversity, the variation in species composition
between areas of alpha diversity, and indicates how closely sites are related (Magurran 1988; Mehmann
1995). It is calculated as: S = 2c I (a+b) , where a is the number of morphotypes in one plot, b is the
number of the other and c is the number of morphotypes in common (Magurran 1988). A value of one

II

indicates total similarity. This qualitative measurement does not take into account species abundance
(Magurran 1988).

To calculate molecular diversity , wh ich presents yet another level of complexity, the Phi index may be
used, which was recently developed by Egger (Baldwin 1999, M.Sc. Thesis) . The index is based on
phylogenetic distances and attempts to resolve the problem of intraspecific variation more adequately
than trad itional diversity indices (Baldwin 1999, M.Sc. Thesis). Following the analysis of RFLP band
patterns, distance values for each root tip, compared with every other root tip, are calculated using the
reciprocal of Dice's index based on shared and unique band patterns. The Phi index value is calculated
based upon the distance matrix (Egger, pers. comm.1999) . The more distantly related species are, the
greater the phylogenetic distances. The larger the value of the Phi index, the more genetically diverse the
site is (Egger, pers. comm. 1999).

1.8 FIRE EFFECTS ON MYCORRHIZAE
1.8.1 Fire effects on soil organisms, soil properties and fungi
Depending on the type of fire, there may be tremendous variation in the soil disturbance and
consequently, in effects on mycorrhizae. Changes vary according to the severity of the fire ; a single
severe fire will likely have greater impacts on mycorrhizae than several light or moderately severe ones
(Agee 1993; Wells eta/. 1979). While examining the impact of fire on soils, it must also be kept in mind
that prescribed burning is often used in conjunction with harvesting and that effects of both disturbances
are often confounded (Hawkes eta/. 1990; Agee 1993).

Fire affects mycorrhizae directly by consuming roots or other sources of fungal inocula in the soil. On
clearcut and prescribed burn sites, fire intensity was positively correlated with the percent colon ization of
ECM roots on outplanted white pine (Pinus strobus L.) , however this was not noted for red pine (Pinus

resinosa Ait. (Herr eta/. 1994). Fungi in chaparral soils were reported to tolerate temperatures of 155°C
in dry soil and 1oooc in wet soil (Dunn and DeBano 1977). In a low severity cool-burn ing prescribed fire
in a mixed conifer forest in California , maximum surface temperatures only reached 1oooc and at 5 em

12

belowground, temperatures were only sooc (DeBano eta/. 1998). The effect of fire on soil temperature
depends on how deep the soil was heated , the maximum temperature that was reached and how long this
temperature was maintained (Agee 1993).

Indirectly, ECM can be affected by changes to the soil or aboveground environment. In a Douglas-fir/larch
forest soil in western Montana, Harvey eta/. ( 1976) reported that in the top 38 em of soil, 95% of the
active ECM were associated with organic material, mainly humus and decayed wood. Consumption of
organic material and woody debris in severe fires should theoretically decrease fungal inocula for
regenerating seedlings and decrease habitats for small mammals (Amaranthus 1991) which disperse
spores of some ectomycorrhizal fungi (Maser eta/. 1978). Phoenicoid fungi (those preferring post-fire
environments) may have an advantage due to their ability to produce hydrolase enzymes and to use
substrates in the postfire environment (Egger 1986). Fire can also alter the dynamics of competition
between ECM and other soil organisms. Bacteria are generally less susceptible to heat than fungi
(Ahlgren 1974). In the increased pH environment, six years after moderately severe fires in subalpine
forests in B.C. and Alberta, bacteria were more abundant than microbial fungi (Bissett and Parkinson
1980). Rhizina undu/ata Fr., a parasitic fungus found on conifer roots such as those of Douglas-fir
seedlings and growing in acid soil in the Pacific Northwest, increased after hot slash burns (Agee 1993;
Wells eta/. 1979). Although not a major problem in B.C., Rhizina root rot can be destructive on postburn
plantations (Baranyay 1972 in Silversides eta/. 1986). Actinomycete bacteria (Streptomyces) were
reported to have produced antibiotics that inhibited mycorrhizal (Laccaria /accata [Scopp. ex Fr.] Bk. & Br.)
and pathogenic ((Phellinus weirii (Murr.) Gilbertson) fungal growth in soils from clearcut and burned sites
(Johnson and Curl 1972 in Perry and Rose 1983). Fire also resulted in a decrease in the activity of soil
invertebrates, from three months to several years afterwards (Metz and Dindal 1980 in Borchers and
Perry 1990). Heat, however, did not seem to be the cause of this decline but rather it was the postfire
changes (drier environment, decreased food supply and greater temperature fluctuations) in the soil
environment that was responsible (Ahlgren 1974).

13

Removal of the aboveground vegetation and the litter layer can lead to increased soil erosion and root
strength loss, increased soil temperature extremes, decreased transpiration and decreased soil moisture
due to the loss of shade (Wells eta/. 1979; Agee 1993; Kimmins 1997). Less visible effects of fire include
the loss of some nutrients, a decrease in soil acidity, decreased bulk density and porosity of the soil, and
changes in water repellency of the soil. In the quick combustion of organic matter, there is an immediate
loss of some nutrients, largely nitrogen and to a lesser extent other elements (Agee 1993; Wells eta/.
1979). Nitrogen is the nutrient most limited in many forest ecosystems, volatilizing at 175°C to 200°C
(White eta/. 1973). In less severe fires , non-volatilized nitrogen can be leached from the system in the
form of nitrate by nitrifying bacteria, that are sensitive to high temperatures (Agee 1993; Wells eta/. 1979).
Total losses in nitrogen caused by fire cannot be immediately replaced by natural sources (from
precipitation and free living nitrogen fixation) , however, over time, levels should return to normal (DeBell
and Ralston 1970; Binkley 1991).

Sulfur, potassium and other nutrients are converted to a more available form in residual organic material if
the fire is less severe (Agee 1993). Calcium, magnesium and sodium are transformed to soluble mineral
forms (Wells eta/. 1979) that are major components of ash (DeByle 1976; Agee 1993). These excess
basic cations increase the pH of the soil, further affecting the availability of nutrients (Ahlgren and Ahlgren
1960; Wells eta/. 1979). For example, chelated iron (Fe 3+}, the form available to plants was less soluble
at a higher pH (6.16) in a broadcast burned soil (Perry eta/. 1984). In addition, leaching of nutrients
becomes greater in soils with low cation exchange capacity than in fertile soils where nutrients tend to
adhere to clay and organic matter particles (Borchers and Perry 1990; Agee 1993).

1.8.2 Effects of fire on mycorrhizal abundance and formation
Although comparison between studies can be difficult, a majority of those reviewed reported decreases in
the number of active mycorrhizal tips following fire disturbance (Table 1). Most studies were conducted in
the Pacific Northwest using Douglas-fir as the preferred host. Different sources of mycorrhizae were
examined , including naturally regenerating seedlings, soil cores, seedlings grown in the greenhouse from
transferred soils and seedlings outplanted in disturbed and transferred soils. Total ectomycorrhizal

14

formation was usually measured by the number of active (live) root tips, and quantitative methods of
assessment varied among investigators (Table 1). Differences found in these studies could be attributed
to variation in the fire regimes, site conditions, host species and experimental protocol. Some studies
reported an increase (Pilz and Perry 1984; Brainerd and Perry 1987), however many studies reported a
decrease in ECM , from undisturbed to clearcut to burned sites (Harvey eta/. 1980; Perry eta/. 1982;
Schoenberger and Perry 1982; Parke eta/. 1984). Some interesting conclusions were also presented.
Harvey eta/. (1980) suggested that in difficult-to-regenerate sites, partial cutting may be less detrimental
than burning in terms of ECM formation . Inoculum potential was examined in greenhouse studies
conducted by Perry eta/. (1982) and Parke eta/. (1984), who found that seedlings grown in disturbed
(clearcut plus burned) soils had decreased numbers of mycorrhizal tips than those in undisturbed (mature
forest) sites. Brainerd and Perry ( 1987) examined mycorrhizae growing in soils in three sites along an
elevation and moisture gradient and concluded that seedlings growing in cold, dry environments appeared
to be more detrimentally affected by disturbance (clearcutting plus burning) than wetter and mesic sites.
Greenhouse studies conducted by Schoenberger and Perry (1987) reported an increase of ECM in
plantation soils previously clearcut plus burned (in Douglas-fir but not in western hemlock). Soil transfers
from previously clearcut plus burned plantation sites were shown by Amaranth us and Perry ( 1987) to
increase mycorrhizae formation in cold , dry sites. The authors speculated that mycorrhizae from the
plantation soils were more compatible with seedlings in the clearcut environment than those in soil
transferred from the mature forest.

15

16

Table 1. Studies on ectomycorrhizae abundance following clearcutting and burning.
Protocol
Mycorrhizae source/ treatment
Host* (age)/
Results: relative mycorrhizal abundance
location
seedlings/ undisturbed (Fd), light,
• %mycorrhizal seedlings (I < 2 yr-olds) in undisturbed> lightly burned~
Fd (1, 2 yr)/
field
and severe clearcut plus bum
Oregon, southseverely burned
central Washington
• mycorrhizae at greater depths (I yr-old) with increase in burn severity.
soil cores/ undisturbed (Fd/larch,
/western Montana
• number of mycorrhizal tips/volume of soil: undisturbed, intensive cut >
field
250 yr), intensive cut (3 yr),
partial cut plus burn.
partial cut plus burn (2 yr)
Fd (4 .5 mo .), Hw (6
• average total mycorrhizal root tips/ seedling: Fd- clearcut > undisturbed I,
soil samples/ undisturbed I (Fd,
green250+ yr; Hw, 100+ yr),
mo.)/ west-central
clearcut plus burn, natural burn > undisturbed 2, plantation; Hwhouse
undisturbed 2 (Fd, 200 yr; Hw,
Oregon, Cascades
undisturbed I and 2, clearcut, natural burn > plantation, clearcut plus
I 00 yr), clearcut (I yr), clearcut
burn
plus burn (< I yr), natural burn
• %mycorrhizal root tips: Fd- undisturbed I, clearcut> clearcut plus burn,
(36-40 yr), plantation previously
natural burn , undisturbed 2> plantation; Hw- natural burn, plantation>
clearcut plus burned ( 18 yr)
undisturbed I and 2, clearcut, clearcut plus burn.
Fd, Se, PI (4-6 mo.)/ • total and mycorrhizal root tips/ seedling: undisturbed > clearcut plus
soil samples I undisturbed (PI ,
greenI 00+ yr), clearcut plus windrow,
south-west Montana
windrow burned, clearcut, clearcut plus windrow
house
windrow burned (13 yr)
• small or no difference in % mycorrhizal tips.
soil samples I undisturbed,
Pp, Fd (14-16 wks)/
green• visual estimation of% mycorrhizal colonization of total root tips:
south-west Oregon,
house
clearcut, clearcut plus burn ( 1-22
undisturbed > clearcut > clearcut plus burn.
yr)
northern California
Fd (lyr 6.5 mo .)/
field
soil samples I undisturbed (Fd/Hw
• mean mycorrhizal tips/ seedling: by aboveground environment clearcut >
west-central
80-250 yr), clearcut (2-3 yr),
clearcut plus burn > undisturbed ; no difference looking at soil origin
Oregon, Cascades
clearcut plus burn (I yr) soils
but greater number of non-mycorrhizal tips in clearcut and clearcut plus
transferred to each undisturbed,
burned soils.
clearcut, clearcut plus burn site
greensoil samples I undisturbed,
Fd, Pp (6 mo.)/
• mycorrhizal % colonization in undisturbed soils: dry montane > moist
clearcut, clearcut plus burn (3-5
house
Oregon
montane > coastal. In disturbance soils, mycorrhizal colonization
yr)/ mesic coastal, moist and dry
significantly increased in the coastal site, and no significant change
montane forest types
occurred in other sites.
field
soil samples I undisturbed (Fw/Fd/
Fd, Ps (lyr 7 mo.)/
• increased mycorrhizal formation in Fd (doubling) from plantation soil
Ps) and plantation (previously
south-west Oregon,
transfers (previously clearcut plus burned) and in Ps from plantation
clearcut and, clearcut plus burn
northern California
soils (previously clearcut); no improvement in soil transfers from
(I 0-15 yr)) soils were transferred
undisturbed sites.
to clearcut plus bum (8-27 yr) sites
*Fd- Douglas-fir, Fw- white fir, Hw- western hemlock, PI- lodgepole pine, Pp- ponderosa pine, Ps- sugar pine, Se- Engelmann spruce

Amaranth us
and Perry
(1987)

Brainerd and
Perry ( 1987)

Pilz and Perry
(1984)

Parke eta/.
(1984)

Perry eta/.
(1982)

Schoenberger
and Perry
( 1982)

Harvey eta/.
(1980)

Wright and
Tarrant (1958)

Reference

1.8.3 Studies on mycorrhizal diversity and fire
Some studies reviewed above discussed mycorrhizal diversity based on general groupings such as white
and brown types and the black Cenococcum geophilum Fr. Using such a broad classification scheme, an
accurate estimate of the mycorrhizal diversity cannot be obtained and the analysis is not very insightful.
In a study describing 12 ectomycorrhizal morphotypes, Pilz and Perry (1984) reported that there were
fewer ECM types found in disturbed compared with the undisturbed sites and that the aboveground
changes caused by disturbance probably influenced mycorrhizal formation more than the soil
environment. Schoenberger and Perry (1982) identified five major ECM groups, and reported that those
associated with Douglas-fir were greater in abundance in unburned , clearcut soil.

Diversity studies on mycorrhizae following disturbance have recently been conducted using traditional
measures of diversity. Visser (1995) studied the effect of time in a successional study on ectomycorrhizal
fungi in 6, 41 , 65 and 122-yr-old jack pine stands following wildfire and reported a significant increase in
mycorrhizal species richness between the 6 and 41-yr-old stands. Simard eta/. (1997) reported a
doubling in mean richness , diversity, and evenness of ECM on outplanted one-year-old Douglas-fir
seedlings in untrenched compared to trenched sites in 90 to 120 year-old Douglas-fir and paper birch
(Betula papyrifera Marsh.) dominated forests. Hagerman et at. (1999) reported reduced ECM richness
and diversity from soil cores collected in clearcut compared to mature forest sites, in a subalpine forest
dominated by 95 to 325 year-old subalpine fir and Engelmann spruce.

17

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septate fungal colonization of bishop pine (Pinus muricata) seedlings in the first 5 months of growth
after wildfire. Mycorrhiza, 8: 11-18.
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and Picea engelmannii. Can . J. Bot. 75: 1843-1850.
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r - - - - - - - - - - - - - - - - -- - - - - MEHMANN, B., EGLI , S., BRAUS, G.H., and BRUNNER, I. 1995. Coincidence between molecularly or
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ScHOENBERGER, M.M., and PERRY, D.A. 1982. The effect of soil disturbance on growth and
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21

SIMARD, S.W ., PERRY, D.A. , JONES, M.D., MYROLD, D. D., DURALL, D.M., MOLINA, R. 1997b. Net transfer of
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1987. Phylogenetic and ecologic aspects of mycotrophy in the angiosperms from an evolutionary
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22

2. Morphological Characterization of Ectomycorrhizae Associated with Hybrid White Spruce
Seedlings in the Aleza Lake Research Forest in the Central Interior of British Columbia

ABSTRACT

Broadcast burning is a forest management practice used for site preparation in British Columbia but its
effects on ectomycorrhizae (ECM), which provide a key link in nutrient and energy cycling within forest
ecosystems, is not well understood. To assess the effects of broadcast burning on ECM abundance and
diversity, 88 outplanted and naturally regenerating hybrid white spruce seedlings growing in two replicate
sites, each of mature, clearcut, and clearcut plus broadcast burned sites in the sub-boreal spruce (SBS)
biogeoclimatic zone were examined. Fungal symbionts were characterized by morphological assessment
and the ECM abundance and diversity were determined for each seedling. A total of 24 distinct ECM
morphotypes were described, 14 of which had known fungal affinities at the genus level. The dominant
types, matching descriptions of E-strain , Cenococcum, MRA , Amphinema, Hebeloma , The/ephora, and a
Russulaceae type showed variable treatment and seedling type differences. The morphotypes E-strain,
MRA , and Amphinema and the lightly colonized or non-mycorrhizal group were significantly more

abundant on planted seedlings in the treated (clearcut and clearcut plus burned) than untreated (mature)
sites. With respect to regenerating seedlings, Russulaceae type 1 was the most abundant morphotype in
the mature forest and Thelephora was the most abundant morphotype in the clearcut sites. The diversity
of ECM on planted seedlings between clearcut and clearcut plus burned sites was not significantly
different, however, Cenococcum was significantly less, and Hebeloma and Russulaceae type 1 were
significantly more abundant in clearcut plus burned sites compared with sites which were only clearcut.
ECM diversity of regenerating seedlings was significantly lower in clearcut sites, compared to those in
adjacent mature sites and compared to planted seedlings in the clearcut sites. Results show that
changes in the occurrence and abundance of some ECM fung i is occurring following clearcutting and
clearcutting plus burning . This suggests that disturbance may be altering the fungal composition on
hybrid white spruce seedlings on these sites towards those types best able to adapt to the changing
environmental cond itions.

23

2.1 INTRODUCTION
In British Columbia, broadcast burning has been commonly used following clearcutting to meet forest
management objectives, one of which is site preparation for seedling establishment after harvesting. This
practice impacts the soil environment, thus affecting site productivity (Wells et at. 1979; Agee 1993).
Studies conducted on the impacts of fire on soil physical and chemical properties have shown that effects
vary according to fire intensity and severity. Furthermore, burning effects are often confounded with
clearcutting, which usually precedes it (Agee 1993). Most broadcast burns, planned to be of low to
medium severity, should have less drastic impacts than severe burns. Burning impacts may include
charring and blackening of the soil surface, removal of competing vegetation , partial consumption of slash
and partial reduction of the organic layer (Silversides et at. 1996). Less obvious effects include increased
soil pH, increased availability of soil nutrients and increased soil temperature extremes (Wells et at. 1979;
Agee 1993). Severe burns may cause more deleterious impacts than less severe burns such as soil
erosion, volatilization of soil nutrients, decreased soil porosity and decreased water infiltration (Wells et at.
1979; Agee 1993).

The effects of broadcast burning on soil organisms have not been extensively studied. One important
group in the soil is mycorrhizal fungi that live in a symbiotic relationship with plant roots , and provide
essential functions such as increased water and nutrient uptake and protection against root pathogens
(Harley and Smith 1983). Mycorrhizal fungi thus are a key link in nutrient and energy cycling within forest
ecosystems (Dighton and Mason 1985). Of the seven currently recognized mycorrhizal classes,
ectomycorrhizae (ECM) are represented by an excess of 5000 fungi which associate with likely more than
2000 plant host species (Kendrick 1992), including commercially important conifer hosts such as spruce
(Picea) and pine (Pinus) in the Central Interior of British Columbia. Ectomycorrhizal fungi have different

physiological and ecological requirements such as optimum pH and temperature for growth (Cline et at.
1987), tolerance to drought (Nilsen et at. 1998) and resistance to root pathogens (Perry and Rose 1983).
It has been proposed that seedlings growing in disturbed soil environments could benefit from having
access to a diversity of fungi; those symbionts best able to function would be favoured and could provide
a buffering capacity for seedlings to adapt to changes in the environment (Perry et at. 1987, Simard eta/.
1997). Furthermore, the loss of fungal species in a functional group might result in a diminished capacity
of the group to work (Staddon et at. 1996) and hence diminished ecosystem function or a reduction in

24

ability of seedlings to successfully grow in disturbed environments. Thus the effect of site treatment on
seedling ECM abundance and diversity should be an important consideration in forest management
practices. One of the main species used in reforestation in the Central Interior of British Columbia is
hybrid white spruce, however, few studies have examined the ECM diversity of these seedlings planted
on broadcast burned sites following harvesting.

Although some studies have been conducted on mycorrhizal formation following fire, less is known
concerning mycorrhizal diversity (Staddon eta/. 1996). Burning might be expected to directly reduce the
number of soil fungal species and sources of inoculum. Obstacles hindering fungal diversity studies
following cutting and burning include the complexity of the soil environment, the difficulty in identifying
fungal symbionts and differences in effects of fire intensity on ECM . The majority of the studies which
examined mycorrhizal formation following harvesting and fire have reported decreased ECM abundance
but comparisons between studies is difficult because of different methods used for assessment (Wright
and Tarrant 1958; Harvey eta/. 1980; Perry eta/. 1982; Schoenberger and Perry 1982; Parke eta/.
1984). For example, Wright and Tarrant (1958) assessed seedlings as being mycorrhizal or nonmycorrhizal, Schoenberger and Perry (1982) examined the percent of mycorrhizal root tips per seedling
and Parke eta/. ( 1984) used visual estimates on a scale of 20% increments. Descriptions of fungal types
in these earlier studies were rudimentary , such as categorizing types into white, brown or black groups
(Wright and Tarrant 1958), or using only macroscopic characteristics to describe types (Schoenberger
and Perry 1982), compared to more recent studies on ECM diversity. The improved resolution and
amount of information available has increased the scope of and confidence in ectomycorrhizae
identification (Agerer 1987-98; lngleby eta/. 1990; Agerer 1991 ; Agerer eta/. 1996-98; Goodman eta/.
1996). For example, Hagerman eta/. (1999) reported 39 mycorrhizal types from soil cores collected in a
three year study of ECM on clearcuts in a subalpine forest in southern British Columbia.

The main objective of the present study was to determine, using morphological characterization, the
effect of broadcast burning following clearcutting on the abundance and diversity of ECM on outplanted
and naturally regenerating hybrid white spruce seedlings growing in the sub-boreal spruce (SBS)
biogeoclimatic zone of central British Columbia. Three different site types were compared : mature
(undisturbed), clearcut only , and clearcut plus broadcast burning (cut plus burned) . A second objective
25

was to compare the ECM abundance and diversity between planted and regenerating seedlings on these
sites to assess seedling difference.

