UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Low soil temperature and efficacy of ectomycorrhizal fungi Husted, Lynn 1991

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1991_A1 H87.pdf [ 11.06MB ]
Metadata
JSON: 831-1.0302170.json
JSON-LD: 831-1.0302170-ld.json
RDF/XML (Pretty): 831-1.0302170-rdf.xml
RDF/JSON: 831-1.0302170-rdf.json
Turtle: 831-1.0302170-turtle.txt
N-Triples: 831-1.0302170-rdf-ntriples.txt
Original Record: 831-1.0302170-source.json
Full Text
831-1.0302170-fulltext.txt
Citation
831-1.0302170.ris

Full Text

LOW SOIL TEMPERATURE AND EFFICACY OF ECTOMYCORRHIZAL FUNGI by LYNN HUSTED B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1969 M.Sc., U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES FOREST SCIENCE DEPARTMENT We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1991 (c) Lynn Husted, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT The influence of root-zone temperature on the efficacy of various ectomycorrhizal fungi, i.e., their a b i l i t y : (1) to colonize roots in a nursery environment, (2) to persist and colonize new roots i n the f i e l d and (3) to improve the growth, nutrition, and physiology of white spruce (Picea  glauca (Moench) Voss) seedlings, was examined i n controlled environment experiments using water baths to regulate root-zone temperature. Eight-week-old non-mycorrhizal seedlings were inoculated with 13 different inocula (1 forest floor inoculum, 12 specific fungi), then transplanted into 6, 16, or 26°C peat:vermiculite mixes for 8 weeks. Maximum root colonization occurred at 16°C for most inocula. The 6°C mix strongly reduced mycorrhiza formation with only 8 of the 13 inocula forming any mycorrhizae during the 8-week test period. Primary infection from ectomycorrhizal propagules (spores and hyphal fragments) was reduced more than was secondary infection from established mycorrhizae; once established, a l l inocula colonized new roots i n 6°C forest s o i l . F a l l - l i f t e d cold-stored seedlings infected with 8 inocula (forest floor, 7 specific fungi) were planted into 6 and 12°C forest s o i l mixtures with or without indigenous ectomycorrhiza inoculum. Survival and colonization of new roots by inoculant fungi was good (> 50%) for the 12-week test duration despite the significant potential for infection by indigenous inoculum. High persistence appeared to be due to successful (>75%) root colonization by the inoculant fungi i n the nursery production phase, to the relative weakness of ectomycorrhizal propagules (spores and hyphal fragments) compared with l i v e ectomycorrhizal attachments, and to the favorable pattern of la t e r a l root egress from the container plug after planting. Colonization of new roots by established mycorrhizae showed an effect of s o i l temperature in the presence, but not the absence, of indigenous inoculum. Percent new root colonization by inoculant fungi was lower in the 12°C forest s o i l . Rapid extension of la t e r a l roots i n the 12°C s o i l increased the likelihood that short roots i n i t i a t e d near the tips of elongating roots would be infected by indigenous fungi. There was no evidence of active or passive interactive replacement between inoculant and indigenous fungi. However, Hebeloma crustuliniforme appeared to inhibit mycorrhizal formation by indigenous fungi; roots not infected by this fungus remained non-mycorrhizal. Application of slow-release f e r t i l i z e r reduced new root colonization by E-strain but had no effect on colonization by H. crustuliniforme or indigenous forest floor fungi. Non-inoculated seedlings (controls) and seedlings inoculated with 5 different inocula (forest floor, 4 specific fungi) were planted i n 6 and 12°C forest s o i l for 3 weeks. Inoculation influenced the rate at which seedlings acclimated to the 6°C s o i l with respect to resistance to water flow and net photosynthetic rate, but had no effect on pre-dawn stomatal conductance. Differences among inoculation treatments were related to the size and nutritional status of seedlings at the time of transplanting. Seedlings infected with Laccaria bicolor or E-strain exhibited the least decrease i n resistance to water flow due to the rela t i v e l y small size (dry weight, short root number) of their root systems at the time of transplanting. Net photosynthetic rate and new foliage production correlated positively with shoot N and P (% dry weight) and the proportion of total seedling N and P contained in shoot tissues at the time of planting. i v