2.2 MATERIALS AND METHODS
2.2.1 Site descriptions
The study area included four treated sites (two clearcut and two cut plus burned) and two adjacent
mature forest sites, located approximately 36 km east of Prince George, British Columbia, near the
Bowron River in the south-western portion of the Aleza Lake Research Forest (Figure 1). This area is in
the SBS biogeoclimatic zone, willow wet, cool (wk1) variant. The SBS zone is situated in the Central
Interior of British Columbia with a latitudinal range of 51 o 30' to 59° N (Meidinger eta/. 1991) and a variant
elevation range of 660 to 1140 m (Delong eta/. 1996). The climate in the SBS zone is continental, with
severe snowy winters, and warm, moist, short summers and is characterized by seasonal temperature
extremes with averages below ooc for four to five months of the year and above 1ooc for two to five
months (Meidinger eta/. 1991 ). Mean annual precipitation is 440 to 900 mm, of which 25 to 50 percent is
snow. Uneven aged and multi-storied canopies have resulted from fire suppression; stand destroying
fires ranging from 20 to 1000 ha in size occur approximately every 200 years (Delong eta/. 1996).
Hybrid white spruce (Picea engelmannii (Parry ex Engelm.) x g/auca (Moench) Voss) and subalpine fir
(Abies /asiocarpa (Hook.) Nutt.) are the climax species and hybrid spruce-oak fern is the zonal
association (Meidinger et a/.1991 ). Dominant understory species at the zonal oak fern site include
Lonicera invo/ucrata (Richards.) Banks ex Spreng ., Ribes lacustre (Pers.) Poir in Lamarck, Vaccinium
membranaceum Dougl. , Rubus parviflorus Nutt., Viburnum edu/e (Michx.) Raf., Oplopanax horridus
(Smith) Miq ., Gymnocarpium dryopteris (L.) Newm., Comus canadensis L. , Orthilia secunda (L.) House
and Rubus pedatus J.E. Sm. (Delong eta/. 1996). Common soils in the Bowron River valley are
Brunisolic Gray Luvisols and Gray Luvisols, formed on loam to clay glaciolacustrine deposits (Delong et
a/. 1996). The humus forms are mor and moder types (Meidinger eta/. 1991 ). The mature forest study
sites were approximately 100 and 200 years old ; vegetation and dominant canopy tree species were
similar (Figure 2). The forest floor in the study area was dominated by both mor and moder types of
humus and the mineral soils were silt loam to clay loam in texture.

26

-------------------,-

1 mature site 1
2 mature site 2
3 clearcut site 1
4 clearcut site 2
5 clearcut burn site 1
6 clearcut burn site 2

;\
N

Figure I. Location of sites in the Aleza Lake Research Forest, Prince George Forest District (insert from Prince
George Forest District recreation map, BC MOF, FRBC, Feb. 1997. Scale approximately I :400 000).

Figure 2. A portion of the forest floor in the mature site located in the Aleza Lake Research Forest, Central Interior
of British Columbia (June 1997)

27

A

B

Figure 3. a) cut plus broadcast burned site and b) clearcut site located in the Aleza Lake Research Forest, Central
Interior of British Columbia (June 1997).

Northwood Pulp and Timber Limited held the license for both the clearcut and cut plus broadcast burned
sites (pre-harvest silviculture data is shown in Appendix A). Table 2 summarizes site treatment and
includes winter harvesting, fall burning and planting data. Planted hybrid white spruce seedlings
consisted of two-year-old (one year greenhouse plus one year nursery) stock. Following harvesting,
estimates of 30 to 46 em of moderate to heavy compact slash were typical of the clearcut sites which
were to be broadcast burned (Ron Jansen, pers. comm. 1997). Using a prescribed fire predictor/planner,
the desired reduction of the slash was targeted at four to six em, which is defined as a moderate fire
severity (Feller 1996). However, no measurements were taken to determine fire severity such as depth of
burn of the litter layer or fuel characterization. Therefore it cannot be certain that these prescribed burn
objectives were met. Furthermore, fire intensity was not measured at the time of burning (Ron Jansen,
pers. comm. 1997).

Table 2. Site descriptions and dates for clearcut (C), and cut plus broadcast burned (CB) treatments in the Aleza
Lake Research Forest.
Elevation
(m)

Harvesting
date/activity

Date/subsequent treatment

Hybrid white
spruce planting

675

1/92-3/92 (96.9 ha)

9/92 windrowing (86.6 ha)
6/93 mounding (1.9 ha)

C2
CB 1

675
686

1/92-3/92 (38.1 ha)
11 /93-3/94 (84.1 ha)

CB2

670

1/94-3/94 (38.2 ha)

9/92 windrowing, windrow burned (36.8 ha)
6/94 bum (56.3 ha), mounding (24.5 ha)
10/94 bum for hazard reduction (80.9 ha)
I 0/94 bum (36.6ha), mounding (6.1 ha)

6/93 (82.9 ha)
6/94 (49.7 ha)
6/95 (90.3 ha)
6/93 (36.8 ha)
6/95 (66.72 ha)

Site

c1

6/95 (18.47 ha)

2.2.2 Seedling sampling

On June 23 and 24, 1997, a 50 m x 50 m block was established in each study site for sampling; each
block was situated at least 10 m inside site boundaries to minimize edge effects. Blocks in the clearcut
sites were situated at least 10 m away from windrows. Within each clearcut or cut plus burned site
(Figure 3a, b), 28 planted hybrid white spruce seedlings were tagged, of which fourteen were randomly
selected (using a computer-generated random number table) and double tagged. In each of the nearby
mature sites, 16 naturally regenerating hybrid white spruce seedlings were tagged and eight were
randomly selected . Half of these double-tagged seedlings were harvested in June and the remaining
seedlings were harvested on August 27 and 28 , 1997. At this time, eight naturally regenerating hybrid
white spruce seedlings were also randomly selected from each of the clearcut sites. Almost no
29

regeneration of hybrid white spruce appeared to be occurring on either of the cut plus burned sites,
though seedlings were found growing on the landings and roads leading into these sites. Unfortunately,
this precluded sampling of regenerating seedlings on cut plus burned sites. A summary of the sampling
design is presented in Table 3.

Table 3. Sampling design for hybrid white spruce seedlings harvested from clearcut, cut plus burned and mature,
sites in the Aleza Lake Research Forest.
Seedling type
Date
Site type
Mature
Clearcut
Cut plus burned
Planted
Spring
14 (7 x 2 sites)
14 (7 x 2 sites)
Fall
14 (7 x 2 sites)
14 (7 x 2 sites)
Naturally regenerating
Spring
8 (4 x 2 sites)
Fall
8 (4 x 2 sites)
16 (8 x 2 sites)
Total
44
16
28
Seedlings were harvested with the surrounding soil to a depth and radius of approximately 20 em, then
bagged and transported in 7 L plant pots to avoid disturbing the root systems. Adjacent to each
harvested seedling (with the exception of regenerating seedlings), soil samples and slash measurements
were taken in both clearcut and cut plus burned sites. In the mature sites, where most seedlings were
growing on a woody substrate, representative soil samples were collected nearby. Local site conditions
including soil horizon thickness, soil moisture, seedling substrate and microtopography as well as
seedling height and leader growth were recorded . The seedlings (88 in total) were stored at soc until
ECM characterization.

2.2.3 Soil and seedling analysis
In the laboratory, soil pH , total carbon and nitrogen of the mineral and organic horizons, and seedling age
and basal diameter were measured . Soil pH was measured by the CaCI 2 method described in
Hendershot et at. (1993) . Air dried , sieved (2 mm mesh) mineral and organic soil samples were mixed
with 0.01 M CaCI 2 in 50 ml conical tubes at 1:2 and 1:10 (soil :solution (g/ml)) ratios , respectively. They
were then shaken mechanically (Eberbach shaker) for half an hour at high speed , allowed to settle for an
hour and measured with a two point calibration pH meter (benchtop pHIISE meter, model 420A). Soil
analysis of total carbon and nitrogen were conducted on a Carlo Erba NA 1500 Elemental Analyser using
the standard Atropine with a detection limit of 0.01 %. Approximately 5 to 10 mg of organic and 30 to 60

30

mg of mineral sieved (0.15 mm mesh) soil were used for analysis. Carbon/nitrogen (C/N) ratios were
then calculated .

Using a dissecting microscope (Olympus SZ-30), seedling age was estimated by counting bud scars on
the stem as well as growth rings on sanded cookies that had been cut just above the root collar. The 16
regenerating seedlings collected in the clearcut were aged by counting bud scars only and were
estimated to be four years old . Basal diameter was averaged over the longest and shortest diameters of
the cookies. To estimate the stand age of the mature sites, each of the five largest diameter hybrid
spruce and subalpine fir trees in each site were cored and the growth rings were counted.

2.2.4 Ectomycorrhizae characterization
For each seedling, root systems were soaked in cold water for several hours, then carefully washed to
eliminate soil particles and organic debris. Occasionally, fine forceps were used to remove remaining soil
particles, under the magnification of the dissecting microscope. The entire root system of naturally
regenerating seedlings was sampled (Figure 4a), however, for planted seedlings, only lateral and
egressed roots growing from the soil plug were selected (Figure 4b). Root systems were floated in water
2

over a grid consisting of 2 cm cells. Root samples, 2 em long, were randomly sampled until
approximately 200 tips were selected. If a cell contained greater than 20 root tips, a sub-cell (1 cm 2 in
size) was randomly sampled . Only healthy root tips (i.e. turgid root and intact meristem) with a length
greater than three times the root width were selected. To avoid confusion with branching forms, an
unbranched tip was considered as one mycorrhiza. If there were fewer than 200 root tips, all healthy tips
were sampled . Initial macroscopic observations of ECM characteristics were made using the dissecting
microscope. Subsequently, root squashes were prepared and viewed using a compound microscope
(Olympus CH-2, 100-1000x). ECM features such as fungal mantle, presence of rhizomorph , emanating
hyphae and other distinguishing characteristics were documented. Permanent slide mounts of squashes
were made, fixed with high viscosity mountant (CMCP-10, Polysciences, Inc.). Macroscopic and
microscopic features of root tips were photographed (Appendix B) using an automatic exposure (PM1OAK) photomicrographic system either attached to a dissecting microscope (Olympus BX-50) or a
compound microscope (Olympus SZ-40) .

31

8

\

'

~

''

I

Figure 4. Root systems of a) naturally regenerating and b) planted hybrid white spruce seedlings harvested from
mature forest and cut plus broadcast burned sites, respectively, in the Aleza Lake Research Forest, Central Interior
of British Columbia.

32

All root tips were categorized as mycorrhizal, non-mycorrhizal or mycorrh izal but lightly colonized
(unidentifiable because of poorly developed features) . Morphological descriptions of ECM were made
with reference to a checklist (Appendix C) adapted from manuals by Agerer (1987-98), lngleby eta/.
(1990), and Goodman eta/. (1996). If an ECM could not be readily identified to genus or species, the
morphotype was given a type name, based on conspicuous features (Appendix D). For each seedling,
the number of ectomycorrhizal morphotypes and the proportional abundance (p) of each were calculated.
If the morphotype was not found on a seedling , a value of 0 was assigned. As well , the site where
morphotypes occurred and the frequency of occurrence of each morphotype (number of seedlings on
which they occurred) were recorded . Non-mycorrhizal and lightly colonized tips were grouped together to
calculate overall ECM abundance or formation (percent colonization) on hybrid white spruce for each type
of study site.

2.2.5 Statistical analysis of morphotype abundance and diversity indices
General seedling , environmental and site characteristics were compared using Students t-test. In
addition, correlations (Pearsons product-moment) of seedling measurements (leader growth, height and
basal diameter) with abundance data were calculated . Differences in ECM abundance for seven of the
most commonly occurring morphotypes (Cenococcum, E-strain , MRA , Amphinema, Hebeloma,
The/ephora and Russulaceae type 1). as well as for the lightly colon ized, unknown category, were
determined using a one-way ANOVA (STATISTICA for Windows Release 5.1 G 1997 edition , Statsoft,
Inc.) for a completely randomized design. Data for replicate sites and for season were pooled as
determined by Students t-test using a Bonferroni correction of a = 0.004 (a I number of comparisons=
0.05 I 13). To compensate for a skewed distribution, the data were transformed by the arcsine .,Jp
function (Sakal and Rohlf 1987), where p is the proportional abundance of a morphotype on a seedling .

Due to an incomplete experimental design (see Table 3 for sampling design), a Bonferroni correction of a
= 0.01 (0.05 1 5) was used for five planned comparisons: 1) between planted seedlings in clearcut and cut
plus burned sites to test for burning effects; 2) between regenerating seedlings in mature and planted
seedlings in clearcut sites to test for cutting and seedlings effects; 3) between regenerating seedlings in
mature and planted seedlings in cut plus burned sites to test for treatment and seedling effects; 4)
,.,,.,

.:u

I

between regenerating seedlings in mature and clearcut sites to test for cutting effects; and 5) between
planted and regenerating seedlings in clearcut sites to test for seedling differences.

The ectomycorrhizal diversity for each seedling was measured using the Shannon and Simpson
composite indices, Shannon evenness and Margalef richness measures (Magurran 1988) (see Appendix
E for sample calculations). Data for lightly colonized and uncolonized tips were excluded when
calculating diversity measures and morphotype data were not transformed. Preliminary analysis (Student
t-test, Bonferroni correction of a =0.004) indicated that replicate sites and seasonal data could be pooled .
One-way ANOVA was used in a completely randomized design to determine treatment or seedling effects
on diversity as stated above.

2.3 RESULTS
2.3.1 Site and seedling characteristics
Results comparing general site and seedling characteristics are presented in Table 4. The slash height in
clearcut sites was significantly less than that on cut plus broadcast burned sites. The LFH layer
thickness, rang ing from 1.6 to 3.3 em, only differed significantly between clearcut site 1 and cut plus
burned site 1. The pH and C/N values were higher in the LFH layer than in the mineral layer. Cut plus
burned site 2 had a higher pH value than other treated sites. No differences were found for the C/N
values between any of the sites. Seedling ages ranged from four to seven years (at the time of sampling)
with the youngest seedlings occurring in the cut plus burned site 2 and clearcut sites (regenerating
seedlings, data not shown) and the oldest seedlings in mature site 1. Planted seedlings were significantly
taller and had significantly greater leader growth and basal diameter than naturally regenerating seedlings
harvested from the mature sites.

34

Table 4. General site, seedlingt and soil characteristics (means ±SE)t for mature, clearcut and cut plus broadcast
burned sites sampled in the Aleza Lake Research Forest.
Mature l
Mature 2
Clearcut ~
Clearcut 2
Cut plus
Cut plus
burned 1
burned 2
Slash (em)
5.9(0.5)a
7. 1(0.8)a
10.4(0.9)b
ll.O(l.l)b
Soil data
LFH layer
thickness (em) 3.3(1.3)ab
2.8(0.3)ab
1.6(0.2)a
2.6(0.4)ab
3.0(0.3)b
2.2(0.3)ab
pH
LFH layer
4.70(0.08)ab
4 .57(0.08)ab
4.57(0.07)a
4.45(0.06)a
4.57(0.15)a
5.09(0.14)b
mineral layer
4.30(0.26)a
4.28(0.12)a
4.27(0.03)a
4. 14(0.03)a
4.11(0.09)a
4.16(0.07)a
C/N
LFH layer
35.48a
32.50a
34.25(8.90)a 25. 99(2.4 7)a 26.00(2.44)a 22.96(0.79)a
mineral layer
11.48(3.31)a
16.8(2.6)a
16.56(2.30)a
14.9(2.2)a
16.62(3 .OO)a 13 .7(1.1)a
Seedling data
6.5(0.7)a
5.8(0. 1)ab
6.0(0.2)ab
5 .5(0. 7)abc
4.9(0.1)bc
4.3(0.2)c
age(yr±)
leader growth
(em)§
3.2(0.3)a
16.6(1.2)b
16.0(1.4)b
3.4(0.2)a
20.1(1.6)b
18.5(1.3)b
height (em)
17.0(2.1)a
13.3( 1.1 )a
67. 1(4.3)b
64.9(2.3)b
69.3(3.0)b
66.5(2.2)b
basal diameter
(em)
0.3(0.l)a
0.3(0. l)a
1.7(0. l)b
1.6(0. l)b
1.5(0. l)b
1.5(0. l)b
tdata does not include measurements for regenerating seedlings harvested from clearcut sites.
~
n rows, means followed by the same letters are not significantly different (p:$;0.05) as determined by one-way
ANOVA.
§leader growth was log transformed. Values presented here are non-transformed.

2.3.2 ECM morphotype occurrence, frequency of occurrence and abundance
Morphotype occurrence for the different treatments as well as other categories is summarized in Table 5.
Overall, a total of 24 ECM morphotypes were described, four of which occurred on fewer than 5% (4) of
the seedlings. More basidiomycete (19) than ascomycete (5) fungal symbionts were described. Fourteen
types had morphological features that could be readily matched to descriptions in the published literature;
the remaining were more difficult to confirm (Appendix F).

Naturally regenerating seedlings from mature sites were associated with the most ECM morphotypes
(20}, whereas regenerating seedlings in clearcut sites had the fewest (12). Planted seedlings from the
clearcut and cut plus burned treatments had a similar number of morphotypes (17 and 18). Regenerating
seedlings were associated with more morphotypes than planted seedlings (22 versus 20) even though
fewer regenerating seedlings were examined in this study. Lightly colonized tips represented 18% of
approximately 17000 tips analyzed.

35

Table 5. Morphotype occurrence on naturally regenerating and planted hybrid white spruce seedlings in treated
(clearcut, and cut plus burned) and untreated (mature forest) sites in the Aleza Lake Research Forest, Central
Interior of British Columbia.
Site/category
Number ofmorphotypes
Occurrence*(%) Meant(±SE)
n
Mature (regenerating seedlings)
16
20
18 (2)
83
Clearcut (regenerating seedlings)
16
12
50
10 (I)
Clearcut (planted seedlings)
28
17
14 (2)
71
Cut plus burned (planted seedlings)
28
18
75
15 (1)
Shared in all sites
7
29
Over all sites
88
24
100
Ascomycetes
5
21
Basidiomycetes
19
79
On less than 4 seedlings
4
17
Regenerating seedlings
32
22
92
Planted seedlings
56
20
83
*number of occurrences(%) out of total number of ECM morphotypes (24).
tmean number ofmorphotypes are pooled over replicate sites (2) and seasons (fall and spring). Regenerating
seedlings in clearcut sites were sampled only in the fall.
The abundance and frequency of occurrence for all ECM morphotypes as well as treatment and seedling
differences for the seven most commonly occurring morphotypes and for lightly colonized tips are shown
in Table 6. Detailed statistical analyses for treatment and seedling differences are provided in Appendix
G. The most common types of ECM included the ascomycetes Cenococcum, E-strain and MRA and the
basidiomycetes Amphinema , Hebeloma, Thelephora and a Russulaceae type. Preliminary data analysis
using the Students t-test indicated that replicate sites as well as seasons could be pooled (using a
Bonferroni correction of a= 0.004), with one exception, E-strain, which showed significant differences for
the cut plus burned replicate sites in the spring. This variation may have been due to the burning of site 1
in the summer as well as in the fall.

Analysis showed that the abundance of Hebeloma (14% versus 6%) and Russulaceae type 1 (5% versus
1%) was significantly greater in the cut plus burned sites compared to the clearcut sites. In contrast, the
abundance of Cenococcum (1% versus 4%) was significantly less.

Comparing treated sites (planted seedlings) to mature (regenerating seedlings), the abundance of Estrain (6 to 13% versus 0.1%), MRA (23 to 26% versus 4%) , and Amphinema (14 to 18% versus 2%) as
well as the lightly colonized tips category (19 to 26% versus 11 %) was significantly greater in both
clearcut and cut plus burned sites. In contrast, Cenococcum (6 versus 1%) was more abundant in mature
than cut plus burned sites but was similar in abundance in mature and clearcut sites. Russulaceae type 1

36

(35% versus 1 to 5%) was significantly more abundant in mature sites than on any of the other sites,
whether seedlings were planted or regenerating. The/ephora (35% versus 3 to 8%) was significantly
more abundant on regenerating seedlings in the clearcut site than in the mature, cut plus burned, or on
planted seedlings in the clearcut sites. Piloderma was only found on regenerating seedlings in both
mature and clearcut sites and did not occur on planted seedlings (Table 6).
Table 6. Mycorrhizae morphotype abundance* (mean percent (±SE)) and frequency of occurrence (%)for planted
(pi) and naturally regenerating (r) hybrid white spruce seedlings in treated (clearcut, and cut plus burned) and
mature sites in the Aleza Lake Research Forest, Central Interior of British Columbia.
Mature-r
Clearcut-r
Clearcut-pl
Morphotype
Cut plus burn-pi
n=28
n=l6
n=l6
n=28
freq
mean
freq
mean
freq
meant
mean
freq
4.1 (0.9)a
1.6 (0 .6)ab 44
1.1 (0.5)b
Cenococcum
5.5 (1.6)a
75
56
36
0.1 (O. I)a
14.1 (7.0)ab 63
6.3 (1.3)b
12.7 (3.4)b 61
E-strain
75
13
4.3 (2 .5)a
11.2 (3.6)a
26.2 (2.8)b 100
38
69
23.3 (4.l)b 89
MRA
0.1 (0 . 1)
Tuber
1.3 (1.3)
5.7 (2.9)
4
6
38
0.3 (0.3)
6
ascomycete unknown
2.0 (0.9)a
14.7 (6.4)ab 44
Amphinema
38
17.7 (4.7)b 79
13.9 (2.8)b 64
7.4 (2.4)a b 69
4.7 (2.6)a
6.2 (1.5)a
Hebeloma
19
61
14.3 (2.3)b 82
/nocybe
0.4 (0.3)
18
2. 1 (1.2)
0.5 (0 .5)
Lace aria
14
0.5 (0.3)
6
21
5.8 (2 .7)
0.5 (0.4)
38
Piloderma
13
35.4 (6.4)a 94
3.8 (3.3)bc
1.0 (0 .6)b
Russulaceae 1
19
21
5.1 (I. 7)c
57
1.4 ( 1.0)
Russulaceae 2
1.6 ( 1.6)
13
0.2(0.1)
7
11
2.7 (1.4)a
8.3 (6 .0)a
44
34.9 (6.8)b 75
29
4.0 (1.7)a
43
Thelephora
Thelephoraceae-Iike
0.1(0.1)
6
0.8 (0.7)
Tomente/la 1
3.1 (1.4)
0.1 (0 .1)
13
4
0.1 (0.1)
50
4
1.0 (0 .9)
1.0 ( 1.0)
Tomente/la 2
13
0.3 (0.3)
7
4
Tomente/la 3
0.1 (0 .1)
6
non-rhizomorphic olive- 0.8 (0.3)
25
0.1 (0.1)
4
green
non-rhizomorphic thin
4.3 (1.4)
2.7(1.3)
50
39
2.7 (1.4)
43
mantled
non-rhizomorphic
0.7 (0 .5)
0.1(0.1)
4
7
undamped
0.1 (0.1)
non-rhizomorphic white 0.4 (0.3)
1.5(1.4)
4
19
7
14
rhizomorphic brown
0.9 (0.9)
0.6 (0.3)
0.4 (0 .3)
13
7
rhizomorphic orange
4.3 (3.9)
0.1 (0.1)
13
4
0. 1 (0.1)
3.0 (1.3)
44
4
rhizomorphic white
lightly colonized
11.0(3 . 1)a 94
7.9 (1.8)a
94
25.8 (2 .9)b 100
19.4 (2 .1)b 100
*percent abundance= number of root tips for each fungal type I total number of root tips sampled per seedling x 100.
twithin rows, means followed by the same letter are not significantly different (Bonferroni correction of a=0.01) as
determined by separate one-way ANOVA comparisons for treatments and two seedling types. Transformed data
(arcsin ..Jp, where p is morphotype abundance) were used. Means (±SE) presented are non-transformed values.

In addition to abundance and frequency differences, ten of the 22 assessed morphotypes were
significantly correlated (ps0.05, data not shown) to several seedling variables: leader growth, basal
diameter and height.

37

I

2.3.3 ECM diversity
For the diversity indices and evenness and richness measures, data for planted and regenerating
seedlings in the mature, clearcut, and cut plus burned sites were not significantly different with respect to
replicate site and season (spring and fall) (Students t-test,

~

Subsequently, replicate site and

season data were pooled for further analysis. Graphical analysis (boxplots) of the pooled data indicated a
few outliers, however these were not removed due to the small sample size. Analysis (one-way ANOVA)
did not indicate violation of the assumptions. The seedling measurements such as leader growth, basal
diameter and height were weakly correlated or non-significant for the diversity indices

~

and were

not included in statistical analysis.

No significant differences were found between clearcut, and cut plus burned sites for planted seedlings
with respect to richness, evenness or the Shannon and Simpson diversity indices (Table 7). However,
ECM diversity of regenerating seedlings in clearcut sites was significantly lower than regenerating
seedlings in mature sites and planted seedlings in the clearcut sites for both the Shannon {p=0.008) and
Margalef (p=0.001) measures (Bonferroni correction of a = 0.01 ). Simpson index values were also low
(p=0.059 and p=0 .022) for these seedlings but were not significant.

Similarity coefficients (Sorenson) supported these results; the least similar values resulted from
comparisons between regenerating seedlings in clearcut sites to those in mature sites (0.63), and to
planted seedlings in clearcut (0.62), and cut plus burned sites (0.53)(Table 8). The similarity coefficient
for planted seedlings in the clearcut treatment versus the cut plus burned treatment was fairly high (0.86).

38

Table 7. Ectomycorrhizae richness, evenness and diversity measures (Shannon 1 and Simpson2 , Shannon Evenness3
and Margalert) showing mean values (±SE). Indices were assessed using one-way ANOV A to test for treatment
effect (clearcut, cut plus burned, and unburned, mature) and to test for seedling differences (naturally regenerating,
(n=16) versus planted (n=28) of hybrid white spruce seedlings growing in the Aleza Lake Research Forest, Central
Interior of British Columbia.
p-valuet
F-statistic
Treatment/ seedling type
df(l, 54)
Cut plus burned/ planted
Clearcut /planted
0.067
1.16 (0.06)
0.797
1.19 (0.08) 1
0.003
0.954
0.61 (0.03)
0.61 (0.03) 2
0.000
0.988
0.71 (0.03)
0.71 (0.03) 3
~
0.90 (0.06)
0.000
0.978
0.90
df(l , 30)
Clearcut/ regenerating
Mature/ regenerating
0.87 (0.08)
8.174
0.008
1.30 (0.52)
0.48 (0.04)
3.844
0.059
0.61 (0.22)
0.62 (0.04)
1.355
0.254
0.69 (0.05)
14.567
1.07 (0.12)
0.59 (0.05)
0.001
df(1, 42)
Mature/ regenerating
Clearcut/ planted
0.579
1.19 (0.08)
0.451
1.30 (0.52)
0.61 (0.03)
0.000
0.991
0.61 (0.22)
0.71 (0.03)
0.058
0.69 (0.05)
0.810
0.142
0.90 (0.06)
1.07 (0. 12)
2.244
df(1 , 42)
Mature/ regenerating
Cut plus burned/ planted
1.054
1.16 (0.06)
0.311
1.30 (0.52)
0.004
0.61 (0.22)
0.61 (0.03)
0.952
0.71 (0.03)
0.776
0.69 (0.05)
0.082
1.07 (0.12)
0.90 (0.06)
1.954
0.169
df(l , 42)
Clearcut/ regenerating
Clearcut/ planted
7.849
1.19 (0.08)
0.87 (0.08)
0.008
5.618
0.48 (0.04)
0.022
0.61 (0.03)
0.62 (0.04)
2.591
0.71 (0.03)
0.115
13.453
0.59 (0.05)
0.001
0.90 (0.06)
tmeans are pooled for both replicate sites and season.
tsignificant differences indicated in bold (p:50.0 1, Bonferroni correction for planned comparisons). P-values
<0.0015 have been designated as 0.00 I.
Table 8. Sorenson similarity coefficients calculated for ectomycorrhizae of naturally regenerating (r) and planted
(pi) hybrid white spruce seedlings from unburned mature, clearcut, and cut plus burned sites in the Aleza Lake
Research Forest, Central Interior of British Columbia.
Treatment/
Similarity
Visualization
seedling comparisons*
coefficient
Mature-r versus clearcut-r
0.63
urr~r
0.87
0.63
Mature-r versus clearcut-pl
0.87
Clearcut-pl
Clearcut-r
Mature-r versus cut plus burned-pi
0.84
~
ft53
Clearcut-r versus clearcut-pl
0.62
Clearcut-r versus cut plus burned-pi
0.53
~
/..
Cut plus burned-pi
C1earcut-pl versus cut plus burned-pi
0.86
*Site data were pooled for treatment/seedling comparisons.

%

2.4 DISCUSSION
2.4.1 ECM morphotype abundance
The 24 ECM types found on hybrid white spruce in the present study , of which 14 were of recognizable
taxonomic affinities, is comparable to the numbers reported in recent studies characterizing ECM .

39

i

Twenty-two morphotypes have been described on regenerating hybrid spruce seedlings growing in the
Stone wildfire site, a Lodgepole pine (Pinus contorta var. latifo/ia Engelm.) dominated stand that was
burned in the summer of 1992 (Egger and Massicotte 1999). On Sitka spruce (Picea sitchensis (Bong .)
Carr.), 25 distinct mycorrhizal types (of which 14 were known) were reported on seedlings and trees
growing in four forest types and four nurseries in the British Isles (Thomas eta/. 1983). However, 13
ECM morphotypes were reported for naturally regenerating Sitka spruce growing in an uneven-aged
plantation forest in southern Scotland that was selectively logged (Flynn eta/. 1998). For young to
mature urban white spruce (Picea glauca (Moench) Voss) and blue spruce (Picea pungens Engelm.)
growing in Calgary, Alberta, 25 mycorrhizal types were reported (Danielson and Pruden 1989). Bruns
(1995) recently reviewed seven studies of small monoculture forests that examined fungal fruitbodies and
mycorrhizae, and reported an average of 20 to 35 species typically found on those sites. In other studies,
20, 22 and 19 ECM types, of which 14, 19 and 14 were identified to genus level or group, were described
by Simard eta/. (1997}, Visser eta/. (1998) and Hagerman eta/. (1999} , respectively.

The 20% ascomycetes and 80% basidiomycetes reported in the present study were also found to be
similar to the percentages for six-year-old jack pine stands (Visser 1995). Danielson and Pruden (1989)
reported different but highly variable values of 47(±27)% ascomycetes and 31 (±21)% basidiomycetes for
urban blue and white spruce.

Mycorrhizal formation , as measured indirectly by lightly colonized, unidentified tips and non-mycorrhizal
tips was increased on planted seedlings in treated sites compared to regenerating seedlings in clearcut
and mature forest sites (Table 6) . Th is was most likely due to seedling differences rather than to
treatment effect: planted seedlings were larger and had correspondingly larger root systems, with more
root tips that could potentially be colonized . Several studies have examined burning effects on ECM
formation , mainly conducted in the Pacific Northwest with Douglas-fir. Sources of mycorrhizae varied and
included naturally regenerating seedlings, soil cores, seedlings grown in the greenhouse on soils
transferred from disturbed sites and seedlings planted in the field growing in disturbed and transferred
soils. Some studies reported an increase (Pilz and Perry 1984; Brainerd and Perry 1987; Richter and
Bruhn 1993) or no decrease (Visser 1995) in ECM abundance following disturbance. However, many

40

studies reported a decrease in ECM , from undisturbed to clearcut to burned sites (Harvey eta/. 1980;
Perry eta/. 1982; Schoenberger and Perry 1982; Parke eta/. 1984).

In a greenhouse bioassay of Douglas-fir and western hemlock (Tsuga heterophylla (Raf.) Sarg .),
Schoenberger and Perry (1982) grew seedlings in soils from central Oregon that had been clearcut, cut
plus burned, naturally burned, and undisturbed (old growth and young growth). Douglas-fir had more
roots and ECM root tips in the unburned clearcut soils, followed by intermediate ECM colonization in the
cut plus burned soils compared to the other sites. Western hemlock had the fewest roots and ECM root
tips in the cut plus burned soils.

In another greenhouse study, Perry eta/. (1982) examined mycorrhizal formation (number of active root
tips per seedling) on Lodgepole pine, Engelmann spruce (Picea enge/mannii Parry ex Engelm.) and
Douglas-fir seedlings grown in soils from 16-year-old clearcut, clearcut plus windrowed, windrow burned
and adjacent mature forest sites in Montana. A significant decrease in the number of total and ECM root
tips occurred in clearcut, clearcut plus windrowed and windrow burned sites compared to undisturbed
forest sites.

Parke eta/. ( 1984) grew Ponderosa pine and Douglas-fir seedlings in soil retrieved from clearcut, cut plus
burned , and undisturbed sites in southwest Oregon and northern California in a greenhouse study to
determine total ECM inoculation potential. Mycorrhizal colonization was quantified by visual estimates
using a scale of zero to five that represented 20% increments. After growing seedlings 14 to 16 weeks,
ECM colonization was greatest in undisturbed forest soils (80 to 100%), followed by clearcut soils (20%
less) and cut plus burned soils (40% less). All treatments were significantly different.

Seasonal differences in ECM abundance were not observed in the present study (no significant
differences in seasonal abundance for the seven common types ). Soil moisture was not measured but
the soil was observed to be drier in August compared to June. Regenerating seedlings in the clearcut
sites, which were only sampled in early fall when soil moisture was low, had fewer lightly colonized tips
compared to planted seedlings in the disturbed sites. Reports in the literature, with respect to season and
soil moisture are variable. An increase in numbers of ECM have been reported at the beginning of the
41

growing season for mature (Harvey eta/. 1978) and clearcut sites (Richter and Bruhn 1993). However,
ECM colon ization was higher for soils in dry montane compared to moist montane and mesic coastal
sites in Oregon (Brainerd and Perry 1987). Brainerd and Perry (1987) suggested that in dry montane
environments with limited moisture and shorter growing season , ECM may be more important in
maintaining tree moisture and nutrient status. In contrast, under drought conditions, ECM was decreased
in studies examining soil cores only (Nilsen eta/. 1998) and studies examining both soil cores and
Norway spruce (Picea abies L. (Karst.)) seedlings (Feil eta/. 1988) compared to control sites and
seedlings that were not subjected to drought.

Many of the morphotypes found in the present study, such as the seven commonly occurring types
discussed here, are considered to be broad host ranging, and match descriptions of those previously
reported for spruce seedlings as well as other conifers, such as Pinus, Pseudotsuga, and Abies.
Danielson and Pruden (1989) reported E-strain as the most common morphotype (38% found on urban
white and blue spruce) , followed by Amphinema byssoides, Hebeloma , Tuber and Tomentella
(approximately 30%). The/ephora and E-strain have also been reported on greenhouse or nursery grown
spruce seedlings (Thomas eta/. 1983; lngleby eta/. 1990). Some of the dominant ECM that have been
reported in clearcut subalpine forest sites include E-strain, Lactarius, Cenococcum, Piloderma,
Hebe/oma, Amphinema and Cortinarius (Hagerman eta/. 1999). Most of these ECM with the exception
of Lactarius were also found in clearcut sites in the present study. In the Eagle wildfire site, located
adjacent to the Aleza Lake Research Forest, the most common ECM morphotypes reported on
regenerating and planted spruce seedlings in burned salvaged-logged , burned unsalvaged and unburned
sites, were similar to the seven most commonly occurring types reported in the present study (Egger and
Massicotte 1998). Morphotypes found on jack pine stands, six years following wildfire, also included
Cenococcum, E-strain , MRA , and Russula spp. and in 122-year-old jack pine stands, Cenococcum,
Hebeloma, MRA , Piloderma , Russula spp. and Tomentella spp. were found (Visser 1995).

In the present study , some differences in ECM abundance occurred between clearcut and cut plus
burned sites, and between treated (regardless of treatment type) and untreated (mature) sites. For
example, Hebeloma and Russu laceae type 1 were more abundant in cut plus burned sites than clearcut
sites but Cenococcum was less abundant. Similarly , Amphinema , E-strain , and MRA were more
42

abundant in all treatment sites (planted seedlings) compared to untreated (mature) sites whereas
Russulaceae type 1 was less abundant.

Cenococcum was less abundant on planted seedlings in cut plus burned sites compared to planted
seedlings in clearcut sites as well as seedlings in mature, undisturbed sites. ECM that successfullly
colonize seedlings in burned sites may need to possess structures or propagules capable of surviving
burns and tolerating adverse conditions in post-fire environments such as moisture stress. Cenococcum,
a common but not abundantly occurring ECM (Wright and Tarrant 1958; lngleby eta/. 1990), is thought to
be drought tolerant (Trappe 1969). It forms sclerotia, vegetative structures that may confer an advantage
by increasing survival in disturbed sites. Cenococcum geophilum sclerotia, collected from soil samples in
Wyoming , were found up to two years after fire in burned sites (Miller et at. 1994). Visser eta/. (1998)
also found Cenococcum sclerotia up to two years following clearcutting in a mixedwood site in Alberta.
Egger and Massicotte (1998) found Cenococcum ECM to be significantly less abundant on regenerating
hybrid spruce in burned salvaged-logged sites than in mature, unburned sites. Schoenberger and Perry
(1982) noted a decrease in the abundance of Cenococcum geophilum Fr. on western hemlock
greenhouse seedlings growing in soils from cut plus burned plantations. Contrary to these reports and
the present study , Pilz and Perry (1984) reported increased abundance of Cenococcum ECM on
Douglas-fir grown in clearcut plus burned areas than in clearcut areas or in the undisturbed forest.
Results by Pilz and Perry (1984) were based on macroscopic ECM characteristics; no Cenococcum
mycorrhizae descriptions were provided in the study by Schoenberger and Perry. Furthermore, it is
possible to confuse Cenococcum with other black mycorrhizae such as some Tomentel/a spp. and MRA
(Danielson 1991 ).

Hebeloma and Russulaceae type 1 were more abundant in the present study, on planted seedlings in cut
plus burned sites than in clearcut sites. One possible reason may be due to the differences in slash
heights and LFH layers in these treated sites. The depth of slash was greater in cut plus burned than in
clearcut sites where windrowing most likely removed much of the larger woody debris. Windrowing may
also have disrupted the LFH layer on the clearcut sites, and removed the nutrient rich forest floor. In
contrast, burning of finer slash would add organic matter to the LFH layer on the cut plus burned sites.
These activities may partially explain the significant differences between clearcut and cut plus burned
43

sites. More slash or a thicker LFH horizon could have beneficial moisture and nutrient effects and the
higher abundance of Hebeloma and Russulaceae type 1 in cut plus burned sites may reflect this. Visser
eta/. ( 1998) reported the presence of Hebeloma only on clearcut sites that received 10 em of wood chips

and its absence on clearcut sites with no added wood chips or with 5 em of added wood chips, in a
mixedwood site in Alberta. The increased abundance of Hebeloma and Russulaceae type 1 in cut plus
burned sites may also be due to an association of these morphotypes with other woody shrubs (willow) or
trees (birch, poplar) present on these sites that perhaps provided a source of inoculum. However, the
relative abundance of vegetation in clearcut and cut plus burned sites was not quantified in the present
study. Danielson (1991) reported Hebeloma fruitbodies under tall willows (Salix) and Visser eta/. (1998)
reported Hebeloma and Russulaceae ECM on aspen (Populus tremuloides) roots. Kernaghan eta/.
(1997) speculated that the Russulaceous ECM they studied were capable of colonizing woody shrubs
such as Betula and Salix.

Although the abundance of Amphinema, E-strain and MRA (as well as lightly colonized tips) was similar
between planted seedlings in clearcut and cut plus burned sites, significant decreases were seen when
comparing regenerating seedlings in the undisturbed mature sites to both types of treatment. Results in
the present study suggest that both seedling type and treatment effect may be influencing colonization by
these fungi for Amphinema and E-strain because intermediate abundance of these types occurred on
regenerating seedlings in clearcut sites. However, for MRA and for lightly colonized tips, seedling type
was likely influencing colonization as no significant differences were found between regenerating
seedlings in mature and clearcut sites. In the Eagle fire study , E-strain , MRA and the lightly colonized
group were more abundant and Amphinema was less abundant on regenerating spruce seedlings in
burned salvaged-logged sites compared to mature sites (Egger and Massicotte 1998). However, on
planted seedlings in that study , Amphinema was more abundant and the lightly colonized group was less
abundant in burned salvaged-logged , than in burned unsalvaged sites. ECM abundance differences
between the Eagle fire and the present study may be due to different fire types (wildfire versus broadcast
burning).

Rhizomorphs are an adaptive feature conferring advantages in disturbed sites and ECM that possess
such mycelial networks may increase access to or storage of soil "nutrients" (Harley and Smith 1983). As

44

well, mycelia emanating from active mycorrhizal roots are thought to be an important source of inoculum
for outplanted seedlings (Hagerman et at. 1999). In the present study, Amphinema was the dominant
rhizomorphic fungi found on planted seedlings in disturbed sites and this may be due to its ability to
increase access to and storage of soil nutrients as well as the ability to spread to and colonize other
seedlings via rhizomorphs . The number of years after disturbance could also be a factor in the
abundance of Amphinema ; Danielson (1991) reported a large increase in abundance of Amphinema on
outplanted white spruce growing on coal mine spoils, four and seven years after treatment with peat,
fertilizer and sewage sludge.

E-strain is believed to consist of a complex group of species (Danielson 1982; lngleby et at. 1990);
possibly including post-fire ascomycetes belonging to the order Pezizales that are commonly found
following burning of forest habitats (Petersen 1970 in Egger and Paden 1986). E-strain has been
reported as a dominant ECM on disturbed sites such as coal spoils (Danielson 1991) and clearcuts
(Hagerman et at. 1999). Additionally, E-strain fungi possess thick walled chlamydospores that may
enhance survival in the soil after disturbance (Thomas eta/. 1983). Perhaps due to its limited mantle
development, it does not compete as well in mature sites with other fungi that have thicker mantles,
rhizomorphs or numerous emanating hyphae, but is able to thrive in extreme environmental conditions
provided by disturbed sites.

MRA occurs globally and is broad host ranging . It has been reported as both ectomycorrhizal and

pathogenic though it is poorly understood in terms of its ecological function (Jumpponen and Trappe
1998). Little is known about the species that comprise MRA and possible candidates include post-fire
ascomycetes. In the present study , MRA varied morphologically: mantles were thin to well developed,
with few to abundant emanating hyphae. While the well developed MRA may be able to persist in
disturbed as well as undisturbed sites, MRA with poorly developed (patchy) mantles might be restricted to
disturbed areas. MRA has been reported as dominant in disturbed sites such as trenched soils (Simard
eta/. 1997) and amended oil sands (Danielson 1991 ).

In the present study , Russulaceae type 1 had the greatest abundance of all ECM in the mature forest. It
has been suggested that Russulaceae species may have a preference for fruiting in decaying wood in
45

North American coniferous forests (Schaffer 1975 in Kernaghan eta/. 1997). Piloderma was also
abundant in mature sites in the present study and absent from all other sites except for a minor
component of regenerating seedlings in clearcut sites. This fungus may prefer organic matter, a condition
found in mature sites and is known to possess proteolytic enzymes to enable it to extract nitrogen from
organic compounds (Dahlberg eta/. 1997). It also prefers fruiting in decayed wood and litter (Visser eta/.
1998). In a 100-year-old Norway spruce stand in southern Sweden , Piloderma croceum Erikss. & Hjortst.
accounted for 19% of the total mycorrhizal tips examined (Dahlberg eta/. 1997). In mixedwood stands of
similar age in Alberta , P. byssinum (Karst.) Jul. was equally abundant (Visser eta/. 1998). In the Eagle
fire study, both Russulaceae type 1 and Piloderma ECM of regenerating spruce were more abundant in
mature than in burned salvaged-logged sites (Egger and Massicotte 1998).