Non-inoculated seedlings (controls) and seedlings inoculated with forest floor or 5 specific fungi were planted i n 6 and 12°C forest s o i l for 12 weeks. The presence of "any" mycorrhiza at the time of transplanting did not improve seedling growth under the experimental conditions (i.e., cool, acidic s o i l s with an indigenous ectomycorrhizal fungal population). On average, mycorrhizal infection increased N and P uptake at 12°C but not at 6°C. Growth response to specific fungi was very variable with some fungi depressing seedlings growth (e.g., E-strain and H. crustuliniforme) and others strongly promoting i t (forest floor inoculum, L. bicolor. Thelephora t e r r e s t r i s ) . Seedling response to the various inocula was not related to the degree of mycorrhizal infection at the time of planting nor to the source of inocula; but was associated with differences i n the content and distribution of nutrients at the time of transplanting and differences i n total nutrient uptake, root efficiency, nutrient-use efficiency and net photosynthetic rate after transplanting. Root efficiency was not proportional to the number of short roots per unit root or to the amount of external mycelium attached to the various mycorrhizae. Implications for applied forestry and research are discussed i n the f i n a l chapter. V TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i LIST OF FIGURES x ACKNOWLEDGEMENTS x i CHAPTERS I INTRODUCTION 1 I I EFFECT OF ROOT-ZONE TEMPERATURE ON ROOT COLONIZATION I n t r o d u c t i o n 9 Methods 12 R e s u l t s 20 D i s c u s s i o n 25 I I I EFFECTS OF COOL SOIL TEMPERATURE AND SLOW-RELEASE FERTILIZER ON THE PERSISTENCE OF ESTABLISHED MYCORRHIZAE I n t r o d u c t i o n 31 Methods 33 R e s u l t s 41 D i s c u s s i o n 50 Summary 58 IV EFFECT OF VARIOUS MYCORRHIZAL FUNGI ON THE ACCLIMATION OF WHITE SPRUCE SEEDLINGS TO LOW SOIL TEMPERATURES I n t r o d u c t i o n Methods R e s u l t s D i s c u s s i o n Summary V EFFECT OF VARIOUS MYCORRHIZAL FUNGI ON THE GROWTH AND NUTRIENT STATUS OF WHITE SPRUCE SEEDLINGS TRANSPLANTED INTO COOL SOILS I n t r o d u c t i o n Methods R e s u l t s D i s c u s s i o n Summary V I IMPLICATIONS FOR APPLIED FORESTRY AND RESEARCH REFERENCES APPENDIX A INDEX OF COMMON AND SCIENTIFIC NAMES B KEY CHARACTERISTICS OF MYCORRHIZA FORMED BY INOCULANT AND INDIGENOUS FUNGI C NUTRIENT CONCENTRATIONS AT 0, 5 AND 12 WEEKS v i i LIST OF TABLES TABLE 2.1 Source of test fungi 13 2.2 Effect of growing mix temperature on mean number of short roots per seedling 20 2.3 Effect of inoculum and root-zone temperature on mean mycorrhiza class 22 3.1 Fungal isolates and inoculum used to study the effects of s o i l temperature and the presence of native ECM fungi on persistence of inoculant fungi 37 3.2 Effect of s o i l temperature on mean seedling root biomass (oven-dry weight), root length and number of short roots for seedlings at 0 and 12 weeks 43 3.3 Effect of slow-release f e r t i l i z e r (NPK) at 12°C s o i l temperature on root parameters at 0 and 12 weeks 44 3.4 Mean mycorrhiza class for roots within original container plug (old roots) and for new roots formed outside the container plug 46 3.5 Degree of short root colonization by inoculant fungi at the time of slow-release f e r t i l i z e r (Osmocote 14-14-14 NPK) application and at 12 weeks after application 48 3.6 Effect of slow-release f e r t i l i z e r (NPK) and inoculation treatments on N and P concentations (% oven-dry weight) i n new foliage at 12 weeks 49 4.1 Effect of inoculation treatment on seedling morphology at the time of transplanting 69 4.2 Effect of inoculation treatment on seedling N and P concentrations (% oven-dry weight) at the time of transplanting 70 4.3 Range in mean mid-day mean xylem pressure potentials (-MPa) of the various inocula at 2, 9 and 21 days 71 4.4 Summary of soil-to-xylem resistance (RSX) estimates at 2, 9 and 21 days 73 v i i i 4.5 Pearson correlation coefficients (r) between 21-day RSX and root morphology and nutrition at the time of transplanting 76 4.6 Effect of inoculation treatment on terminal bud break 78 4.7 ANOVA of pre-dawn stomatal data 79 4.8 ANOVA of log-transformed Pn (/xmoles C02/m2s) data 81 4.9 Interaction of s o i l temperature and inoculation on mean values of net photosynthetic rate 2 days after transplanting 82 o 4.10 Comparison of net photosynthesis (/imoles C02/m s) 21 days after transplanting with shoot N (% oven-dry weight), seedling N (mg/seedling) and the ratio of shoot N to root N at the time of transplanting 83 5.1 Equations used to calculate growth analysis indices 106 5.2 Mycorrhizal infection of new roots formed outside the container plug in 6°C and 12°C s o i l 109 5 . 3 Analyses of covariance for total, shoot and root biomass 112 5.4 Total seedling, mature shoot (MS) and new foliage (NF) biomass (oven-dry weight) averaged across time and temperature compared with seedling nitrogen status at the time of transplanting 116 5.5 Interaction of s o i l temperature and inocula on mean root biomass (g oven-dry weight/seedling) 118 5.6 Analysis of (co)variance for root morphology data 119 5.7 Effect of inocula on root morphology 121 5.8 Effect of s o i l temperature and inoculation treatment on the allometric coefficient (k) and shoot/root ratios 122 5.9 . Probabilities that inoculation or mycorrhizal infection present at the time of transplanting influenced the growth and morphology of transplanted seedlings 124 Effect of s o i l temperature on shoot concentrations ' Concentration (% oven-dry weight) and content (mg) of N and P i n new foliage biomass at 1 2 weeks I n i t i a l seedling N and P content and N and P uptake between 0 and 1 2 weeks Effect of inocula on the ratio of shoot N to root N Pearson correlation coefficients (r) relating seedling i n i t i a l shoot nutrition and nutrient uptake to caliper growth ( 0 - 1 2 weeks), shoot-to-root allometry (k), 12-week new foliage and root biomass Effect of s o i l temperature on growth analysis indices i n the 5 to 1 2 week period Relative growth rate, RGR, [(g growth/(g seedling/week)] by inoculation treatment for the 0 - 5 and 5 - 1 2 week periods Effect of inoculation treatment on relative root ratio, RWR, (g root/g seedling) Specific absorption rate for nitrogen, AN, for the 0 - 5 and 5 - 1 2 week periods Effect of inocula on the relative increase in nitrogen, RN, [g N/(g N seedling/week] for the 0 - 5 and 5 - 1 2 week periods Effect of inocula on nitrogen-use efficiency estimated on the basis of absorbed N (NUEa) and on the basis of i n i t i a l N content (NUEi) for the 0 - 5 week period; and on the basis of absorbed N for the 5 - 1 2 week period Analysis of variance for Pn data at 5 , 1 0 and 1 2 weeks Effect of inoculation treatments on the rate of Pn (pmol C02/m 2s) and WUE (/xmol C02/mmol H 2 O ) Effect of s o i l temperature and time on specific leaf dry weight (mg/cm ) Effect of inoculation treatment on mean xylem pressure potential (-MPa) 5 weeks after transplanting X LIST OF FIGURES FIGURE 2.1. Effect of root-zone temperature on mycorrhiza formation by three inocula: T. terrestris 2088, L,. bicolor 5313 and forest floor 24 4.1 Effect of s o i l temperature on RSX [MPa/log (/ig H20/cm2s)] at 2, 9 and 21 days 72 4.2 Relationship between seedling resistance to water flow (RSX) at 21 days and i n i t i a l root biomass 75 5.1. Mean biomass of mature shoot, new foliage and root tissues at 0, 5, 10 and 12 weeks at 6 and 12°C s o i l temperatures 115 ACKNOWLEDGEMENTS This study was funded by Forestry Canada under the Canada-British Columbia Forest Resource Development Agreement (1985-1990), a National Science and Engineering Research of Canada scholarship and a Science Council of B r i t i s h Columbia G.R.E.A.T award. I would like to thank the Canadian taxpayers for supporting these programs. Many people from Forestry Canada provided invaluable assistance during the research and analysis phase of the dissertation. I would li k e to acknowledge Clarence Simmons for his advice and programs for conducting non-parametric analyses; Rob Hagel for seedling culture advice; George Edwards for providing growth chamber space; Tony Trofymow, Tad Woods and Charles Dorworth for lending microscopes and other laboratory equipment. At U.B.C, Fred Lewis, Farida Bishay and Jeff Glaubitz completed hundreds of tedious root and leaf measurements with care and good humour. Chemical analyses were conducted by MacMillan Bloedel Limited, Woodlands Services Division, Nanaimo, B.C. Soilcon Laboratories, Richmond, B.C. provided s o i l water retention data for the various s o i l mixtures. I owe a great deal to three dissertation advisors Drs. Tony Trofymow, Bob Danielson and Shannon Berch and to my thesis supervisor Denis Lavender. Tony Trofymow was not only very generous with advice but also provided the means (laboratory space and equipment) for me to conduct a major portion of my research i n Vict o r i a when my husband was transferred there i n 1987. Bob Danielson and Shannon Berch introduced me to mycorrhizal fungi and the techniques for