A possible difference in ECM colonization between regenerating and planted seedlings is that fungi
commonly found in greenhouses (such as Thelephora) might be an additional source of inoculum for
outplanted seedlings. Richter and Bruhn (1993) reported that Thelephora terrestris colonized Pinus roots
for all three years after outplanting. In the present study, seedlings were not assessed for ECM before
planting. However, Thelephora was found on all sites, and was most abundant on regenerating seedlings
in the clearcuts. It may be likely that some of the Thelephora tips reported on regenerating seedlings in
clearcuts were a Laccaria species, as these types have similar mantle and hyphal characteristics. Both

The/ephora and Laccaria are reported to occur in a variety of habitats (lngleby eta/. 1990). In addition,
Thelephora possesses rhizomorphs, and therefore may be better able to colonize roots of regenerating
seedlings than non-rhizomorphic types.

2.4.2 Treatment effects or seedling type differences on ectomycorrhizal diversity
In the present study , broadcast burning did not appear to affect the ECM diversity of planted seedlings
when clearcut sites were compared to cut plus burned sites. This is in contrast to the few burn studies
that have examined ECM diversity . Pilz and Perry (1984) examined ECM on Douglas-fir seedlings grown
in three western Cascade Mountain sites in undisturbed , mature (80 to 250 year old Douglas-fir/western
hemlock), clearcut, and cut plus burned soils that had been transferred to each of the undisturbed ,
clearcut, and clearcut plus burned sites. They found more types of ECM in undisturbed than disturbed
ones. Similarly , in a study of ectomycorrhizal fungal succession following wildfire in northeastern Alberta ,

46

Visser (1995) reported a significant increase in mycorrhizal species richness between the six- and 122year old stands, using soil cores for ECM assessment. In a greenhouse bioassay , Brainerd and Perry
(1987) examined the diversity of six-month-old Douglas-fir and ponderosa pine seedlings grown in soil
from disturbed (three- to five-year-old clearcut plus burned) and undisturbed forest sites in Oregon.
These sites represented a moisture/elevation gradient. Diversity (Shannon index) was highest in the dry
montane site and lowest in the mesic coastal sites for undisturbed soils. Diversity decreased in disturbed
soils in all site types. ECM morphotype information was not provided in this study .

In the present study, a significant decrease in ECM diversity for the naturally regenerating seedlings in
clearcut compared to mature sites was noted. Other studies have reported decreased diversity following
disturbance. Simard eta/. (1997) conducted a trenching study in 90 to 120 year-old Douglas-fir and
paper birch (Betula papyrifera Marsh.) dominated forests in the southern interior of British Columbia to
determine the effect of ECM occurrence on one-year-old Douglas-fir seedlings outplanted for six to 16
months. They reported a doubling in mean richness, diversity, and evenness of ECM per seedling in the
untrenched versus trenched treatment. Hagerman eta/. ( 1999) examined clearcut size effects on ECM
diversity and persistence in a subalpine forest dominated by 95 to 325 year-old subalpine fir and
Engelmann spruce in southern British Columbia. They reported reduced ECM richness and diversity as
well as reduced numbers of active fine roots in soil cores from clearcut compared to mature forest sites,
sampled two and three growing seasons after logging.

One reason for the difference in ECM diversity noted in the present study could be that the majority of
hybrid spruce seedlings found in mature sites were rooted in woody substrates. Similarly, in
approximately 100 to 200 year-old sub-boreal spruce stands in central British Columbia, 48% of hybrid
spruce seedlings were found on rotting wood substrates (Kneeshaw and Burton 1997). Woody
substrates provide a source of moisture (Harvey eta/. 1978), a haven for possible animal vectors that
help to disperse mycorrhizal inocula (Maser eta/. 1978) and possibly better access to sunlight than
seedlings on the forest floor. In a 250 year-old Douglas-fir/larch forest in western Montana, Harvey eta/.
(1976) found that in the top 38 em of soil, 95% of the active ECM were associated with organic material,
mainly humus and decayed wood . In a later study (Harvey eta/. 1981 ), they reported increased ECM
numbers with increases in organic matter (up to 45% by volume) in the top 30 em of the soil, with more

47

tips in decayed wood than in humus. This could partly account for differences seen in the present study:
the regenerating seedlings in the mature sites could benefit from the nutrient rich , moist, and shaded
environment and from less stress associated with drought and temperature extremes. Another possible
reason for differences in ECM diversity may be related to seedling age. Regenerating seedlings in
mature sites were approximately two years older than those on clearcut sites.

Regenerating seedlings in clearcut sites in the present study were also significantly less diverse than
planted seedlings in the same site. Seedling measurements showed that planted seedlings were larger
than regenerating seedlings and were therefore able to support the formation of a larger root system
which could exploit larger soil volumes and reach more fungal propagules than regenerating seedlings.
As well, planted seedlings had two years initial growth in the greenhouse and nursery soils and may have
been colonized by fungi such as Thelephora and E-strain , respectively. Fewer regenerating seedlings
were sampled on clearcut sites compared to planted seedlings. This, in combination with smaller root
systems, may account for some of the lower species richness : rare, less abundant species comprised
most of the ECM missing on regenerating seedlings.

The fact that very few regenerating seedlings were found on cut plus burned sites was unexpected. It is
unlikely that the absence of seedlings was related to the availability of mycorrhizal inoculum (planted
seedlings in the same sites were colonized) but rather was related to seed source. High seedling
regeneration may have occurred after 1993, a time when the seed crop was rated as good (John Revel
pers. comm. 1999). At this time, clearcut sites were one year old and were potentially ideal for seedling
regeneration. However, on the cut plus burned sites, cones and seedlings surviving the clearcutting (in
the winter of 1993) would have been burned in 1994. The optimum conditions for Engelmann spruce
(Picea enge/mannit) regeneration in the Engelmann Spruce-Subalpine Fir biogeoclimatic subzone are

seedbeds created by clearcutting with exposure of mineral soil compared to a seedbed of undisturbed or
burned forest floor (Feller 1998). Seedbeds created by low severity burns supported the largest number
of living spruce seedlings after three growing seasons (Feller 1998). If the seed source is insufficient,
white spruce does not readily regenerate after logging , requiring the outplanting of one- or two-year-old
nursery (or greenhouse) grown seedlings (Silversides eta/. 1986).

48

Some differences in ECM diversity and abundance may be attributed to the general site, seedlings and
soil characteristics. Those discussed previously include slash height and LFH layer thickness. Soil
characteristics such as pH and C/N ratios did not appear to differ greatly and cannot be correlated to
ECM abundance and diversity in this study as these measurements may not be representative of
rhizosphere conditions. However, they are useful for general site descriptions and determining variability
within and between sites. Carbon/nitrogen ratios were within the range of reported values in the
literature; the minimum C/N ratios for organic and mineral soils are 20, and 10 to 12, respectively (Brady
1974). A higher ratio represents a lower rate of decomposition and less readily available nitrogen and
this would be expected for the LFH layer, where nitrogen would be bound and less available than in the
mineral layer (Brady 1974). Seedling age varied among sites (four to seven years), generally being lower
in cut plus burned sites due to a later planting date than those in clearcut sites and lower in regenerating
seedlings in clearcut sites. Seedlings were older in the mature sites compared to all other sites. Finally,
although the diversity indices in this study were not strongly correlated with the measured seedling
parameters, the abundance values of nearly half of the assessed morphotypes were significantly
correlated with leader growth , seedling height and basal diameter. For example, MRA abundance
positively increased with increase in leader growth. More rigorous examination of these measurements
(soil and site characteristics and seedling parameters) followed by other types of analysis such as
canonical correspondence analysis may explain differences in ECM abundance.

Difference in diversity may not have been detected due to the uncertainties in resolving and identifying
some morphotypes, in particular some of the lightly colonized types. In the present study, the most
abundant morphotypes appeared to be fairly easy to distinguish. The Shannon and Simpson indices
assume that all species are known in the sample (Magurran 1988). Some ECM types were identifiable to
the species level but others could only be resolved to the genus or family level. Walker (1987) discussed
classification of Sitka spruce ECM claiming it was possible but not easy to determine mycorrhizae to the
genus level. Use of traditional diversity indices (Shannon and Simpson) may not reflect real differences
in diversity if a morphotype does not represent a species. The absence of some of the rare ECM on
regenerating seedlings in the clearcut sites is probably the cause of significant differences seen in the
richness measure and Shannon index, both which are sensitive to changes in the number of rare species.

49

The Shannon and Simpson indices may also be sensitive to sample size (Magurran 1988), which varied
in the present study between the naturally regenerating and planted seedlings.

In conclusion , results in the present study suggest that broadcast burning following clearcutting does not
appear to be affecting ECM diversity. However, some changes in ECM abundance occurred as a result
of both types of disturbance. The impact of these changes in abundance to seedling establishment and
growth are unknown and require further studies. Although broadcast burning did not appear to have
more adverse effects on mycorrhizal diversity than clearcutting , fungal diversity was reduced in
regenerating seedlings in clearcut sites. Applications of results in the present study to forest
management is limited to hybrid white spruce seedlings in the SBS biogeoclimatic zone willow wet, cool
(wk1) variant. ECM abundance and diversity could not be correlated to a specific burn severity without
depth of burn measurements, although a general estimate of a moderate severity was made. Future
studies should endeavor to accurately measure fire severity and intensity. It should be noted that very
few hybrid spruce seedlings regenerated on cut plus burned sites, reaffirming the current practice to
replant these sites.

50

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3. Molecular Characterization of Mycorrhizae Associated with Hybrid White Spruce Seedlings in
the Aleza Lake Research Forest, Central Interior of British Columbia
ABSTRACT

Broadcast burning is a common form of site preparation following clearcutting in coniferous forests of
British Columbia. Associated with many conifer and deciduous species, ectomycorrhizae (ECM) provide
a key link in nutrient and energy cycling within forest ecosystems. To assess the impacts of broadcast
burning practices on ectomycorrhizae (ECM) diversity , planted and naturally regenerating hybrid white
spruce seedlings, growing in the sub-boreal spruce (SBS) biogeoclimatic zone in the Central Interior of
British Columbia, were sampled from two treated (clearcut, and clearcut plus burned) as well as adjacent
mature forest sites (uncut and unburned). During an initial study on ECM morphology, seedling root
systems were characterized for mycorrhizae and approximately 1800 tips were subsampled for molecular
analysis (PCR-RFLP). RFLP analysis of eight commonly occurring ECM morphotypes as well as lightly
colonized tips revealed 12 genotypes (those that shared one or no band patterns of three restriction
endonucleases) with 18 variants (those sharing similar band patterns for two of the three restriction
endonucleases). Analysis of the commonly occurring morphotypes revealed that four (Cenococcum,
Tuber, Hebeloma and The/ephora) exhibited only one molecular genotype. However, Hebeloma and
The/ephora had several variants. The other four morphotypes (Amphinema, E-strain , MRA, and

Russulaceae type 1) possessed two or three genotypes, some with several variants. The number of
genotypes and variants appeared to increase with increasing disturbance from regenerating seedlings in
mature sites (6 genotypes, 9 variants) to planted seedlings in clearcut plus burned sites (11 genotypes,
17 variants). However, ECM molecular diversity, assessed using the Phi index, was not significantly
different between treatments or for planted versus naturally regenerating seedlings. This suggests that
the molecular diversity of ECM on hybrid white spruce seedlings was not affected by clearcutting or
clearcutting plus broadcast burning . Molecular characterization provided a more comprehensive estimate
of diversity, specifically for total species richness , in combination with morphological methods and
increased understanding of inter- and intra-specific variation with respect to ectomycorrhizal associations.

55

3.1 INTRODUCTION
Broadcast burning on clearcut sites has been used as a common method of site preparation prior to
outplanting of seedlings (Hawkes eta/. 1990). The impacts of this practice have been summarized in
reviews of numerous studies examining changes in soil chemical, physical and biological characteristics
(Wells eta/. 1979; Agee 1993). Results vary, but in general more severe fires cause the most deleterious
effects (e.g. increased soil erosion, decreased soil porosity, increased volatilization of plant nutrients and
a decreased number of soil organisms). A growing number of studies have focussed on burning effects
on important soil components such as mycorrhizae. Mycorrhizae are symbiotic fungal-plant root
associations in which the fungal partner enhances moisture and nutrient uptake in exchange for plant
carbohydrates (Harley and Smith 1983). They are believed to be essential to seedling growth and
survival. Ectomycorrhizae (ECM), one of several types of symbioses, are associated with angiosperm
and gymnosperm hosts; many of these are important commercial forest species (e.g. Picea, Pinus, Abies)
in British Columbia.

In general, previous studies of burning effects have reported decreases in ECM abundance, however,
comparisons and interpretations are sometimes difficult due to different methods of assessment. For
example, on clearcut plus burned sites in the field , Wright and Tarrant (1958) only assessed seedlings as
being mycorrhizal or non-mycorrhizal, Pilz and Perry (1984) examined the mean number of mycorrhizal
root tips per seedling and Parke eta/. ( 1984) used visual estimates on a scale of 20% increments.
Descriptions of fungal types in these studies were rudimentary , such as categorizing types into white,
brown or black groups (Wright and Tarrant 1958). In the last decade, the resolution of morphological
identification has constantly improved, due to the publication of description standards (lngleby eta/. 1990;
Agerer 1987-98; Agerer eta/. 1996-98; Goodman eta/. 1996). However, morphological identification
requires considerable skill and this may be hampered by problems such as the phenotypic variation of
ECM on different hosts and under varying environmental conditions (Egger 1995). In contrast, molecular
characterization of ECM is in theory easier to learn, is less time consuming in the processing of tips, and
examines ECM genotypes that are independent of environmental variation (Egger 1995; Gardes and
Bruns 1996). However, in order to make inferences from molecular data, molecular analysis still relies on
comparisons with identified mycorrhizal tips, based on morphological data (Horton and Bruns 1998;

56

Varga 1998, M.Sc. Thesis; Egger and Massicotte 1999); on sporocarp data (Karen eta/. 1997;
Kernaghan eta/. 1997}, or on cultures from identified root tips (Mehmann eta/. 1995).

Molecular assessment includes DNA amplification by the polymerase chain reaction (PCR, Mullis and
Faloona 1987), digestion of selected targeted sequences by restriction endonucleases and analysis of
the restriction fragment length polymorphism (RFLP) band patterns (Gardes and Bruns 1996). The
internal transcribed spacer (ITS) region of the ribosomal RNA gene unit of DNA (rONA) has been widely
used for amplification . This region lies between the 18S and 25S rRNA coding genes and contains the
5.8S rRNA gene flanked by two non-coding spacers called ITS1 and ITS2 (Gardes and Bruns 1996).
Due to its relatively rapid rate of evolution , the ITS region is suitable for identification to the species or
species group for most fungi (White eta/. 1990; Gardes and Bruns 1996). Several studies on ECM fungi
have examined this region using the PCR-RFLP method . A study by Kraigher eta/. (1995) attempted to
distinguish two species of Lactarius that are difficult to separate by morphological typing . Another study
by Kernaghan eta/. (1997) compared morphological characterization of Russulaceae mycorrhizae to
sporocarp tissue to confirm identification. Recently, Horton and Bruns (1998) examined fungal
associates for possible linkages between Douglas-fir (Pseudotsuga menziesii D. Don) and bishop pine
(Pinus muricata D. Don) and Horton eta/. (1998) examined ECM and dark septate fungal colonization on

bishop pine after wildfi re. Jonsson eta/. (1999) examined mycorrhizae and sporocarps in a
chronosequence study of ECM community and composition following wildfire in Scots pine (Pinus
sylvestris) stands. Thus, PCR-RFLP analysis of the ITS region is currently being used in a variety of

applications to address questions concerning ECM .

The Shannon and Simpson indices are commonly used diversity indices that have been applied to ECM
morphological data (Brainerd and Perry 1987; Simard eta/. 1997; Houston eta/. 1998). However, using
these indices for ECM may be problematic. For example, for some ECM , it is possible that one
morphological type (morphotype) could represent more than one species; for other ECM, one species
may simply have more than one assemblage of morphological characteristics depending on its growth
stage or on environmental conditions (Mehmann eta/. 1995). This problem of species uncertainty could
violate the assumption of the diversity ind ices: that all species in a sample are known (Magurran 1988).
However, these ind ices currently appear to be the best measure of diversity for morphological data. To
57

assess diversity using molecula r data, the Phi index has been derived by Egger (Baldwin 1999, M.Sc.
Thesis) . Using the Phi index, ECM root tips are matched with every other tip in the sample and their
distances (representative of their relatedness) are used instead of species richness and abundance data,
in calculating mycorrhizal molecular diversity of the entire sample (Egger, pers. comm. 1999).

The main objective of this study was to determine, using molecular characterization (PCR-RFLP
methods) , the effect of broadcast burning following clearcutting on the diversity of ECM on planted and
regenerating hybrid wh ite spruce growing in mature, clearcut, and cut plus burned sites in the SBS
biogeoclimatic zone of central British Columbia. In addition , the study was to explore differences in ECM
diversity between planted and regenerating seedlings and to compare molecular results with previous
morphological assessments. The study further examined the Phi index as a useful measure of diversity
for molecular analysis in place of traditional methods.

3.2 MATERIALS AND METHODS
3.2.1 Ectomycorrhizae sampling
The study area included four treated (two clearcut and two cut plus burned) and two adjacent mature
forest sites, located approximately 36 km east of Prince George, British Columbia, near the Bowron River
in the south-western portion of the Aleza Lake Research Forest. The study area is part of the SBS
biogeoclimatic zone, willow wet, cool (wk1) variant; climax tree species are hybrid white spruce (Picea
engelmannii (Parry ex Engelm.) x glauca (Moench) Voss) and subalpine fir (Abies /asiocarpa (Hook.)
Nutt.) and the hybrid spruce-oak fern is the zonal association (Meidinger eta/. 1991 ). A total of 88 hybrid
white spruce seedlings were harvested , half in late June and half in late August 1997, from 50 m x 50 m
blocks. Sampling included 16 regenerating seedlings from mature sites, 28 planted and 16 regenerating
seedlings (only sampled in the fall) from clearcut sites and 28 planted seedlings from cut plus burned
sites. Roots systems were washed and floated over a grid in water and 200 healthy (i.e. turgid roots with
intact meristems) tips were randomly sampled for each seedling . Ten percent of the tips representing
each ECM morphotype for each seedling were selected for molecular analysis (approximately 1800 root
tips) and stored in 1 ml microcentrifuge tubes at -20°C until processed . Pre-harvest silviculture
prescriptions are presented in Appendix A and morphological characterization and descriptions are
presented in Chapter 2 and Appendices B, C and D.

58

3.2.2 DNA extraction
Isolation of fungal DNA was conducted using a modified CTAB protocol (Zolan and Pukkila 1986). A thin
section of the apical end of each root tip (approximately 2 mm) was excised , using a sterile dissecting
blade for each morphotype, and was placed in a separate glass micromortar (Mandel Scientific) on ice
(-20°C) for at least 15 minutes. The frozen tips were then quickly ground and suspended in 175 111 of 2X
CTAB buffer (8 .6 ml autoclaved , millipore water, 3.54 ml 5M NaCI, 1.41 ml 1M Tris-HCI (12.1 g Tris
(hydroxymethylamino-methane, Trizma® Base, Sigma Chemical Co.) and approximately 4.2 ml HCI to
pH 8.0), 578 111 of 0.5M EDTA (Ethylenediaminetetraacetic acid) at pH 8.0, 2.89 ml of 10% CTAB
(Hexadecyltrimethly-ammonium bromide, Sigma Chemical Co.), and 28.9 111 of 2-mercapto-ethanol
solution. Tips were then reground and then transferred to sterile 1 ml microcentrifuge tubes. A final 175
111 rinse with 2X CTAB was added to the micromortars and transferred to the microcentrifuge tubes. The
contents of the tubes were incubated at 60°C in a heatblock (VWR Scientific) for 45 to 60 minutes.
Following incubation , an equal amount (approximately 350 111) of 24:1 chloroform:isoamyl alcohol solution
was added to the tubes, which were spin vortexed , then centrifuged for 10 minutes at 13000 rpm.

To precipitate the DNA, the top aqueous layer was pi petted into a new microcentrifuge tube and an equal
amount (approximately 350 11l) of absolute isopropanol (stored at -5°C) was added. The tubes were
mixed by inverting for 1 minute, then stored at -5°C for 10 minutes before being centrifuged at 13000
rpm. Contents of the tubes were removed by air suction , leaving the pellet and approximately 100 111 of
liquid. To remove salts from the pellet, about 175 f.tl of 70% ethanol (stored at -5°C) was added and the
tubes were finger vortexed a few times and then centrifuged for 3 to 5 minutes at 13000 rpm. Two more
washes with ethanol followed , then the remaining liquid was removed by air suction , leaving
approximately 50 111 of solution , which was removed by placing the tubes in a dessicator (VWR Scientific)
overnight. The remaining pellet was used for DNA amplification .