isolating and culturing specific fungi, identifying morphological types and assessing root colonization. While I s t i l l have much to learn in this regard, I am very grateful for the opportunity to share their enthusiasm and expertise. Denis Lavender shared his enthusiasm for s i l v i c u l t u r e and applied research and guided my selection and formulation of a research problem. The planning and writing of my thesis dissertation benefited from the ideas, suggestions and criticisms of a l l members of my thesis committee: Drs. I.E.P. Taylor, B. van der Kamp, T. Ballard and the above-mentioned advisors. Finally, my family provided emotional support and a balanced perspective on l i f e throughout my studies; thank you Eileen, Jack, Pauline, Tom, Matthew, Janice and Ron. And a special thanks to my husband, Terry Berscheid, for his constant encouragement and help with computer glitches, especially during the f i n a l year of my program. 1 CHAPTER I INTRODUCTION The Problem My thesis research was funded by the Canada-British Columbia Forest Agreement 1985-1990. This program was implemented to improve reforestation success, particularly on backlog sites (i.e., those logged more than 10 years ago and not reforested). The early growth of outplanted conifer seedlings on many northern sites is less than expected due i n part to the cool s o i l s of planting sites which are not mechanically site-prepared (Butt 1986). The average mid-summer temperature of northern planting sites at a s o i l depth of 10 cm is approximately 10°C; i n spring and early summer i t is closer to 4 or 5°C (Tyron and Chapin 1983, Binder et a l . 1987). These s o i l temperatures are well below the optimal range (18-24°C) for most conifer species (Ritchie and Dunlap 1980). Regardless of s o i l temperature, the degree to which nursery seedlings are preconditioned to the planting site has a great influence on seedling f i e l d performance (Burdett 1983). Preconditioning seedlings, by inoculating them with specific ectomycorrhizal (ECM) fungi, has been shown to improve f i e l d performance (Castellano and Trappe 1985, Kropp et a l . 1985, Last et a l . 1984, Molina and Trappe 1982, Wilson et a l . 1987). Mycorrhizal seedlings may benefit from enhanced nutrient or water uptake, increased resistance to pathogens or greater root longevity (Harley and Smith 1983). However, mycorrhizal inoculation does not always improve seedling performance (Bledsoe et a l . 1982, Shaw et a l . 1987b). Trofymow and van den Driessche (1991) summarized the results of 84 outplanting t r i a l s with a r t i f i c i a l l y inoculated conifer seedlings. Inoculation increased seedling 2 performance on only 43% of the t r i a l s located on routine reforestation sites. More research is needed to determine the conditions (host species, site and s i l v i c u l t u r a l system) under which benefits are most l i k e l y to occur. Use of mycorrhizal inoculation to improve seedling performance is severely limited by a lack of knowledge of the ecology of ECM fungi. "Mycorrhizal fungi occur in a large diversity of plant communities and their adaptation to extremes of environment is widely acknowledged; at the same time, we are s t i l l in the "Dark Ages" with regard to applying these features of ecological specialization to agriculture, forestry or i n restoring native vegetation. In spite of enormous interest i n "ta i l o r i n g " host-fungus combinations for specific planting sites, we are limited by our understanding of the environmental characteristics of the site, the range of tolerance of the mycorrhizal symbiont, and the a b i l i t y of the fungus to benefit the host under natural conditions" (Parke 1985, p. 107). The applied forestry objective of the funding agency presented an opportunity to examine the influence of an environmental factor, s o i l temperature, on the growth and physiology of conifer seedlings; the modifications of these effects by ectomycorrhizal inoculation; and the influence of s o i l temperature on the efficacy of various ECM fungi. Although i t is known that temperature alters the growth of ECM fungi in culture (Dennis 1985, Hacskaylo et a l . 1965, Marx et a l . 1970, Samson and Fortin 1986, Theodorou and Bowen 1971) and in forest soi l s (Heninger and White 1974, Marais and Kotze 1978, Parke et a l . 1983b), the influence of temperature on the efficacy of ECM fungi, in particular their a b i l i t y to persist in forest soi l s and to promote seedling growth, is poorly understood (Perry et a l . 1987). Trappe (1977) stressed the importance of s o i l temperature on the efficacy of various mycorrhizal fungi and the need for more knowledge of the adaptation of ECM isolates to the s o i l 3 temperatures at the planting s i t e . Inoculation with mycorrhizal fungi has been shown to reduce the adverse effects of high root-zone temperatures (Marx and Bryan 1971, Borges and Chaney 1989). Inoculation might have the reverse effect, however, in sub-optimal root temperatures. Research with vesicular-arbuscular mycorrhizae (VAM) has shown that cool temperatures eliminate the benefits normally derived from mycorrhizal infection (Moawad 1978, Smith and Roncadori 1986). The growth of VAM plants may even be less than that of non-mycorrhizal plants at low temperatures (Furlan and Fortin 1973, Hayman 1974, Schenck and Schroder 1974). Similar research has not been conducted for ectomycorrhizae. Many studies have shown that some species or strains of mycorrhizal fungi are more e f f i c i e n t than others i n promoting host plant growth (Benecke and Gobi 1974, Trappe 1977, Marx 1979). A fungus which increases growth of a host plant in one set of study conditions may not i n another set (Trappe 1977). Much research on mycorrhizal efficacy has focussed on physiological attributes of ECM fungi, such as their a b i l i t y to produce plant growth regulators or absorb nutrients from solutions. More research on the ecology of mycorrhizal fungi should also contribute to our understanding of differences i n efficacy. White spruce''" seedlings were selected as host plants. The survival and growth of planted white spruce seedlings is less than satisfactory on many backlog reforestation sites in the northern interior of B r i t i s h Columbia (Butt 1986) . Planting check, a period of slow shoot growth after planting, is common and results in reduced shoot growth (50% of expected) ^ The s c i e n t i f i c name is Picea glauca (Moench) Voss. Common names of vascular plants are used in the dissertation with Appendix A l i s t i n g both common and s c i e n t i f i c names. 4 for several years after planting. This increases the susceptibility of spruce seedlings to brush competition. The potential benefits of a nursery inoculation program are greatest when the growth of outplanted seedlings is limited by water or nutrient a v a i l a b i l i t y i n the f i r s t growing season, the inoculum potential of indigenous fungi at the planting site i s low, and the inoculant fungus is adapted to the planting site environment (Trappe 1977, Parke 1985). Based on these c r i t e r i a , mycorrhizal inoculation has good potential for improving the reforestation of white spruce on backlog sites. Planting check of white spruce results, at least i n part, from poor root growth and reduced water or nutrient uptake in the f i r s t growing season (Burdett et a l . 1984, Butt 1986). Furthermore, the native inoculum potential of backlog sites may be lower and less predictable compared to recently logged sites or more southern reforestation sites where most mycorrhizal research has been conducted. Danielson (1985) found that new roots formed outside the original root mass of bare-root seedlings planted on a backlog site near Prince George, B.C. were not colonized by indigenous fungi i n the f i r s t growing season. Finally, 70% of interior spruce seedlings are grown in southern nurseries. White spruce seedlings often become mycorrhizal in container nurseries. It is not known i f the nursery ECM fungi persist or benefit seedling growth at northern planting sites. Fungi colonizing seedlings in warm (15 to 35°C), f e r t i l e container growing mixes may not be adapted to the cool (5 to 10°C), less f e r t i l e s o i l s of northern planting sites. 5 Experimental Approach The thesis research was conducted in a controlled environment chamber so that s o i l temperature could be varied independently of other environmental conditions, including s o i l f e r t i l i t y and indigenous ECM inoculum. Controlled environment studies have been c r i t i c i z e d repeatedly, especially by foresters operating i n the "real world" of fluctuating environments. This c r i t i c i s m is valid, especially i f one wishes to calculate gains i n growth due to various inoculation treatments. However, this was not the primary goal of the research. So i l temperature effects on plant growth are influenced by many unrelated factors including s o i l f e r t i l i t y , water content, bulk density and ai r temperature (Sutton 1969, Nielson 1971, Ritchie and Dunlap 1980, Kuhns et a l . 