3.2.3 DNA amplification
The DNA pellet was resuspended with 50 111 of 8 mM of NaOH and heated to 60°C for 10 minutes in a
heatblock. Subsequently, 4 111 was added to 27 111 of master mix (17.2 111 autoclaved , millipore water, 3.0
11110X DNA Polymerase Buffer (BIO/CAN Scientific) , 3.0 1112mM dNTP stock solution (2mM each of

59

dATP, dCTP, dGTP and dTTP, Pharmacia Biotech), 2.4 J..! I25mM MgC1 2 , 1.2J..!I each of 10 J..!M
oligonucleotide primers ITS 1 (TCC GTA GGT GAA CCT GCG G) (White eta/. 1990) and NL6Bmun
(CAA GCG TTT CCC TTT CAA CA) (Egger 1995), and 0.08 Il l of 5un its/lll pure or 1:1 UltraTherm™ DNA
Polymerase to Polymerase Buffer (BIO/CAN) and put into 0.6 ml microcentrifuge tubes kept on ice. A
drop of mineral oil (Sigma Chemical Co.) was added to prevent evaporation and the tubes were briefly
spun to 10000 rpm . The Perkin Elmer Cetus Thermocycler was used for DNA amplification at two
settings: 1) for robust, well colonized roots , denaturation at 94°C for 45 sec., annealing temperature of
48°C for 45 sec., and an extension step at n oc starting at 130 sec., increasing 1 second per cycle for 35
cycles and ; 2) for lightly colonized roots or roots which were weakly amplified using the first protocol,
similar settings as above except cycles were extended to 40, and annealing temperature was decreased
to 46°C to increase possibility of amplification . As well, pure and 1:1 diluted DNA Polymerase were used
for lightly colonized and robust tips, respectively.

To determine whether there was sufficient DNA and to visualize double amplifications (double bands
indicating the presence of two types of fungal DNA, called doublets), about 4 f.ll of loading buffer (0.003%
bromophenol blue and 0.45% glycerol) was added to 4 f.ll of PCR product, loaded and then run with a 1
Kb ladder standard (Life Technologies) on a 0.7% agarose (Sigma Chemical Co.) gel (0.7 g agarose in
100ml of 10X TBE (108 g TRIZMA® Base, Sigma Chemical Co.), 55 g Boric acid and 40 ml 0.5 M EDTA
and deionized water to make 1 litre). To stain the resulting bands, 22 f.ll of ethidium bromide was added
to the gel when it was poured . Moderate to strong bands, ranging in size from 800-1200 base pairs, were
selected for digestion. Doublets were stored for analysis in a future study.

3.2.4 Digestion, gel electrophoresis and photography
For digestion of PCR products, three restriction endonucleases were used. Approximately 7.4 f.ll of
amplified DNA was added to 0.2 ml microcentrifuge tubes, each containing an endonuclease (0.5, 0.3
and 0.4 f.ll of Alu I, Hinf I and Rsa I (Pharmacia Biotech) , respectively) , 2 f.ll of the corresponding buffer
solution (Pharmacia Biotech) and 5 f.ll autoclaved filtered (millipore) water. These tubes were placed in
an incubator at 3rC for a minimum of 5 hrs and then centrifuged briefly to remove condensation from the
caps (if products were refrigerated) and 4 f.ll of loading buffer was added to the solution . The contents of

60

the tube were loaded and ru n on 2.5% high resolution gel (1.0% NuSieve agarose, 1.5% agarose, 1OX
TBE buffer) at approximately 90mV. Ethidium bromide (approximately 7 ,. dl100 ml) was added before
pouring the gel , enabling band patterns to be viewed under the UV light. Digital images of gels were
taken using the Gel Print 2000i photographic system (BioPhotonic Corp.) and were saved on disk as well
as printed on Mitsubishi thermal paper (K65H Mitsubishi Electric Corp.).

3 . 2.5 Analysis of molecular data
Band patterns were assessed using the RFLP analysis application software RFLPscan Plus, Version 3.0,
(© 1990-1996 Scanalytics). Band size was calibrated using the Desmile calibration method with log
piecewise linear curve fitting and bands in all lanes were matched simultaneously at a 2% tolerance level;
banding patterns across different gels were compared at a 6% variation level to compensate for
differences in gels. Fragments less than 75 base pairs were not counted to reduce the possibility of
including primer dimer products. Using RFLPscan Database, Versions 2.1 and 3.0 (© 1990-1996
Scanalytics) , 14 databases were created , separated by replicate site and season for each treatment. In
addition, databases for eight commonly occurring morphotypes (Amphinema , Cenococcum, E-strain,
Hebeloma, MRA , Russulaceae type 1, Thelephora , and Tuber) and for the group of lightly colonized but
unidentified tips were created.

Pairwise comparisons of all banding patterns were compiled for each database. Pairs of tips were
matched using a modification of Dice's index; the modification was that Dice's index was converted to a
distance value (i.e. 1-Dice's index). As calculated using the RFLPscan software, modified Dice's index=

L (polymorphic bands) I (shared bands+ total bands) I 3 (Egger unpublished). Once all the possible
pairwise combinations were examined , a distance matrix was created . Cluster analysis using the
unweighted pair-group method with arithmetic means (UPGMA) of the distance matrix was done using
the Neighbor-JoiningiUPGMA module in PHYLIP (Phylogeny Inference Package) Version 3.5c (©19861993 Joseph Felsenstein ). The UPGMA phenograms produced were viewed using TreeView, Version
Win 3.2 (© 1998 Roderic DM Page).

61

From each phenogram , clusters of tips were selected to determine intra- or inter-specific variation by
comparing the band patterns for all three enzymes. If the band size differed by more than 6% of the total
molecular weight, the variation was considered a polymorphism. Tips with similar band patterns for two
enzymes but differing for the third enzyme were classified as intraspecific variants (Gardes and Bruns
1996). If only one or no enzyme band pattern was shared , then tips were considered to be different
interspecific genotypes. Due to the large sample size, not all band patterns for all the tips were
considered when reporting the major genotypes and variants; excluded tips were those that occurred
infrequently (less than 10 in a cluster) or tips that were separated by a large distance (greater than 20%)
in the phenogram.

The newly derived Phi index (see Appendix H for calculations) was used to assess genetic diversity
between treatments and for each of the eight commonly occurring morphotypes using band patterns from
all successfully amplified and digested tips. One-way ANOVA (STATISTICA for Windows Release 5.1 G
1997 edition, Statsoft, Inc.) was used to determine significant differences between treatments and
between seedling types using a Bonferroni correction of alpha = 0.01 (a I number of comparisons =0.05 I
5) for five planned comparisons due to an incomplete experimental design. Comparisons included: 1)
between planted seedlings in clearcut and cut plus burned sites to test for burning effects; 2) between
regenerating seedlings in mature and planted seedlings in clearcut sites to test for cutting and seedlings
effects; 3) between regenerating seedlings in mature and planted seedlings in cut plus burned sites to
test for treatment and seedling effects; 4) between regenerating seedlings in mature and clearcut sites to
test for cutting effects; and 5) between planted and regenerating seedlings in clearcut sites to test for
seedling differences.

62

3.3 RESULTS
3.3.1 Amplification and digestion success rates
Of all tips selected for morpholog ical characterization , 69% (1276) were successfully amplified and
digested for RFLP analysis (Table 9). The eight commonly occurring morphotypes (Cenococcum , Estrain , MRA, Tuber, Amphinema , Hebeloma , Russulaceae type 1 and Thelephora ) plus the lightly
colonized category, were further examined for band patterns. These comprised 91% of all successfully
amplified tips. The level of amplification varied among types but in general was high among some of the
commonly occurring ECM (e.g. Cenococcum, E-strain , Amphinema , Hebe/oma and Russulaceae type 1).
For the well colonized ECM Tomentella type 1 and Piloderma , amplification rates were expected to be
higher but a number of tips were lost in early extractions.

For most well colonized tips favourable settings for amplification included denaturation at 94°C for 45
sec., annealing temperature of 48°C for 45 sec., and an extension step at

n oc starting at 130 sec.,

increasing 1 second per cycle for 35 cycles. The amplification success rate for E-strain , MRA , and nonrhizomorphic thin mantled and unclamped types as well as for lightly colonized tips was improved by
using a lower annealing temperature (48°C} and increasing the number of cycles from 35 to 40 cycles.

Unsuccessful amplification included weak bands (showing insufficient DNA for further processing) or
doublets. The morphotypes MRA, The/ephora and the category of lightly colonized tips had the highest
percentage of doublets (8, 9 and 7%, respectively) followed by E-strain (5%) .

63

Table 9. Summary of DNA amplification (PCR*) ofmycorrhizal root tips from naturally regenerating and planted
hybrid white spruce seedlings growing in the Aleza Lake Research Forest, Central Interior of British Columbia.
Morphotype
Type
Doublets
Doublets
Total
Tips
Amplification
rate(%)
code
tips
amplifiedt
%total
2
68
Cenococcum
53
77.9
I
1.5
£-strain*
149
124
83 .2
8
5.4
MRA*
204
62.6
3
326
27
8.3
4
17
58.6
Tuber
29
Ascomycete unknown
5
I
Amphinema
9
258
221
85 .7
1.2
3
I
145
115
79.3
5
3.4
Hebeloma
H
4
4
100.0
Jnocybe
14
Lacearia
E
17
82.4
A
10
50.0
Piloderma
20
164
139
84.8
Russulaceae I
F
0.6
Russulaceae 2
G
23
15
65.2
Thelephora
8
176
113
64 .2
15
8.5
p
I
I
100.0
Thelephoraceae-like
..,
.)
Tomentella I
6
17
17.6
Tomentella 2
7
9
7
77.8
Tomentella 3
100.0
I
0
I
Non-rhizomorphic olive-green
J
3
2
66.7
Non-rhizomorphic thin mantled*
L
44
32
72.7
2
4.5
Non-rhizomorphic undamped*
M
3
2
66.7
K
Non- rhizomorphic white
9
8
88 .9
Rhizomorphic brown undamped
D
10
6
60.0
Rhizomorphic orange undamped
c
15
8
53.3
12
Rhizomorphic white
B
8
66.7
lightly colonized*
NIX
339
169
49.9
25
7.4
Totals I meanst
1843
1276
69.2t
87
4.7t
*settings include denaturation at 94°C for 45 sec., annealing temperature of 46°C for 45 sec., and an extension step
at 72°C starting at 130 sec., increasing I second per cycle for 40 cycles. Otherwise, different annealing temperature
(48°C) and number of cycles (35) were used .
tincludes tips which were further digested and analysed for RFLP patterns and excludes those tips showing weak or
double bands.

64

3.3.2 Band patterns of selected ECM morphotypes
Molecular band patterns for eight commonly occurring ECM morphotypes and the lightly colonized ,
unknown group are presented in Tables 10 to 12. Table 10 shows the genotype (band pattern difference
in more than one endonuclease) and variant (band pattern difference in only one enzyme) patterns for the
four ascomycetes: Cenococcum, E-strain , MRA and Tuber. Band patterns of Cenococcum were
represented by one major genotype. E-strain showed two genotypes; for genotype 2, band patterns of
variants differed by the addition of a band in Hinf I, as well as a restriction site for Rsa I. For MRA ,
although genotype 2 only differed in the Rsa I endonuclease, it had more and different restriction sites
and was distinctly larger in size than genotype 1. The distance on the phenogram was also sufficient
(approximately 20%) to justify designating the two groups as separate genotypes. Like Cenococcum,

Tuber showed one genotype . For ascomycetes, the total band size was highest for Tuber and lowest for
Cenococcum. Most of the variants occurred on both regenerating and planted seedlings and in more
than one treatment. MRA was an exception where genotype 2, occurred only on planted seedlings.

Band patterns for the four basidiomycetes (Amphinema, Hebeloma , Russulaceae type 1 and The/ephora)
are presented in Table 11 . Three different sets of band patterns for Amphinema, varying for Alu I and

Rsa I, resulted in three genotypes. Of these, restriction sites varied from none (genotype 2, variant 1) to
three (genotype 3) for Rsa I and from two to three sites for Alu I, producing a total of six variants.
Interestingly, all band patterns for Hebeloma were similar to those of genotype 1 for Amphinema,
including its three variants. Two genotypes were defined for Russulaceae type 1: genotype 2 differed
from genotype 1 for all endonucleases in band pattern (having fewer restriction sites) but not for total
band size for Alu I and Rsa I. Variants of genotype 1 differed in total band size for Hinfl. For

The/ephora , only one genotype was resolved with two variants, differing in band patterns for A/u I.

Most band patterns were seen in more than one treatment or type of seedling (Table 11 ). Exceptions
included all three genotypes for Amphinema. Genotype 1, variant 3 for Amphinema , only occurred on
regenerating seedlings in clearcut sites, genotype 2, variant 1, occurred on cut plus burned sites only,
and genotype 3 only occurred on planted seedlings.

65

Table 10. RFLP band patterns of four ascomycete morphotypes amplified (PCR *) from naturally regenerating and
planted hybrid white spruce seedlings in the Aleza Lake Research Forest, Central Interior of British Columbia.
Genotype (no. tips)
Band patterns using
Type
Genotypet (no. tips)
Band
ern ~ using
Variant (no. tips)
three endonucleases
(no. amplified Variant:j: (no. tips)
three endonucleases
Alu I Hinfi Rsa I
Alu I Hinfi Rsa I Occurrence
Occurrence§
tips)
Gen. I (45)
438 270
261
Cenococcum
149
159
182
Var. I (45)
(53)
M-r/ C-p ll CB-pl
127
137
l!.Q
697 _..21
__22
650
679
E-strain
Gen . I (65)
686 507
983
923 Gen. I (65)
693 493
Var. 2 (29)
(124)
Var. I (36)
186
160
185
I6I
C-pll C-r/ CB-pl
C-pl/ C-r/ CB-pl
ill ill
ill I44
8I2
992 799
989
Gen. 2 (30)
672 490
886
177
I83
Var. I (30) .
_.2£
C-pl/ C-r/ CB-pl
978
ill 166
97I
ill
98I
Gen. I (108)
620 428
558
MRA
(204)
Var. I (I08)
I47 248
ill.
M-r/ C-p l/ C-r/ CB-pl
733
ill I68
880 844
Gen. 2 (32)
442
635 437
Var. I (32)
I47 248
I 55
C-pl/ CB-pl
I42
ill I64
849
I24
895
863
Tuber
Gen. I (IO)
587 327
357
(17)
Var. I (IO)
I84 307
303
M-r/ C-pl/ C-r
I44
I76
255
__!11 ill
___2.!.
I029 935
I006
* primers used for the ITS region of rDNA were ITS I and NL6Bmun.
t genotypes were defined as tips having band pattern differences in more than one enzyme (Alu I, Hinfi, or Rsa 1).
t variants were defined as tips having band pattern differences in only one enzyme. Genotypes and variants
reported here include clusters on phenograms with :2:10 tips.
§ M- mature, C- clearcut, CB- cut plus burned, r-regenerating seedling, pi-planted seedling.
~b nd patterns presented were taken from a representative tip within each variant cluster in the phenogram.

66

Table II. RFLP band patterns of four basidiomycete morphotypes amplified (PCR *)from naturally regenerating
and planted hybrid white spruce seedlings in the Aleza Lake Research Forest, Central Interior of British Columbia.
Genotype (no. tips)
Band patterns using
Type
ern ~ using
Genotypet (no. tips)
Band
Variant (no. tips)
three endonucleases
three endonucleases
(no. amplified Variantt (no. tips)
Alu I Hinf I Rsa I Occurrence
Alu I Hinfi Rsa I
tips)
Occurrence§
Gen. I (117)
562
316 763 Gen . I (124)
360
321
Amphinema
779
191
285 1!.Q Var. 2 (60)
189
289
176
Var. I (42)
(221)
M-r/ C-pll CB-pl
117
158 943 M-r/ C-pl/ C-r/
165
955
ill
CB-pl
661
152
...21 146
967
905
927
Gen . I (124)
651
322
750
Var. 3 (15)
189
282
176
C-r
167
926
.!l.Q
960
ill
924
Gen. 2 (24)
361
321
988
Var. I (24)
189
290
CB-pl
ill 165
661
150
926
320 323 Gen. 3 (37)
Gen . 3 (37)
362
319
321
579
Var. I (18)
189
286 292 Var.2(19)
190
292
286
C-pl/ CB-pl
116
169 150 C-pl/ CB-pl
166
151
ill
_2Q
670
153
.21. ill 136
981
932 901
930
848
Gen. I (77)
577
320 764 Gen . I (77)
312
Hebeloma
357
770
193
293 ill Var. 2 (3 I)
189
285
(115)
Var. I (20)
174
121
158 942 M-r/ C-pll CB-pl
M-r/ C-pll CB-pl
944
160
llQ
149
99
656
146
990
920
903
Gen . I (77)
648
749
319
Yar. 3 (26)
195
275
178
M-r/ C-pll CB-pl
165
927
ill
956
150
909
Russulaceae 1 Gen. 1 (108)
527
791
313 783 Gen. I (108)
526
258
(139)
Var. I (78)
182
258 ill Var. 2 (30)
188
217
179
M-r/ C-pll CB-pl
150
169 956 M-r/ C-r/ CB-pl
159
165
970
109
151
ill !54
970
982
_5g
..21.
988
883
Gen . 2 (23)
670
337 567
Var. I (23)
187
283
199
M-r/ C-pll C-r/ CB-pl
ill 169 ill
968
.!21 947
943
Gen. I (101)
Thelephora
576
310 783 Gen. I (101)
602
317
823
Var. I (45)
(113)
185
254 201 Var. 2 (56)
191
261
206
C-pl/ C-r/ CB-pl
984 C-r/ CB-pl
1029
159
167
118
161
123
154
ill 147
992
104
1075
107
976
1006
*primers used for the ITS region of rONA were ITS I and NL6Bmun.
tgenotypes were defined as tips having band pattern differences in more than one enzyme (Alu I, Hinfl, or Rsa 1).
tvariants were defined as tips having band pattern differences in only one enzyme. Genotypes and variants reported
here include clusters on phenograms with 2: I 0 tips.
§M- mature, C- clearcut, CB- cut plus burned, r-regenerating seedling, pi-planted seedling.
~ nd patterns presented were taken from a representative tip within each variant cluster in the phenogram.
67

Table 12 shows the analysis of band patterns for the lightly colonized category. A total of three
genotypes were resolved (one variant each) and all patterns were similar to those of four previously
described morphotypes (E-strain , MRA , Amphinema and Hebeloma) (Tables 10 and 11 ). The first band
pattern closely resembled that of E-strain (genotype 1, variant 2) . The second band pattern, representing
the largest cluster of tips in the phenogram for the lightly colonized group, matched the most common
band pattern for MRA (genotype 1, variant 1). The third band pattern matched the most common band
pattern of Amphinema as well as that of Hebeloma (genotype 1, variant 2) . All band patterns for the
lightly colonized group were found in sites where the matched morphotype also occurred . No new band
patterns were resolved for the lightly colonized groups using the criterion (a minimum of 10 tips per
cluster in the phenogram) for reporting band patterns.

Table 12. Comparison of RFLP band patterns of lightly colonized but unknown ECM with known morphological
types amplified (PCR*) from naturally regenerating and planted hybrid white spruce seedlings in the Aleza Lake
Research Forest, Central Interior of British Columbia.
Lightly colonized group
Matched reference type
Genotypet (no. tips)
Band patterns for
Genotype (no. tips)
Band patterns for
Variantt (no. tips)
unknown type using
Variant (no. tips)
reference type using
three endonucleases
Occurrence§
Occurrence
three endonucleases
Alu I Hinfl Rsa I
Alu I Hinfl Rsa I
Lightly colonized a
661
504 967 E-strain
507 983
686
Gen. 1 (65)
182
Gen. a (II)
164
185
161
Var. a (11)
Var.
2
(29)
148
144
ill
ill
C-pll CB-pl
957
816
C-pl/ C-r/ CB-pl
989
812
Lightly colonized b
634
444 559 MRA
620
428 558
149
246 ill Gen . I (108)
147
Gen. b (60)
248
175
I 13
Var. b (60)
162 732 Var. I (108)
168 733
ill
M-r/ C-pl/ C-r/ CB-pl
896
852
M-rl C-pll C-r/ CB-pl
880
844
318 760 Amphinema
Lightly colonized c
359
360
321
779
Gen. c (11)
190
288
177 Gen. I (124)
189
289 176
Ill
Var. c (11)
160 937 Var. 2 (60)
165 955
ill
M-r/ C-pll CB-pl
660
148
M-r/ C-pll C-r/ CB-pl
661
152
914
927
Hebel om a
312 770
357
Gen. I (71)
189
285
174
Var.2(31)
160 944
llQ
M-r/ C-pll CB-pl
146
656
903
*primers used for the ITS region of rONA were ITS 1 and NL6Bmun.
tgenotypes were defined as tips having band pattern differences in more than one enzyme (Alu I, Hinfl, or Rsa 1).
tvariants were defined as tips having band pattern differences in only one enzyme. Genotypes and variants reported
here include clusters on phenograms with ;;o:IO tips.
§M- mature, C- clearcut, CB- cut plus burned, r-regenerating seedling, pi-planted seedling.
~ nd patterns presented are taken from a representative tip within each variant cluster in the phenogram.

68

3.3.3 Molecular diversity for commonly occurring morphotypes
For the eight morphotypes examined plus the lightly colonized , unknown group (representing 74% of the
1155 tips amplified}, 16 genotypes and 24 variants were identified , of which four genotypes and six
variants appeared to be duplicates (Tables 10 to 13). These included the band patterns for Hebeloma
and the lightly colonized group. Combining these similar band patterns left 12 distinct genotypes and 18
variants. Cenococcum and Tuber were the least diverse, each with one genotype and variant
representing 85% and 59% of tips amplified , respectively, for these types. The/ephora and Hebeloma
also had only one genotype each , but had two and three variants, respectively . E-strain, MRA and
Russulaceae type 1 were moderately diverse, with two genotypes and two to three variants, while
Amphinema and the lightly colonized group were the most diverse, with three genotypes and three to six

variants. Morphotypes with the three lowest and three highest Phi index values were Tuber, Thelephora,
Cenococcum and the lightly colonized group, MRA and E-strain , respectively.

Table 13. Molecular genotype and variant occurrence and diversity (Phi index)* for commonly occurring ECM
morphotypes found on naturally regenerating and planted hybrid white spruce seedlings growing in treated (clearcut,
and cut plus burned) and untreated (mature) sites in the Aleza Lake Research Forest, Central Interior of British
Columbia.
Morphotype
n
Genotypest
Variantst
Variants(% tips)
Phi
Cenococcum
53
I
I
0.056
85
E-strain
124
2
3
77
0.135
MRA
204
2
2
69
0.158
Tuber
17
I
1
59
0.036
Amphinema
221
3
81
6
0.098
Hebeloma
115
I
67
3
0.063
2
94
Russulaceae I
139
3
0.074
Thelephora
113
I
2
89
0.055
Lightly
169
3
49
3
0.267
colonized§
e n~
1155
16
24
~
*all amplified tips (n) are included in diversity analysis for each morphotype.
tgenotypes were defmed as tips having band pattern differences in more than one enzyme (Alu I, Hinfl, or Rsa 1).
tvariants were defined as tips having band pattern differences in only one enzyme. Genotypes and variants reported
here include clusters on phenogram s with :::: I 0 tips.
§lightly colonized tips possessed Hartig nets but were not identifiable.

Table 14 shows an assessment of genotype and variant occurrence for treatment and for regenerating
versus planted seedlings for the eight ECM morphotypes and lightly colonized group. Previous
morphotyping showed that morphotypes were found on all sites and types of seedlings. Blank cells refer
to tips that either were not similar to any of the reported band patterns (Tables 10 to 14) and did not meet
the criterion or did not successfully amplify to determine band patterns. Tuber, an exception, did not
69

occur on cut plus burned sites. MRA and Amphinema had twice as many genotypes and variants on
planted compared to regenerating seedlings (Table 14). A trend of increasing genotypes and variants
occurred for commonly occurring morphotypes as disturbance increased . Naturally regenerating
seedlings in mature sites had the lowest number of distinctive genotypes (six and nine) and planted
seedlings in cut plus burned sites had the most (11 and 17). Regenerating seedlings in clearcut sites had
intermediate numbers (8 genotypes and 11 variants) . Planted seedlings in both treated sites had similar
numbers of genotypes and variants (Table 14).

Table 14. Number of molecular genotypes and variants for ECM found on naturally regenerating and planted hybrid
white spruce seedlings growing in treated (clearcut, and cut plus burned) and untreated (mature) sites in the Aleza
Lake Research Forest, Central Interior of British Columbia.
Morphotype
Mature/regenerating
Clearcut/regenerating
Clearcut/planted
Cut plus burned/
planted
I genotype*
I genotype
1 genotype
Cenococcum
I variant
I variantt
1 variant
E-strain
2 genotypes
2 genotypes
2 genotypes
3 variants
3 variants
3 variants
I genotype
I genotype
2 genotypes
2 genotypes
MRA
I variant
I variant
2 variants
2 variants
I genotype
I genotype
Tuber
1 genotype
I variant
I variant
I variant
I genotype
1 genotype
2 genotypes
3 genotypes
Amphinema
2 variants
2 variants
4 variants
5 variants
Hebeloma
I genotype
I genotype
1 genotype
3 variants
3 variants
3 variants
Russulaceae 1
2 genotypes
2 genotypes
2 genotypes
2 genotypes
3 variants
2 variants
2 variants
3 variants
Thelephora
I genotype
I genotype
l genotype
2 variants
I variant
2 variants
Lightly colonizedt
2 genotypes
I genotype
3 genotypes
3 genotypes
2 variants
I variant
3 variants
3 variants
II genotypes
11 genotypes
Total distinct
6 genotypes
8 genotypes
types§
9 variants
II variants
15 variants
17 variants
*genotypes were defined as tips having band pattern differences in more than one enzyme (Alu I, Hinfl, or Rsa 1).
tvariants were defined as tips having band pattern differences in only one enzyme. Genotypes and variants reported
here include clusters on trees with 2 10 tips.
tlightly colonized tips possessed Hartig nets but were not identifiable.
§all Hebeloma band patterns matched those of Amphinema; lightly colonized tips matched band patterns ofE-strain,
MRA, Amphinema and Hebeloma.

3.3.4 Treatment effects on ECM molecular diversity using the Phi, Shannon and Simpson indices
Phenograms (a total of 14 (see Appendix I for example)) generated for each site database separated by
season (spring and fall) and replicate (1 and 2) generally displayed large, distinct groups both for putative
ascomycetes (Types 1 to 5, Table 9) and basidiomycetes (Types 6 toP, Table 9). These groups were
separated by large distances (greater than approximately 30%) .
70

Preliminary analyses on Phi index values (Student's t-test, Bonferroni correction of a =0.01) showed that
neither season nor replicate site values differed significantly and that those databases could be pooled .
However, pooling both repl icate sites and season would have reduced the sample size of Phi values
(calculated for one database) to 1, precluding ANOVA. As a result, only season databases were pooled
and analysis was done on both unpooled and pooled databases to examine the outcome of pooling on
diversity assessment. No significant differences were found for treatment effect (clearcut, cut plus burned
and undisturbed) or for seedling type (naturally regenerating versus planted) using the Phi index values
as a measure of molecular diversity (one way ANOVA, a=0 .01 using a Bonferroni correction) for either
pooled or unpooled databases (Table 15). Pooling of the databases for season resulted in an increase in
the Phi values for clearcut as well as cut plus burned sites and a decrease for the mature sites but this
did not change ANOVA results.

One way ANOVA (a =0.01 using a Bonferroni correction) of Shannon and Simpson composite indices
were not significant for either treatment or seedling effect. Databases were kept separate for replicate
site and season but Shannon and Simpson values were pooled for these variables, after conducting
preliminary Student t-tests (Bonferroni correction of a=0 .01 ).

71

Table 15 . Statistical summ ation' for treatm ent effect and seedling type on molecular diversity assessed using Phi,
Shannon and Simpson index valu es (mean±SE)) for ECM assoc iated with naturally regenerating and planted hybrid
white spruce seedlings grow ing in treated (clearcut, and cut plus burn ed) and untreated (mature) sites in the Aleza
Lake Research Forest, Central Interi or of British Co lumbia.
F-statistic (dt), p-value
Treatment/seedling type comparison
Clearcut/planted
Cut plus burned/planted
F( I ,6)=3 .664, p=O. l 04
0.240(0.0 13)
0.268(0.006) 1
F( I ,2)=2.667, p=0.244
0.253(0.008)
0.270(0.006) 2
F( I ,6)=0.036, p=0.856
3.245(0.107)
3.221(0.061 )3
F( I ,6)=0.295 , p=0.606
0.942(0.007)
0.937(0.008) 4
Mature/regenerating
Clearcut/regenerating
F( I ,4)=0.877, p=0.402
0.220(0.027)
0.276(0.038)
F( I ,2)=0.322, p=0.628
0.220(0.027)
0.191(0.044)
F( I ,4)=0.032, p=0.866
3. 135(0.1 39)
3.079(0.199)
F( I ,4)=0.005, p=0.948
0.936(0.01 4)
0.935(0.0 17)
Clearcu t/planted
Mature/regenerating
F( I ,6)=0.037, p=0.854
0.268(0.006)
0.276(0.038)
F( l ,2)=3 .199, p=0.2 16
0.270(0.006)
0.191(0.044)
F( I,6)=0.470, p=0.519
3.22 1(0.061 )
3.079(0.199)
F( I,6)=0.011 , p=0.919
0.93 7(0.008 )
0.935(0.0 17)
Cut plus burned/planted
Mature/regenerating
0.240(0.013 )
F(l ,6)=0.783 , p=0.410
0.276(0.038)
0.253(0.008)
F( l ,2)= 1.961 , p=0.296
0.191(0.044)
3 .245(0.107)
F( l ,6)=0.538, p=0.491
3.079(0.199)
0.942(0.007)
F( I ,6)=0. 173, p=0.692
0.935(0.017)
Clearcut/regenerating
Clearcut/planted
F( I ,4 )=6.425 , p=0.064
0.220(0.027)
0.268(0.006)
F( I ,2)=3.285, p=0.212
0.220(0.027)
0.270(0.006)
F( l,4)=0.483 , p=0.525
3. 135(0.139)
3.221(0.061)
F( 1,4)=0.001 , p=0.994
0. 936(0.014)
0.937(0.008)
I, Bonferroni correction for planned comparisons.
One-way ANOV A, ~
1
Phi values, databases with season and replicate site data kept separate (unpooled) .
2
Phi values, databases with season data (pooled).
3
Shannon index, databases with season and replicate site data kept separate (unpooled).
4
Simpson index, databases with season and replicate site data kept separate (unpooled).

3.4 DISCUSSION
3.4.1 Genetic diversity between treatments and between seedling type
In the present study , the genetic diversity of ECM associated with hybrid white spruce, as calculated by
the Phi, Shannon and Simpson indices, did not appear to be significantly affected by treatment or
seedling type. Similarly , Baldwin (1999, M.Sc. Thesis) also found no difference in ECM diversity (Phi,
Shannon and Simpson indices) for regenerating black spruce (Picea mariana) seedlings growing in
clearcut and cut plus burned sites (of low and high intensity burns) in a mixedwood paper birch (Betula
papyrifera)-black spruce forest. In a recent study examining wildfire and salvage-logging effects on
planted and naturally regenerating hybrid white spruce seedlings, the Phi, Shannon and Simpson indices
showed no significant effects of treatment on ECM diversity (Egger and Massicotte 1999).

72

Traditional diversity indices assume that all species in the sample are known and this may be difficult to
determine with ECM . Molecular analysis differs from morphological assessment (which uses species
counts based on descriptions) because the band patterns produced could represent interspecific or
intraspecific variation of ECM . In this respect, the Phi index is a more appropriate measure than
traditional diversity indices because it uses phylogenetic distance, which may be less variable than
species descriptions, as a measurement of species relatedness. However, the accuracy of the Phi as a
measure of molecular diversity depends on the number of tips and types that are successfully amplified.

3.4.2 Genetic variation of hybrid wh ite spruce ectomycorrhizae
The 12 genotypes and 18 variants found on hybrid white spruce compares favourably to numbers
reported in other studies on ECM diversity. Varga (1998, MSc. Thesis) found 14 and 31 distinct ECM
RFLP topologies (or variants) for Sitka alder (Alnus sinuata [Regel] Rydb) and Lodgepole pine (Pinus
contorta Dougl. ex Loud . var. /atifo/ia Engelm.), respectively , in the Central Interior of British Columbia,

SBS biogeoclimatic zone. Mehmann eta/. (1995) reported 23 RFLP types (or variants) from fungal
sporocarps and cultures originating from a 40-year-old Norway spruce stand in Switzerland and Horton et
a/. (1998) reported 14 ECM molecular types (or variants) on Bishop pine seedlings five months after
wildfire. Phenograms generated in the present study were very large and one consequence of this size
was that RFLP patterns for the less abundant types as well as clusters of less than 10 tips were not
examined. Therefore the number of genotypes and variants reported in the present study is most likely a
conservative estimate.

In the present study , the morphotypes Cenococcum and Tuber were each composed of one genotype
and one variant. These ECM also had the lowest Phi values, suggesting a high degree of similarity
among their isolates. Other studies on Cenococcum have reported variable levels of genetic variation.
LoBuglio eta/. (1991 ), examining numerous isolates of Cenococcum over a large geographic area, found
high genetic variation . However, studies (more limited in geographic range) by Varga (1998, M.Sc.
Thesis}, Baldwin (1999, M.Sc. Thesis) and Egger and Massicotte (1998) reported two, four, and six types
(or variants) respectively , for Cenococcum ECM .

73

The MRA, and E-strain types are believed to consist of complexes of fungal species (lngleby et at. 1990).
These morphotypes had intermediate numbers of genotypes and variants (represented by approximately
70 and 80% of the amplified tips for these types) but had the highest Phi values (which included all tips in
the analysis). As well , MRA had more genotypes and variants on planted compared to regenerating
seedlings; morphological results reported an increase in the abundance of this morphotype as well as Estrain on planted compared to regenerating seedlings (Chapter 2) . Although individual morphotypes by
treatment using the Phi values was not assessed , high index values suggest that these more abundant
types may also be more diverse, having more genotypes or variants. Similar to the present study, Varga
(1998, M.Sc. Thesis) reported two genotypes and three variants for MRA on Lodgepole pine. Egger and
Massicotte ( 1998) reported five variants each for both E-strain and MRA.

The Russulaceae species are often difficult to separate morphologically due to similar features (Horton
and Bruns 1998). Horton and Bruns (1998) found three different Russulaceae ECM RFLP variants and
Egger and Massicotte (1998) reported four variants. In the present study, Russulaceae type 1 was
abundant in mature sites (morphologically) and amplified well for molecular analyses. Although it
possessed similar numbers of genotypes and variants (accounting for 94% of amplified tips for this type),
it had a lower Phi value and may have been less variable genetically. Phi index values are partially
determined by distance; a low value may indicate that although several genotypes exist, these may be
genetically quite similar. If there were many tips of closely related species having small distances and
only few diverse species having larger distances, Phi values might be expected to remain low, although
diverse species contribute more to the Phi index on average than closely related species (Egger pers.
comm. 1999).

Amphinema and Hebeloma ECM are also difficult to distinguish morphologically (lngleby et at. 1990).

The molecular band patterns for Hebeloma were identical to those for one genotype and its variants for
Amphinema. It is possible that Hebeloma was not found in the present study or that some Hebeloma tips

were mistaken for Amphinema . If the second scenario were true, the number of genotypes and variants
for Amphinema should be lower. Similar to the present study , Egger and Massicotte (1998) found that
Amphinema and Hebeloma had a variant in common. On Sitka alder, 2 genotypes were reported for
Hebeloma (Varga 1998, M.Sc. Thesis). Egger and Massicotte (1998) reported many variants for

74

Amphinema and eight variants for Hebeloma . Amphinema and Hebeloma had intermediate Phi values in

the present study .

The/ephora had only one genotype and two variants and these were found on both planted and

regenerating seedlings. This suggests that Thelephora occurred independently of greenhouse inoculum,
though some may have been on planted seedlings initially. This was also true for all E-strain variants,
suggesting its occurrence was not solely due to nursery inoculum. In contrast, eight variants were
reported for The/ephora from mature forest, burned-salvaged-logged , and burned-unsalvaged sites
(Egger and Massicotte 1998).

One of the most interesting groups was that of the lightly colonized , unknown group. The three
genotypes resolved were all previously characterized as belonging to MRA , E-strain, and the similar
Amphinema and Hebeloma band patterns. This suggests that lightly colonized tips may often belong to

ECM that have been characterized . Scoring these as a group may be affecting abundance rather than
richness measures as only three distinct morphotypes were found examining 50% of the tips.

With respect to the molecular variation for the commonly occurring morphotypes, the total number of
genotypes and variants appeared to increase with increase in disturbance (from mature to clearcut to cut
plus burned sites) as well as increase from regenerating to planted seedlings. Jonsson eta/. (1999) did
not find any changes in community composition following wildfire, but they did note an increased
dominance in the commonly occurring types in fire-disturbed sites. In the present study (Chapter 2, Table
14), an increase in the number of some commonly occurring types (MRA and Amphinema) occurred in
disturbed sites.

Caution should be used when interpreting intraspecific variation using the ITS region for amplification. In
some instances, the ITS region may not be variable enough to determine differences in closely related
species that other regions , such as the intergenic region (IGR) may detect (Gardes and Bruns 1996). In
other situations, some fungi isolates may exhibit so much variation in the ITS region that they would be
classified as different species (Gardes and Bruns 1996). Thus, the use of the ITS region may not reflect
real differences in intraspecific variation of some morphotypes.
75

The percentage of tips available for RFLP analysis after amplification and digestion in the present study
was 69% . This compares favourably with other studies (49% for alder and 63% for lodgepole pine (Varga
1998, M.Sc. Thesis) ; 65% for black spruce (Baldwin 1999, M.Sc. Thesis) ; 60% for hybrid white spruce
(Egger and Massicotte 1999); 56% for soil cores collected in one to 62 year-old Scots pine stands
(Jonsson eta/. 1999). Amplification rates in the present study for commonly occurring morphotypes were
more variable than those in the study by Varga (1998 M.Sc. Thesis) , who reported 67, 75 and 75% for
Cenococcum, MRA and Amphinema , respectively.

The percentage of doublets in the present study (approximately 5%) was similar to other studies (6%
(Baldwin 1999 M.Sc. Thesis); 7% (Egger and Massicotte 1999) however, a higher rate of doublets (15%)
was reported by Jonsson eta/. (1999). One of the explanations for doublet formation could be due to
heteroduplex DNA products formed in the PCR reaction (Jonsson eta/. 1999). The authors further
suggested that heteroduplexes could be attributed to heterogeneity in the amplified segment or from
cross-hybridization between slightly different amplifications products in the PCR reaction (Jonsson eta/.
1999). Another reason that doublets might occur is if ECM tips are additionally colonized with another
ECM fungi or if fungal endophytes are present and are amplified . Endophytes grow within plant root cells
(unlike ECM which grow between cells), are widely and abundantly distributed and easy to isolate.
However, they are poorly understood in terms of their ecological function (Jumpponen and Trappe 1998).
MRA has been reported to vary from being non-pathogenic (called dark septate endophytes) to

pathogenic; little is known about which species or functional groups comprise this morphotype
(Jumpponen and Trappe 1998). Nevertheless, endophytic types could have been inadvertently amplified
during molecular analysis of MRA. In the present study , MRA had one of the highest rates (8%) of
doublets. Thelephora also had a high rate of doublets (9%) as did the group of lightly colonized tips (7%).
Interestingly, a recent study examining doublets from post-fire ECM also found a high percentage in
The/ephora (34%) and MRA (10%) morphotypes as well as in the lightly colonized group (25%) (Rosling
eta/. unpublished).

3.4.3 Comparison between molecular and morphotyping characterization
Both molecular and morphological characterization methods have advantages and disadvantages in the
identification of ECM . With respect to morphological techniques, a problem often reported is the
76

tremendous environmental variation of ECM morphotypes. Types that look and are reported as different
may actually be the same, resulting in over-representation of species. In some instances, less
conspicuous or common looking (e.g. white rhizomorphic or Russulaceae) mycorrhizae are lumped as
similar morphotypes, resulting in under-representation of species. Molecular analyses appears to
partially address the concern of environmental variation . In the present study, three situations occurred:
1) one morphotype represented one RFLP genotype (e.g. Cenococcum, Tuber) ; 2) one morphotype had
one RFLP genotype but several variants (e.g. Hebeloma, Thelephora) ; and 3) one morphotype had more
than one RFLP genotype and variant (E-strain , Amphinema , Russulaceae type 1). In some instances,
morphotyping methods appeared to agree with molecular assessments whereas in others it may have
underestimated the ECM diversity.

Morphotyping is a process that requires considerable time to examine root systems, especially when
large samples need to be analysed . Molecular analysis (e.g. amplification , digestion, database matching)
does not require a lot of time beyond the initial preparation, however, considerable time can be spent on
analysing phenograms and determining the number of genotypes and variants.

The task of identifying ECM using morphological techniques can be subjective. For example,
differentiation between morphotypes is based on the observer's opinion of root colour, texture and mantle
pattern . Differences in researcher interpretation may have contributed to some of the differences in
diversity (Shannon and Margalef index) with respect to regenerating seedlings in the clearcut sites. Eight
unknown, rare types were absent on these regenerating seedlings while two other types were unique to
them (Chapter 2) . Some errors attributed to morphotyping differences might be restricted to those rare
types. However, many broad host-ranging types have been consistently reported in studies possibly
because these types may be easier to identify (such as Amphinema). Molecular data in the present study
confirm this: tips of commonly occurring morphotypes found on the same regenerating seedlings in
clearcut sites had similar band patterns when compared to tips from planted seedlings from the same site
as well as from different sites. Similar to morphological techniques, analysis of band patterns is also
subjective, both in determining band presence when examining gels, as well as determining whether a
partial digestion or a double amplification has occurred . It was observed, however, that when tips of one
morphotype were combined on one gel , differences were minimized and comparisons within a type were
77

easier to assess. Determining the number of genotypes and variants from the phenograms also requires
individual interpretation. Using the criterion of ten tips per band pattern to determine whether genotypes
or variants could be reported appeared to work well for large ECM databases, however, it may not have
assessed smaller ECM databases (e.g. Tuber) as well.

Differences in scientific terminology and methods are common problems for both morphological and
molecular techniques (Mehmann eta/. 1995). This makes comparisons between studies challenging.
Differences in morphological methods were discussed previously (see Introduction). With respect to
molecular methods, different protocols, primers or endonucleases make it difficult to compare band
patterns from different studies . ITS1-F (Gardes and Bruns 1991) and ITS4 (White eta/. 1990) have been
frequently used (Karen eta!. 1997; Kernaghan eta!. 1997; Horton and Bruns 1998) but few studies have
used NL6Bmun. The primer NL6Bmun was used in the present study as it is preferential for
basidiomycetes but it also amplifies ascomycetes. The use of different endonucleases results in different
fragment sizes and band patterns. In addition , bands may be matched at different levels of tolerance and
different fragment size calculation algorithms may be used in other studies. This can result in different
numbers of variants being reported .