1985). In f i e l d experiments, i t is very d i f f i c u l t to separate the effects of s o i l temperature from those of site preparation technique, organic matter content of the rooting zone and s o i l drainage (e.g, Binder et a l . 1987, Brand and Janas 1988). The formation and persistence of mycorrhizae depend to a significant degree on the physiology of the host plant which i n turn i s affected by i t s environment (Trappe 1977, Harley and Smith 1983). In a series of experiments examining the interactions between s o i l temperature, soybean growth, and mycorrhizal development, Schenck and Smith (1982) found that their results varied from year to year possibly due to uncontrolled variation in the shoot environment, i.e., photoperiod, l i g h t intensity, a i r temperature and humidity. Physiological measurements, such as the net photosynthetic rate and resistance to water flow, are also a function of many factors including a i r temperature, radiation, relative humidity, s o i l water content, temperature, and f e r t i l i t y . Given a l l these potential 6 interactions, i n a "real world" environment, the effects of s o i l temperature on the mycorrhizal symbiosis may be confounded by many variable factors. In a controlled environment, a constant shoot environment can be maintained for a l l inoculation and s o i l temperature treatments. The confounding of a i r - s o i l temperature differentials with s o i l temperature treatments was recognized, but not considered to be cause for concern. Examination of data presented by Lavender and Overton (1972) summarizing root and shoot growth of Douglas-fir seedlings over a range of s o i l , a i r and s o i l - a i r d i f f e r e n t i a l s v e r i f i e d this assumption. A degree change i n s o i l temperature had a much greater effect on these growth data than did a degree change in a i r temperature over a wide range of shoot-air temperature differ e n t i a l s (2-20°C). Similarly, data from Lawrence and Oechel (1983b) show a consistent s o i l temperature effect on the photosynthetic rate of four deciduous taiga trees over a 5 to 15°C range i n s o i l - a i r d i f f e r e n t i a l s . Relationships developed in a controlled environment are not reliable predictors of absolute plant growth in natural environments; however, these have proven useful during the interpretation of f i e l d studies involving root pathogens (Salt 1979), water relations of plant communities (Jarvis 1976) and seedling physiology (Lavender 1988). In addition, the results of controlled environment studies can be used to develop models of f i e l d responses (Jarvis 1976) or to screen mycorrhizal fungi for particular f i e l d environments. 7 Scope of Thesis Determining how to measure efficacy of ECM fungi was the f i r s t research question. According to Trappe (1977), the efficacy of a particular ECM fungus w i l l depend on: (1) i t s aggressiveness i n conifer nurseries (degree of root colonization), (2) i t s survival and colonization of new roots of outplanted seedlings and (3) i t s inherent a b i l i t y to benefit the host plant. It i s the third aspect of Trappe's definition that is problematic. Although the benefits most often measured are host plant survival and growth, particularly shoot growth, there are numerous other equally v a l i d measurements, e.g., nutrient uptake, drought tolerance, resistance to specific pathogens (Smith 1985). Parke (1985) stressed that efficacy should be defined by the factors most limiting to conifer seedling growth on particular reforestation sites. Improved nutrient uptake may not benefit seedlings growing on nutrient rich sites or on extremely droughty sites where nutrients are not the major factor limiting plant growth. A fungus which increases host plant drought resistance may not be effective in well-watered s o i l s . Bjorkman (1962, cited in Kropp and Langlois 1990) suggested that alleviation of transplant shock is an important benefit of mycorrhizal infection to boreal spruce seedlings. I decided to use four c r i t e r i a to compare the efficacy of mycorrhizal fungi at different s o i l temperatures. The f i r s t and second were as defined by Trappe (1977), the third was the a b i l i t y to reduce transplant shock of cold-stored spruce seedlings, and the fourth was the a b i l i t y to improve their nutrient uptake and growth (both root and shoot) during a 12-week simulated growing season. Chapters II, III, IV, and V respectively address these four c r i t e r i a . Finally Chapter VI discusses the implications of the 8 main results of this work for applied forestry and research. 