A problem unique to molecular methods was the difficulty of adequately amplifying the lightly colonized
tips, and some thin-mantled MRA , thereby possibly biasing diversity analysis towards the types that
amplified well. Sometimes even well colonized tips did not amplify due to unforeseen difficulties with
extractions. However, as discussed earlier, lightly colonized tips appeared to be well represented by
previously identified species, suggesting that failure to amplify tips may not greatly affect species richness
results (genotypes and variants). Nevertheless, Phi values and diversity results that are influenced in
part by the numbers of tips, may still be affected by the inability of some tips to be amplified. Failure to
amplify some tips also made it difficult to calculate species abundance. For diversity analysis, the
variable success rate in PCR amplification meant that databases could not be created for individual
seedlings as was done with the morphological data. As a result, only one number could be calculated for
each site treatment database using the Phi index. Morphological comparisons (ANOVA) based on 16 to
28 diversity values for each site appeared to be a stronger statistical test than those based on two Phi
values.
78

Despite the differences in number of diversity values, diversity measures obtained for molecular (Phi,
Shannon and Simpson indices) and morphological (Shannon and Simpson values, evenness and
richness measures) analytical techniques were mostly in agreement. Both studies showed no differences
in ECM on planted seedlings growing in clearcut and cut plus burned sites. However, results for the
morphology assessment between regenerating seedlings in mature and clearcut sites and between
planted and naturally regenerating seedlings in the clearcut site (see Chapter 2) differed from molecular
assessment, which showed no differences for these sites. This may partly be explained by the fact that
rare types that were characterized morphologically, did not always successfully amplify. The Shannon
composite index and Margalef richness measure, both sensitive to the number of morphotypes (Magurran
1988), were perhaps better able to detect differences whereas the Phi index may not have been as
sensitive to either increases or decreases in the number of types. Rare types counted in morphological
analysis also may not have been included in molecular analysis because they did not amplify. The Phi
index may be likened to the molecular counterpart of the Simpson index, as it appeared to be more
sensitive to the most abundant species (genotypes and variants) whose distances contribute to the total
distance more than do the less abundant ones. In the morphology assessment, the Simpson index was
not found to be significant for either treatment or seedling effects in terms of diversity.

Studies using species diversity and similarity indices to measure fungal community composition in the
past, have not directly addressed community dynamics such as function and stability (Zak 1992).
However, in a recent greenhouse study conducted by van der Heijden eta/. (1998), it was shown that
arbuscular mycorrrhizal diversity was a major factor in contributing to the maintenance of plant
biodiversity. Shoot and root biomass, hyphal length, and plant phosphorus showed increasing trends
with increase in the number of mycorrhizal fungal species. Similar studies conducted for ECM would lend
support to the belief that an increase in ECM diversity is beneficial for ecosystem functioning . Zak (1992)
also suggests that examining species-abundance distributions will help to determine changes in fungal
communities over time and to predict return times as well as determine better the role of disturbance
events. In the present study however, treated sites were only compared for one growing season. In the
assessment of using two methods of ECM characterization, it was found that morphological
characterization provided a portrait of the fungal community, including rare types which may be missed or

79

may not amplify well using molecular methods. An assessment of morphotyping accuracy as well as the
variation within morphotypes (species or isolate difference) can be obtained from molecular analysis.
Research methods that choose several characterization techniques that complement each other provide
a more comprehensive view of ECM abundance and diversity.

3.4.4 Conclusions
In conclusion , both morphological and molecular techniques showed no differences in ECM diversity for
planted hybrid white spruce seedlings in the clearcut plus broadcast burned treatment compared to the
clearcut treatment. However, morphological results showed a significant treatment effect between
regenerating seedlings in clearcut and mature forest sites as well as a seedling effect between naturally
regenerating and planted seedlings in clearcut sites. Morphologically, a total of 24 distinct morphotypes
were described , 14 which were of known fungal affinities. Molecular analysis produced 12 genotypes and
18 variants for eight common ECM types plus the lightly colonized, unknown group. Differences in the
distribution and in the inter- and intra- specific variation of the commonly occurring morphotypes
(Amphinema , Cenococcum, E-strain, Hebeloma , MRA, Russulaceae type 1, The/ephora , and Tuber)

were also shown by morphological and molecular techniques. These results are limited to hybrid white
spruce seedlings, growing in mature forest sites in the SBS biogeoclimatic zone willow wet, cool (wk1)
variant as well as to windrowed clearcuts and to burns estimated to be of moderate severity. The
limitations of morphological and molecular characterization techniques seem to be shared; they both
have some subjective aspects and comparisons with other studies are difficult due to differences in
materials and protocols. Using both methods together provides a more comprehensive view of ECM
diversity as well as a verification or validation on the accuracy of each method . Improved standardization
of both methods would facilitate the ability to assess ECM diversity in complex forest ecosystems.

80

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descriptions of North American ectomycorrhizae. Mycologue Publications, Sidney, B.C.
HARLEY, J.L. , and SMITH , S.E. 1983. Mycorrhizal Symbiosis. Academic Press, London .
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HORTON, T.R. , CAzARES, E. , and BRUNS, T.D. 1998. Ectomycorrhizal , vesicular-arbuscular and dark
septate fungal colonization of bishop pine (Pinus muricata) seedlings in the first 5 months of growth
after wildfire. Mycorrhiza 8: 11-18.
HOUSTON, AP.C. , VISSER, S., and LAUTENSCHLAGER, R.A 1998. Microbial processes and fungal
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INGLEBY, K., MASON , P.A. , LAST, F.T. , and FLEMING, L.V. 1990. Identification of ectomycorrhizas. ITE
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HMSO, London , England .
JONSSON, l. , DAHLBERG, A. , NILSSON , M-C., ZACKRISSON , 0 ., and KAREN , 0. 1999. Ectomycorrhizal fungal
communities in late-successional Swedish boreal forests , and their composition following wildfire.
Molecular Ecol. 8: 205-215.
JUMPPONEN , A. , and TRAPPE, J.M. 1998. The profusion of dark septate endophytic fungi in nonectomycorrhizal fine roots of forest trees and shrubs. New Phytol. 132: 295-310.
KAREN , 0 ., HOGBERG, N., DAHLBERG, A. , JONSSON , L. , and NYLUND, J-E. 1997. Inter- and intraspecific
variation in the ITS region of rONA of ectomycorrhizal fungi in Fennoscandia as detected by
endonuclease analysis. New Phytol. 136: 313-325.
KERNAGHAN , G ., CuRRAH , R.S ., and BAYER, R.J . 1997. Russulaceous ectomycorrhizae of Abies
lasiocarpa and Picea engelmannii. Can . J. Bot. 75: 1843-1850.
KRAIGHER, H., AGERER, R. , and JAVORNIK, B. 1995. Ectomycorrhizae of Lactarius lignyotus on Norway
spruce, characterized by anatomical and molecular tools. Mycorrhiza, 5: 175-180.
LOBUGLIO, K.F., ROGERS, S.O., and WANG , C.J.K. 1991 . Variation in ribosomal DNA among isolates of
the mycorrhizal fungus Cenococcum geophilum. Can . J. Bot. 69: 2331-2343.
MAGURRAN, A.E. 1988. Ecological diversity and its measurement. Princeton University Press, Princeton,
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MEHMANN, B., EGLI , S., BRAUS, G.H., and BRUNNER, I. 1995. Coincidence between molecularly or
morphologically classified ectomycorrhizal morphotypes and fruitbodies in a spruce forest. In
Biotechnology of Ectomycorrhizae. Edited byV. Stocchi, P. Bonfante and M. Nuti. Plenum Press, New
York. pp. 41-52.
MEIDINGER D., POJAR, J., and HARPER, W .L. 1991. Sub-Boreal Spruce Zone. In Ecosystems of B.C.
Edited by D. Meidinger and J. Pojar. B.C. Ministry of Forests, Victoria, B.C. pp. 209-221 .
MULLIS, K.B ., and FALOONA, F.A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalysed
chain reaction . Methods in Enzymology, 155: 335-350.
PAGE, R.D.M. 1998. TreeView: An application to display phylogenetic trees on personal computers.
Computer Applications in the Biosciences, 12: 357-358.
PARKE, J.L. , LINDERMAN, R.G., and TRAPPE, J.M. 1984. Inoculum potential of ectomycorrhizal fungi in
forest soils of southwest Oregon and northern California . For. Sci. 30: 300-304.
PILZ, D.P., and PERRY, D.A. 1984. Impact of clearcutting and slash burning on ectomycorrhizal
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I

WELLS, C.G. , CAMPBELL, R.E., DEBANO, L.F., LEWIS, C.E. , FREDRIKSEN, R.L. , FRANKLIN , E.C., FROELICH,
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WHITE, T.J ., BRUNS T.D., LEE, S., and TAYLOR, J. 1990. Amplification and direct sequencing of fungal
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Douglas-fir forest type. US Forest Serv., Pac. Northwest Forest Range Exp. Sta. , Res. Notes 160.
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organization and role in the ecosystem 2"d ed . Edited by G.C. Carroll and D.T. Wicklow. Marcel
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6:195-200.

83

84

Appendix A. Pre-harvest characteristics of treated sites in the Aleza Lake Research Forest (from pre-harvest site prescription forms prepared by Northwood
Pulp and Timber Limited for the B.C. Ministry of Forests).
Site characteristic
Burn site 1
Burn site 2
Clearcut site 1
Clearcut site 2
Ecology:
Sxw*-oak
Sxw-devil's club Sxw - pink spirea- Sxw-oak fern
association
Sxw-devil's club
Sxw-oak fern/
Sxw-oak fern /
fern (21.90)
(43.28)
oak fern (10.60)
size (ha)
(3.88)
(29 .06)
devil's club (86.7) devil's club (36.6)
moisture
mesic
subhygric
subhygric
mesic
subhygric
subhygric
subhygric
nutrition
mesotrophic
mesotrophicsubmesotrophic mesotrophic
mesotrophicmesotrophic
mesotrophic
permesotrophic
mesotrophic
permesotrophic
Geography:
elevation (m)
686
670
- 670
675
0
0
0-5
slope%
0
0-5
n/a
19
n/a
n/a
aspect
n/a
SE
n/a
n/a
s
Soils:
mor
moder
mor
humus form
moder
moder
n/a
n/a
8-10
8-14
5-8
humus depth (em)
5
5
silty loam
silty loam
silty loam
n/a
silty loam to silty
texture
clay loam
0
0
% coarse fragments
0
0
0
total depth (em)
>50
>50
>50
>50
>50
Soil protection:
high
high
compaction
n/a
n/a
low
medium
displacement
high
medium
erosion
low
low
mass wasting
medium
high
forest floor
medium
medium
sensitivity rating
13.7
13.0
Site degradation(%) =
6
6
1.8
2.0
block road
3.0
3.9
landings
8.0
bladed structure
8.0
*Sxw- hybrid white spruce

M

85

h) outer mantle of Cenococcum showing stellate pattern; i) outer mantle of Tomentella type 1 showing regular synenchyma pattern and mounding of cells; j)
Amphinema inner mantle showing net synenchyma pattern as well as a clamped hypha in the bottom left corner (arrow); k) E-strain Hartig net; 1) Cenococcum
undamped, septate (arrows), emanating hypha; and m) Thelephora rhizomorph (bar is equal to 10 J..Lm).

L

Ascomycetes (a-b) and basidiomycetes (c-g) ectomycorrhizae (bar is equal to I mm): a) Cenococcum with abundant emanating hyphae; b) E-strain with few emanating
hyphae and hyaline apex; c) Amphinema with patchy yellow mantle and dark yellow rhizomorphs; d) Hebeloma with patchy white mantle and abundant loosely attached
emanating hyphae; e) Inocybe opaque coloured smooth mantle; f) Russulaceae type 1 smooth mantle and robust tip; and g) Tomentella type 1 with warty appearance.

Appendix B. Macroscopic and microscopic characteristics of selected ectomycorrhizae found on naturally regenerating and planted hybrid white spruce
seedlings in mature forest, clearcut, and cut plus burned sites in the Aleza Lake Research Forest, Central Interior of British Columbia.

Appendix C: Checklist for ectomycorrhizae morphological data (adapted from Goodman et al. 1996).
Date:
Location :

Fungus:
Host:
Dissecting Microscope:
Colour:
Texture:

smooth/ finel y grainy/ felty/ velvety/ warty/ woolly/ cottony/ stringy/ short spiny/ long spiny/
other_ _ _ __ __ __

Lustre:

matte/ shiny/ reflective

Branching:

monopodia! pinnate/ monopodia( pyramidal/ dichotomous/ irregular/ coralloid/ tuberculate/ not
branched/ other_ _ _ _ _ _ _ __

Tip shape:

straight/ beaded/ club-shaped/ tortuous/ bent

Dimensions:

length of system :_ _mm tip length: _ _mm

tip width:_ _mm

Rhizomorphs: no yes (attachment and abundance)_ _ _ _ _ __
Notes:

Compound Microscope:
Mantle:
Outer: felt prosenchymal net prosenchymal net synenchymal interlocking irregular synenchymal non-interlocking
irregular synenchymal regular synenchyma
Inner: felt prosenchymal net prosenchymal net synenchymal interlocking irregular synenchymal non-interlocking
irregular synenchymal regular synenchyma
Thickness: _ _fl m Cell Width :_ _ Jlm
Hartig net:

yes

no

Emanating Hyphae:
Type:
cystidial indeterminant
Colour: _ _ _ _ _ _ _ __
Width:_ _ Jlm
Length: _ _ Jlm
Clamps (location): no yes_ _ _ _ _ __
Septa: yes
no
none/ crystalline/ verrucose/ globular/ other _ _ _ __
Ornamentation:
Notes :
Mycelial Strands:
Type:
loose, undifferentiated/ smooth, undifferentiated/ slightly undifferentiated/ differentiated, random hyphae/
differentiated, central core/ highly differentiated
Hyphae: as per emanating hyphae

86

---

outer mantle (OM)/ inner mantle (IM): net
synench yma stellar pattern , both spherical and
elongated cells (3.5-5 !Jm wide), mantle 20-40
~
thick, Hartig net (HN) labyrinthic

Mantle and Hartig net
Emanating hyphae (EH)
Rhizomorph

----

87

none observed
EH: rare to frequent, 3.5-6 !Jm wide, thickwalled (- 1 !Jm wide), sometimes verrucose
(with ornaments to 0.5 !Jm), dark brown to
black, sometimes branching but usuall y
straight, septate, no clamps seen
E-strain: single ( 1-3 mm long, 0 .2
OM: net prosenchyma, hyphae can be
none observed
EH: often not seen , 5-l 0 !Jm wide, smooth to
mm wide), smooth to pinnate ly
IM :
strong ly verrucose, occas iona lly branched,
constricted at septa (3-15 ~ x 5-35 ~
branched, gray, yellow, or rusty
net synenchyma, patchy, typically large cells (to hyaline to light tan to reddish-brown, no
brown, apex sometimes appearing
clamps seen
14 !Jm wide, to 34 !Jm long), walls 0.5- I !Jm
uncolonized
w ide; mantle not always covering tip, thin , 2 to 3
cell layers (12-22 !Jm) thick, HN labyrinthic
Mycelium radicis atrovirens (MRA): OM : net prosenchyma to net synenchyma ( 1.5-9 EH: rare to abundant, 1.5-4 !Jm wide, smooth none observed
mostly single (1 .5-4 mm long, 0.2
!Jm wide to 20 !Jm long); IM: net synenchyma, to finely verrucose, hyaline, reddish-brown , or
mm wide), finely grainy, thin to well mantle 10-15 !Jm thick, often incomplete but
grey-black, no clamps seen
developed mantle, dark brown,
may be well colonized, inflated cells and
reddish brown, brown-black, greyconstricted septa, septa sometimes lens-shaped,
black or black, sometimes with a
HN labyrinthic
hyaline apex
Tuber: sing le to pyramidal ( 1-3 mm OM : variable, net prosenchyma connecting with EH : frequ ent to abundant, tapering bristle-like none observed
long, 0.4 mm wide), finel y grainy or cystidia, to interlocking irregular synenchyma
cystidia (45-130 !Jm long), I !Jm at tip, 3-5 !Jm
bristle-like to short spiny, robu st, pale (3-8 !Jm x I 0-15 ~
sometimes nonwide at base, enlarged basal cell at mantle
yellow, sandy or yellow-brown
interlocking or regular synenchyma; IM : net
edge, often lower basal septa plus one or more
colour to reddish-gold or rusty-brown synenchyma (3-5 !Jm x 10-25 !Jm) to irregular
above, thick-walled, hyaline, no clamps seen,
occasionally
very large hyaline hyphae seen
interlocking, mantle 12-40 !Jm thick, HN
near root tips. Variation : occasionally with
Iabyrinthic
fusiform cells (- 15 !Jm wide at base x 25 !Jm
long), or short hyphae (-4 x 30 !Jm)
ascomycete unknown: single (I mm OM: non-interlocking irregular synenchyma to EH: rare, 8-13 !Jm wide, walls 1.5-2 !Jm thick, none observed
long) to pinnate (2 mm long, 0.4mm regular synenchyma (4-15 x 7-30 !Jm); IM : net strongly verrucose, branched, yellow-brown, no
wide), smooth, finely grainy, or
clamps seen
synenchyma, mantle 12-23 !Jm thick, HN
warty, robust, pale green, or reddish- Iabyrinthic
brown

Ectomycorrhizae (macroscopic
features)
Ascomycetes
Cenococcum geophilum: mostly
sing le (1-4 mm long, 0.25-0.4 mm
wide), some pinnate, fin e ly grainy,
bl ack, robust, sclerotia sometimes
present

M-r

M- r,
C-r,
C-pl

M-r,
C-r,
C-pl ,
CB-pl

M-r,
C-r,
C-pl ,
CB-pl

M-r,
C-r,
C-pl ,
C B-pl

Site*

Appendix D. Ectomycorrhizae descriptions of naturally regenerating and planted hybrid white spruce seedlings growing in treated (clearcut, and cut plus
burned) and untreated (mature) sites in the Aleza Lake Research Forest, Central Interior of British Columbia.

OM: felt to net prosenchyma, elongated cells;
IM: net synenchyma (1 .5-3 ~ wide), mantle
17-19 ~un thick, HN labyrinthic

88

M-r,
C-pl,
CB-pl

none observed

M-r,
C-pl,
CB-pl

M-r,
strands frequent,
loose, undifferenC-r
tiated, 18-30 Jlm wide
or more, branching,
white to dark yellow
M-r,
none observed
C-r,
C-pl,
CB-pl

none observed

strands few to
M-r,
abundant, loose,
C-r,
undifferentiated, 20- C-pl ,
CB-pl
30 Jlm wide, white,
cream, straw or
yellow colour, some
possibly differentiated
(5 Jlm wide)
M-r,
EH : often very abundant, 2-3 Jlm wide, smooth occasional rhizomorphs observed,
C-r,
to finely verrucose, branched , hyaline to
C-pl ,
occasionally pale straw colour, round clamps at appear undifferenmost septa, anastomosis 'H' most with clamps tiated hyphae similar CB-pl
to mantle EH
none observed
CB-pl
EH: rare to frequent, 1.5-2 Jlm wide, septa
often close together (banded), hyaline, clamped

EH: often very abundant, I .5-5 Jlm wide,
sometimes slightly thick-walled, usually finely
verrucose, branched, anastomosis 'H', hyaline
to pale yellow, large clamps at most septa

OM: net prosenchyma; IM : net synenchyma
lnocybe: single (1.5 mm long) to
pinnate (6 mm long, 0.25 mm wide), (1.5-2 Jlm wide), mantle 10-20 f..tm thick, HN
smooth, milky-white to buff,
labyrinthic
somewhat robust
Laccaria: single (1-3 mm long, 0.2- OM: net prosenchyma; IM: net synenchyma (2-4 EH: frequent, determinant (I 00-180 Jlm long)
0.25 mm wide), smooth, occasionally Jlm wide), mantle I I-I8 Jlm thick, septa
as well as indeterminant hyphae, 2-5 Jlm wide,
robust, buff, cream colour to tan,
possibly thicker and closer together, mantle with thin-walled, smooth, infrequently branched,
often hyaline at tip
debris or deposits, HN labyrinthic
hyaline, flattened clamps
Piloderma: single to pinnate (3 mm OM: felt to net prosenchyma (1 .5-3 Jlm wide)
EH: very abundant, 1-3 Jlm wide, abundant
long, 0.2 mm wide), finely grainy,
with needle-like crystals, numerous parallel
crystals (sometimes needle-like (2-4 pm lon g)
woolly, hyaline to white or pale to
hyphae on surface; IM : net synenchyma (I .5-3 or round), hyaline to pale tan-yellow,
bright yellow
Jlm wide), mantle 15-34 Jlm thick, HN
anastomosis septate, 'H' type or short 'H' or
labyrinthic
almost contact type, no clamps seen
OM:
net
prosenchyma
to
irregular
(interlocking)
Russulaceae I: single to pinnate ( I-3
EH: rare to abundant, 2-4 Jlm wide, thickmm long, 0.2-0.25 mm wide), smooth synenchyma to net synenchyma (I-I2 x 3-25
walled (to I Jlm), heavily verrucose (large
to cottony, pale to dark brown, to pale Jlm), thick walls; IM: irregular to net
encrustations), hyaline, hypha! attachment
yellow-cream, robust
often towards base of mycorrhizae,
synenchyma (2-3 Jlm wide), mantle I5-20 Jlm
thick, HN labyrinthic
anastomosis 'H', large round clamps at most
septa
Russulaceae 2: single (1-3 mm long, OM: net prosenchyma to non-interlocking
EH: rare, fusiform cystidia, -2 wide x 6 Jlm
irregular synenchyma to regular synenchyma (2- long, as well as indeterminant, lightly
0.2 mm wide), smooth, greyishbrown to reddish-brown, robust
7 x 7-13 Jlm), IM: net synenchyma (2-3 Jlm
verrucose, 2-3 Jlm wide, hyaline hyphae,
wide), mantle I7-20 Jlm thick, HN Iabyrinthic
clamped

Hebeloma: single (3 mm long) to
pinnate (7 mm long, 0.2 mm wide),
cottony, dark ye llow-brown , pale
yellow or white, often patchy.

Basidiomycetes
Amphinema: single (0.25 mm wide) OM: felt prosenchyma; IM: net synenchyma
to pinnate (7 mm long), tips only
(hyphae 2-3 Jlm wide), mantle 20-23 Jlm thick,
slightly enlarged, cottony, patchy
variable, HN labyrinthic
yellow or white, few to many loosely
associated EH

OM: felt prosenchyma to net prosenchyma or
interlocking synenchyma (2-10 x 12-30 Jlm);
IM: net to non-interlocking irregular
synenchyma (2-5 Jlm wide), mantle 14-23 Jlm
thick, HN labyrinthic

89

Tomentella 2: single (2-7 mm long) to OM : non-interlocking irregular synenchyma to
pinnate (II mm long, 0.3-0.4 mm
regularsynenchyma(3-10x 7-15 Jlm); IM : net
wide), finely grainy, red-brown ,
synenchyma (3-5 Jlm wide), mantle 13-20 Jlm
robust
thick, black, HN labyrinthic
OM: variable, net prosenchyma to regular
Tomentella 3: single to pinnate,
sometimes grainy surface charcoal to isodiametric or angular synenchyma with
radiating large cells (can be interlocking
grey-greenish black, to dark blackirregular synenchyma); IM: net synenchyma,
brown
HN labyrinthic
non-rhizomorphic olive-green: single OM: net prosenchyma to net synenchyma (2-3
(1-2 mm long, 0.25 mm wide), finely Jlm wide), IM: net synenchyma (2-3 Jlm wide),
grainy, robust, olive-green,
mantle I 0-15 Jlm thick, HN labyrinthic
sometimes mottled black apically
OM: net prosenchyma, IM: net synenchyma
non-rhizomorphic thin mantled:
single (1-2 mm long, 0.2 mm wide) to (1.5-2 Jlm), 10 Jlm thick, HN Iabyrinthic
pinnate, smooth, tan/yellow to brown

Thelephoraceae-like: buff, cream,
OM : net synenchyma (or towards net
whitish-grey to slight yellowish ,
prosenchyma), rather large, branching mantle
robust, dull , rough texture, possibly
cells (3-7 Jlm wide), some elongated but not as
crystalline deposits, no EH or
obvious as lacticifers, septate; IM : net
rhizomorphs obvious
synenchyma, HN labyrinthic
Tomentella I: single (1-5 mm long) to OM : non-interlocking irregular synenchyma to
pinnate (6-1 0 mm long, 0.4-0.6 mm regular synenchyma ( 4-11 x 9-26 ~
up to 40
wide), sometimes densely clustered, Jlm on longest angle), sometimes forming
grainy to warty, dark brown to black, clusters of raised roundish cells; IM : net
some with silvery-fawn colour on
synenchyma (2-4 Jlm wide), mantle 20-30 Jlm
darker brown, robust
thick, HN labyrinthic

Thelephora: single to pinnate (1-5
mm long, 0.25 mm wide), smooth to
long spiny, frequently robust, beige
or fawn or grey to darker brown or
rusty brown

M-r,
CB-pl

M-r,
C-pl,
CB-pl

none observed

none observed

EH: frequent, 1.5-3 Jlm wide, thin-walled,
finely verrucose, olive-green, clamped

EH: rare, 1.5-2 Jlm wide, thin-walled, hyaline,
branched, no clamps seen

EH: rare to frequent, several types may be seen : not always present,
M-r,
medium to dark
C-r,
I) most abundant type, 4-7 Jlm wide, thickC-pl ,
walled, branched, dark brown, small clamps as brown, strands may
CB-pl
well as 2-5 Jlm wide, thin-walled, unbranched , have ' rope-like ',
knotted
appearance,
tan, no clamps; 2) pale to medium brown
tapering cystidia (to 120 Jlm long, 5 Jlm wide at branching, compact,
base, 1.5 Jlm at tip), thick-walled, mostly single with hyphae - 2-5 Jlm
wide, differentiated
but sometimes branching, some with basal
clamp; 3) large dark brown EH (6 Jlm wide),
thick walls (- I ~ wide), often present but
attachment not always seen, no clamps seen
M-r,
strands infrequent,
EH: abundant, 2.5-3 .5 Jlm wide, thin-walled ,
smooth , undifferentia- C-pL
branched, yellow-brown, round clamps
ted, 16-30 ~ wide, CB-pl
dark brown
C-r
EH : few, 3-4 Jlm wide, some short (30-70 Jlm none observed
long) to longer, rounded hyphal tips, smooth,
septate, brown, no clamps seen

EH: rare to very abundant, bristle (common) as strands not always
M-r,
well as whip-like (infrequent) cystidia (60-400 seen, loose to smooth, C-r,
Jlm long), sometimes tapering, 1.5-5 Jlm wide, undifferentiated, 18- C-pl,
CB-pl
thin (infrequent) to thick-walled (common),
30 Jlm wide or
sometimes retraction septa, tan to hyaline, basal greater, hyaline to tan
clamps, usually thinner walled below clamp
C-r
none observed
EH: few, 3-5 Jlm wide, some short mantle
hyphae or slightly longer (to 50 Jlm), hyaline,
smooth , no clamps seen

90

C-pl ,
EH: rare, short mantle cells (15-60 !-(m long), or none observed
CB-pl
frequent, whip-like, bristle-like cystidia (60I 00 !-(m long), 2.5-5 !-(m wide, occasionally
other finely verrucose, indeterminant, hyaline
hyphae (2-4 !-(m wide), no clamps seen
non-rhizomorphic white: single (2-4 OM: net prosenchyma, IM : net synenchyma (3-6 EH: frequent, 3-5 !-(m wide, thin-walled,
M-r,
none observed
mm long, 0.2 mm wide), smooth ,
C-pl ,
!-(m wide), mantle 13-15 !-(m thick, HN
restricted septa, branched, hyaline, flattened
yellow-brown to patchy-white
CB-pl
labyrinthic
clamps
OM : felt to net prosenchyma; IM: net
rhizomorphic brown undamped :
M-r,
strands rare to
EH : frequent, 1-2.5 !-(m wide, thin-walled ,
single (5 mm long, 0.25 mm wide),
frequent, loose to
C-pl ,
synenchyma (2-2.5 !-(m wide), mantle I 0-20 !-(m sometimes finely verrucose, rarely branched ,
finely grainy, black-brown or pale
smooth , undifferentia- CB-pl
thick, sometimes mottled black appearance, HN hyaline, no clamps seen
olive-green
labyrinthic
ted, 8-20 !-(m wide,
brown-black
OM: felt to net prosenchyma, IM: net
rhizomorphic orange undamped:
EH : frequent to abundant, tapering bristle-like strands rare to
M-r,
single (2-3 mm long, 0.3 mm wide), synenchyma (2-3 !-(m wide), mantle 15-22 !-(m
CB-pl
frequent, loose, to
cystidia (20-70 !-(m long), also indeterm in ant
finely grainy to short spiny, robust,
thick, HN labyrinthic
hyphae, 2-2.5 !-(m wide, tan colour, no clamps smooth , undifferenolder tips patchy rusty orange/brown,
tiated, 13-15 !-(m
seen
sclerotia
wide, light orangebrown
M-r,
strands abundant,
rhizomorphic white: single (2-4 mm OM: net prosenchyma (2-5 !-(m wide), IM: net
EH: frequent, 2-5 !-(m wide, thin-walled,
long, 0.2 mm wide), woolly to
restricted septa, branched, hyaline, anastomoses loose, undifferentia- C-pl
synenchyma (3-5 !-(m wide), mantle 10-20 !-(m
cottony, robust, patchy to uniformly thick, HN labyrinthic
present, clamped
ted, 20-30 !-(m wide,
white
white
*r=regenerating, pl=planted, M=mature forest site, C=clearcut site, CB=cut plus burned site

non-rhizomorphic undamped: single OM: felt to net prosenchyma, IM: net
(2-4 mm long, 0.2 mm wide), smooth synenchyma (2-4 ~ wide), mantle 20-26 !-(m
to finely grainy, reddish-brown at
thick, HN Iabyrinthic
base to yellow-brown at tip, as well
as patchy yellow or white

Appendix E. An example of calculations for richness (Margalet), evenness (Shannon) and composite
(Shannon and Simpson) index measures using ectomycorrhizae morphological abundance data for
seedlings growing in mature site I in the Aleza Lake Research Forest, Central Interior of British
Columbia.
Seedling
ECM fungus*
4
2
3
5
6
7
8
0.01 I
0.010
E-strain
0.315
0.167
0.144
0.098
Cenococcum
0.054
0.106
MRA
0.016
Tuber
Ascomycete unknown
0.053
0.021
0.137
0.063
Amphinema
0.109
0.326
0.126
0.323
0.021
Hebeloma
lnocybe
Lacearia
0.098
0.118
0.183
0.404
0.209
Piloderma
0.030
0.250
0.478
0.575
0.112
0.3 88
0.017
Russulaceae I
0.135
Russu1aceae 2
0.135
Thelephora
0.970
0.092
Thelephoraceae-like
0.054
0.085
0.273
0.021
0.048
0.011
Tomentella I
Tomentel/a 2
0.167
Tomentella 3
Non-rhizomorphic olive-green
0.027
0.043
0.028
Non-rhizomorphic thin mantled
0.076
0.133
0.026
0.086
0.115
Non-rhizomorphic undamped
Non-rhizomophic white
0.041
0.005
0.011
Rhizomorphic brown undamped
Rhizomorphic orange undamped
Rhizomorphic white
0.130
0.070
0.043
0.213
1.000
Total proportional abundance
1.000
1.000
1.000
1.000
1.000
1.000
1.000
Number of tips (n)t
Number of species (S)
Marga1eft
Shannon§

188
199
161
92
180
193
187
174
10
6
2
6
8
6
8
10
0.955
0.189
0.984
1.548
0.963
1.338
1.744
1.710
1.617
1.779
1.381
1.438
1.330
0.135
1.787
2.108
0.673
0.058
0.775
0.797
0.692
0.629
n~
0.802
0.866
0.742
0.903
0.856
0.771
0.624
0.859
Shannon evenness#
0.195
0.915
*All types in this study are listed although they may not all be found on seedlings in this site.
tLightly colonized tips were excluded from the original sample of -200 tips assessed per seedling.
tMargalef index = (S-1 )/In n, n being the total number of identified mycorrhizal tips.
§Shannon= -L Pi In Pi, p being the proportional abundance of each morphotype/n, or the number in each cell.
~
n = 1 - L pt
#Shannon evenness = Shannon/ In S.

91

Appendix F. Ectomycorrhizae morphotypes found on naturally regenerating (r) and planted (pi) hybrid
white spruce seedlings in treated (clearcut, and cut plus burned) and mature sites in the Aleza Lake
Research Forest. Table shows known or suspected genera or species from comparisons made with the
published literature.
Reference
Morphotype
Code Suspected Genera/species
Agerer 1987-1998 (plate 23);
Amphinema spp.
9 Amphinema byssoides (Pers.: Fr.) Erikss.
lngleby eta/. 1990; Danielson 1991 ;
Goodman eta/. 1996 (CDE 6),
Massicotte et a/. 1998 1•
Amphinema-like
Hagennan eta/. 1999 (OUC 020).
Agerer 1987-1998 (plate II);
Cenococcum spp.
2 Cenococcum geophilum Fr.
Ingleby eta/. 1990; Danielson 1991;
Goodman eta/. 1996 (CDE 10);
Visser eta/. 1998.
Cenococcum
Hagennan eta/. 1999 (OUC 030);
Massicotte et a/. 1998.
Humaria hemisphaerica (Wigg.: Fr) Fuckel lngleby eta/. 1990.
E-strain
E-strain
Danielson 1982; Visser eta/. 1998;
Massicotte et a/. 1998.
lngleby eta/. 1990; Visser eta/.
Hebeloma mesophaeum (Pers.) Que!
Hebe/oma
1998.
Danielson 1991 ; Massicotte et a/.
Hebeloma-like
1998; Hagerman eta/. 1999 (OUC
080).
H Inocybe p etiginosa (Fr.:Fr.) Gillet
Ingleby eta/. 1990.
Inocybe
Inocybe appendiculata KUhn.
Beenken et a/. in Agerer et a/ . 1996982 ; Agerer 1987-1998 (plate 94).
E Laccaria proxima (Boud.) Pat.
Ingleby eta/. 1990.
Laccaria
Gomphidius glutinosus (Schaeff.: Fr.) Fr. Agerer 1987-1998 (plate 58).
Massicotte eta/. 1998; Visser eta/.
Mycelium radicis
3 MRA Melin
atrovirens
1998.
Type ITE.3
Piceirhiza bicolorata
Ingleby eta/. 1990.
Agerer 1987-1998 (plate 73).
A Piloderma byssinum (Karst.) Jiil.
Piloderma
Visser eta/. 1998.
Piloderma croceum
Agerer 1987-1998 (plate 62).
Piloderma-like
Hagennan eta/. 1999 (OUC 200).
Agerer 1987-1998 (plates 30).
Russulaceae l
F Piceirhiza gelatinosa
Piceirhiza guttata
Agerer 1987-1998 (plate 32).
Russula xerampe/ina
Agerer 1987-1998 (plate 2).
Visser e t al. 1998.
Russula spp. Type 2
G n/a
Russulaceae 2
8 Thelephora terrestris (Ehrh.) Fr.
Thelephora
Ingleby eta/. 1990; Danielson 1991.
Agerer 1987-1998 (plate 48).
Thelephora terrestris Pers.
Thelephora-like
Massicotte eta/. 1998.
Lactarius deterrimus Groger.
Agerer 1987-1998 (plate 3).
Thelephoraceae
P n/a
6 Type ITE.5
Ingleby eta/. 1990; Visser eta/.
Tomentella 1
1998.
Agerer 1987-1998 (plate 19).
Piceirhiza nigra
Goodman eta/. 1996 (CDE 2).
Tomente//a-1 ike
7 n/a
Tomentella 2
Tomentella 3
0 n/a
4 Tuber sp.
Ingleby 1990; Massicotte eta/. 1998;
Tuber
Visser eta/. 1998.
Tuber puberulum Berk.
Agerer 1987-1998 (plate 22).
olive-green
J nla
rhizomorph, brown
D n/a
c n/a
rhizomorph, gold
92

I

rhizomorph, white
undamped, thin mantle
undamped, yellow
unknown ascomycete

8

L
M
5

Cortinarius obtusus Fr.
n/a
n/a
Genea verrucosa Vitt.

Agerer 1987-1998 (plate 12).
Jakucs et al. in Agerer et al. 199698 ; Agerer 1987-1998 (plate 120).

white rhizomorph-like
K n/a
Massicotte, H.B ., Tackaberry, L.E., Ingham , E.R., and Thies, W.G. 1998. Ectomycorrhizae establishment on
Douglas-fir seedlings following chloropicrin treatment to control laminated-root rot disease: assessment 4 and 5
years after outplanting. Applied Soil Ecology I 0: 117-125.
2
Beenken, L., Agerer, R. and Bahnweg, G. 1996. lnocybe appendiculata KUhn+ Picea abies (L.) Karst. In
Descriptions of ectomycorrhizae. Edited by R. Agerer, R.M. Danielson, S. Egli, K. Ingleby, D. Luoma, and R. Treu.
Einhorn-Verlag, Schwabisch Gmiind, Germany. pp. 35-40.
3
Jakucs, E., Bratek, Z., and Agerer, R. 1998. Genea verrucosa Vitt + Quercus spp. In Descriptions of
ectomycorrhizae. Edited by R. Agerer, R.M . Danielson, S. Egli, K. Ingleby, D. Luoma, and R. Treu. EinhomVerlag, Schwabisch Gmiind, Germany. pp. 19-23. Plant hosts for all other morphotypes are reported to be Picea
spp.
1

93

94

Appendix G. Statistical summation (one-way ANOV A*) for treatment (mature, clearcut and cut plus burned) and seedling effect (naturally regenerating (n=l6)
and planted (n=28)) on mean proportional abundance (±SE) for seven commonly occurring ectomycorrhizae associated with hybrid white spruce growing in
the Aleza Lake Research Forest, Central Interior of British Columbia.
Morphotype
Clearcut-planted
Mature-regenerating
Mature-regenerating
Mature-regenerating
Clearcut-regenerating
Cut plus burned-planted
Clearcut-planted
Cut plus burned-planted Clearcut-regenerating
Clearcut-planted
p-valuet
ue~
ue~
p-value§
ue~
4.06 (0.94)
5.55 (1.65)
Cenococcum
5.55 ( 1.65)
5.55 (1.65)
1.59 (0.64)
1.13 (0.52)
4.06 (0.94)
1.13 (0.52)
1.59 (0.64)
4.06 (0.94)
0.002
0.758
0.006
0.072
0.045
6.26 ( 1.30)
0.11 (0.07)
E-strain
0.11 (0.07)
0.11 (0.07)
14. 11(7.01)
6.26 (1.30)
12.73 (3 .37)
12. 73 (3.37)
14. 11 (7.01)
6.26 (1 .30)
0.001
0.001
0.333
0.013
0.411
26. 16 (2.78)
4.34 (2.52)
4.34 (2.52)
4.34 (2 .52)
MRA
11 .22 (3.59)
23 .27 (4.13)
26.16 (2.78)
11 .22 (3 .59)
23.27 (4.13)
26.16 (2.78)
0.242
0.001
0.001
0.075
0.001
17.69 (4.70)
2.01 (0.87)
2.01 (0.87)
Amphinema
2.01 (0.87)
14.66(6.41)
13 .89 (2.83)
17.69 (4.70)
13 .89 (2.83)
14.66 (6.41)
17.69 (4.70)
0.559
0.006
0.004
0.089
0.420
6.19(1.46)
7.38(2.37)
7.38 (2.37)
7.38 (2.37)
4.72 (2.62)
Hebeloma
4.72 (2.62)
14.34 (2.30)
6.19 ( 1.46)
14.34 (2.30)
6.19 (1.46)
0.122
0.005
0.638
0.057
0.160
1.02 (0.56)
35 .38 (6.44)
35.38 (6.44)
Russulaceae 1
35.38 (6.44)
3.79 (3.27)
3.79 (3.27)
5.09 (1.71)
1.02 (0.56)
5.09 (1.71)
1.02 (0.56)
0.008
0.001
0.001
0.001
0.451
8.31 (5.95)
2.68 (1.36)
8.31 (5 .95)
8.31 (5 .95)
Thelephora
34.85 (6.81)
4.02 ( 1.69)
2.68 ( 1.36)
4.02 ( 1.69)
34.85 (6.81)
2.68 ( 1.36)
0.431
0.200
0.431
0.006
0.001
11.04 (3 .05)
25.79 (2.94)
11.04 (3.05)
11.04 (3.05)
7.92 (1.78)
lightly colonized
19.39 (2.13)
25 .79 (2.94)
7.92 (1.78)
19.39(2.13)
25. 79 (2.94)
0.106
0.001
0.020
0.006
0.001
*significant values (Bonferroni correction of a=0.01) are in bold and are based on transformed data (arcsin --./p, where pis proportional morphotype abundance). Means
are presented as non-transformed data. P-values <0.0015 have been designated 0.001.
tdf(l, 54).
Nf(I, 42).
§df(l , 30).

Appendix H. An example of a Phi index calculation for ectomycorrhizae molecular data (PCR-RFLP) after
PHYLIP analysis.
Tip I
4
6
7
8
9
10
2
3
5
0.644
0.086
0.039
0.644
0.600
0.545
0.528
0.590
I
0.000
0.086
0.492
0.048
0.000
0.609
0.609
0.630
0.662
0.581
2
0.086
0.000
..,
.)
0.758
0.630
0.662
0.644
0.556
0.048
0.000
0.048
0.758
0.086
0.662
0.581
0.492
0.609
0.609
0.63 0
0.000
0.048
0.000
4
0.039
0.719
0.481
0.481
0.000
0.000
0.630
0.644
0.609
0.758
0.609
5
0. 758
0.609
0.000
0.000
0.630
0.867
0.630
0.644
0.609
0.481
6
0.630
0.630
0.000
0.300
0.073
0.630
0.630
0.202
7
0.600
0.630
0.300
0.000
0.662
0.662
0.719
0.867
0.257
0.321
8
0.545
0.662
0.644
0.581
0.481
0.630
0.073
0.257
0.000
9
0.528
0.581
0.270
0.202
10
0.492
0.556
0.492
0.481
0.481
0.321
0.270
0.000
0.590
2.479
2. 718
2.160
3. 108
3.508
3. 136
2. 142
A
2.131
2. 166
1.821
0.240
0.345
0.275
0.348
0.241
0.302
0.390
0.238
0.202
B
0.237

c

0.282

n- 10
D = cell matrix value
A= I: (D 2)
B =A I (n- I)
C=I:B i n
Protocol
I . Databases from RFLP patterns were created for each treatment or morphotype using all tips that were
successfully amplified and digested. The software packages used included RFLP analysis application RFLPscan
Plus, Version 3.0, (© 1990-1996 Scanalytics) and RFLPscan Database Versions 2.1 and 3.0 (©1990-1996
Scanalytics).
2. Pairs of tips were matched for shared and unique bands at a 2% tolerance level within gels and a 6% variation
level between gels to compensate for gel differences. The modified Dice's index (!-Dice's index) was used
(sum (polymorphic bands) I (shared bands + total bands) I 3 restriction enzymes) to convert the resulting matrix
of similarity values to distances).
3. Clustal analysis using the unweighted pair-group method with arithmetic means (UPGMA) of the distance
matrix was done using the Neighbor-Joining/UPGMA module in PHYLIP (Phylogeny Inference Package)
Version 3.5c (© 1986-1995 Joseph Felsenstein).
4. Each cell (D) in the matrix was squared and the columns were added (A), then divided by the sample size (n)I. The resulting value (B) for each column (tip) was summed and the final value was divided by n. Databases
of various sizes can be compared as the sample size is taken into account in the calculation. Higher phi values
represent a more genetically diverse site: smaller distances represent more closely related organisms.

95

I

Appendix I. Sample phenogram of MRA

~
~

~

G
E
N

1.....
r-

0

T
y

r-

p

E

-

1
1......

'----

~

=1
G
E
N
0
T
y
p
E

;--

2

I
l

I
I

~

D.l

Genotypes 1 and 2 are indicated by clusters contained within the lines. All other clusters were excluded.
96