9 CHAPTER II EFFECT OF ROOT-ZONE TEMPERATURE ON ROOT COLONIZATION Introduction The rate at which ECM fungi colonize root systems affects their a b i l i t y to enhance host plant growth and physiology. The most effective fungi are often those that rapidly and extensively colonize seedling root systems (Abbott and Robson 1984, Perry et a l . 1987). Temperature influences the growth of ECM fungi i n pure culture (Hacskaylo et a l . 1965, Marx et a l . 1970, Theodorou and Bowen 1971, Dennis 1985, Samson and Fortin 1986) and the rate of mycorrhiza formation when seedlings are planted in forest s o i l (Heninger and White 1974, Marais and Kotze 1978, Parke et a l . 1983b). These studies suggest that the optimum temperature for fungal growth in culture and ectomycorrhiza formation i n forest s o i l s l i e s between 18 and 35°C, i.e., temperatures that are well above the s o i l temperatures measured at northern planting sites. However, significant intra-and interspecific variation in fungal response to temperature in the culture experiments has been reported (Dennis 1985, Samson and Fortin 1986) with some fungal isolates more tolerant of temperature extremes than others. Parke (1983) hypothesized that the response of ECM fungi to temperature was related to their geographic origin. Optimum temperatures for ECM fungi native to hot climates (e.g., southeastern U.S.A., Australia and Southern Africa) are higher, between 30 and 35°C (Marx et a l . 1970, Theodorou and Bowen 1971, Marais and Kotze 1978), than optimum temperatures (between 18 and 24°C) for fungi native to the Pacific Northwest (Parke 1983). In container nurseries, rapid colonization by selected inoculant fungi i s necessary i f contamination by indigenous nursery fungi (e.g., Thelephora terrestris) is to be prevented. It would be useful to know i f fungi indigenous to cool forest sites can form mycorrhizae rapidly i n peat:vermiculite growing mixes that can reach temperatures as high as 35°C during the f i r s t 3 months after sowing (Husted and Barnes 1987). In temperate and boreal forests, adaptation of ECM fungi to cool s o i l temperatures i s more important than tolerance of high temperatures. Poor adaptation to cool so i l s could be a major cause of the observed i n a b i l i t y of many mycorrhizal fungi established in container nurseries to persist and benefit outplanted conifer seedlings (Trappe 1977). In B r i t i s h Columbia, Pisolithus tinctorius. a fungus with a high optimum temperature for root infection (Marx and Bryan 1971) was the f i r s t ECM fungus tested in an outplanting t r i a l ^ " . Possibly because i t was adapted to warmer forest soils than occur in B r i t i s h Columbia, P^ . tinctorius f a i l e d to persist oh a southern Vancouver Island clear-cut, a relatively warm forest site for Br i t i s h Columbia. The objective of the work reported in this chapter was to determine the effect of three growing mix temperatures, 6, 16, and 26°C on root colonization by a variety of fungal isolates collected from forest and container nursery environments. It was hypothesized that isolates collected from nursery environments (with temperatures reaching 35°C) would form mycorrhizae more rapidly at 26°C and more slowly at 6°C than isolates collected from Pacific Northwest forest sites. I or i g i n a l l y planned to compare nursery and northern forest isolates of two or more fungal species, such as T. terrestris or Laccaria bicolor naturally found in both ^ Personal communication with J. Dennis, Forestry Canada, Victoria, B.C. environments. However, i t was not possible with the time available to obtain this experimental material. B r i t i s h Columbia does not have a permanent culture collection of the mycorrhizal fungi commonly found in nursery and forest environments. Approach The inoculum potential of ECM fungal cultures varies with isolate, species, and culture conditions. To ensure that each test fungus had a high inoculum potential, I saturated the seedling growing medium with slurries containing higher concentrations of mycelium than reported in previous studies (Danielson et a l . 1984b, Boyle et a l . 1987). Seedlings were grown for 8 weeks before inoculation. This delay increased the likelihood of contamination from air-borne spores of indigenous nursery fungi. In fact the f i r s t attempt at this experiment was a failure because of contamination. However, the delay had two important advantages. F i r s t , root colonization by mycorrhizal fungi is strongly influenced by the size and physiology of the host plant, especially i t s production of carbohydrates. During the 8-week growth period, the germinants were culled and thinned to minimize variation in seedling size. Second, the delay allowed some distinction between the effects of temperature on the a v a i l a b i l i t y of potential infection sites ( i . e . , short root number) and the effects of temperature on fungal growth and intensity of colonization. 12 Methods Source of seed, fungi and forest s o i l inoculum White spruce seed from a central interior (approx. 55°N, 123°W) seedlot (No. 29144) was obtained from the B.C. Ministry of Forests. Five ECM fungal isolates were collected from container nurseries; seven from forest sites. The origins of these twelve isolates are summarized in Table 2.1. T. terrestris i s the most common mycorrhizal fungus identified on nursery seedlings in North America, but the others have also been reported in nurseries (Castellano and Molina 1989, Danielson and Visser 1990). Forest floor inoculum was obtained from three vigorous white spruce plantations established near Mackenzie, B.C (approx. 55°N). Production of tree seedlings Seeds were surface s t e r i l i z e d 15 min i n 30% H2O2, rinsed i n d i s t i l l e d water and refrigerated for 24 h at 4-5°C; then sown, 3 per cavity, i n 40 cm"* Spencer-LeMaire Rootrainers f i l l e d with a pasteurized (70°C, 60 min) peat:vermiculite growing mix prepared from: 110 L (a bale) of Premier brand 'Forestry Peat', 57 L medium grade horticulture vermiculite, 175 g Micromax Micronutrients (Sierra Chemical Co., Milpitas, CA) and 750 g coarse dolomite lime. Seedlings were grown in a controlled environment chamber for 8 weeks: 18 h photoperiod, 20°C day/night a i r temperature and 70-90% relative humidity. Three weeks after germination, the seedlings were thinned to one per cavity with the largest and smallest seedlings removed to minimize between seedling variation i n size. 13 Table 2.1. Source of test fungi Amphinema bvssoides (Fr.) J. Erikss. (0288) Author. Isolated in 1988 from surface-sterilized ectomycorrhizae of container spruce seedlings grown at the Balco Canfor Reforestation Centre, Kamloops, B.C. E-strain (or Complexipes moniliformis Walker) (947) Dr. R.M. Danielson's collection at the University of Calgary. Isolated in 1978 from white spruce seedlings growing in a f e r t i l i z e d subalpine s o i l . E-strain (0188) Author. Isolated in 1988 from surface-sterilized ectomycorrhizae of container Douglas-fir seedlings grown at the Harrop Nursery, B.C. Forest Floor Author. Bulked sample collected in 1987 from three juvenile spruce plantations near Mackenzie, B.C. Hebeloma crustuliniforme (Bull, ex St. Amans) Quelet (5247,5249) University of Alberta, Microfungus Collection. Collected in 1985 from white spruce (Picea glauca) woods, Lac La Biche forest, AB. Hebeloma crustuliniforme (Bull, ex St. Amans) (5) University of Washington Collection (Dr. Bledsoe). Isolated in 1984 from a mixed conifer forest at 550 m in Wenatchee National Forest, WA. 14 Table 2.1. (continued) Hebeloma crustuliniforme (Bull.-ex St. Amans) (8) University of Washington Collection (Dr. Bledsoe). Isolated i n 1971 from a Douglas-fir forest, Benton County, Oregon. Hebeloma crustuliniforme (Bull, ex St. Amans) (125) From Dr. G. Hunt, Balco Canfor Reforestation Centre, Kamloops, B.C. Isolated i n 1985 from a mixed spruce, f i r and cedar forest near Barrier Lake, B.C. (1400m). Laccaria bicolor (R. Mre.) Orton (5268) University of Alberta Microfungus Collection. Isolated i n 1985 from a pine-spruce forest, Slave Lake Forest, Alberta. Laccaria bicolor (R. Mre.) Orton (5313) University of Alberta Microfungus Collection. Isolated i n 1976 from container seedlings i n FSL greenhouses, Corvallis, Oregon. Laccaria laccata (Scop, ex Fr.) Berk.& Br. (101C.D) From Dr. G. Hunt, Balco Canfor Reforestation Centre, Kamloops, B.C. Isolated from sporocarps in the nursery. Thelephora terrestris Ehrhart ex Fr. (2088) Author. Isolated i n 1988 from surface-sterilized ectomycorrhizae of spruce seedlings grown at CIP nursery, Saanichton, B.C. Thelephora terrestris Ehrhart ex Fr.(Laval) University of Laval CRBF Culture Collection. Isolated i n 1980 from a young poplar stand near Quebec City. 15 Once a week, seedling blocks were rerandomized to minimize effects of block position within the growth chamber, and the seedlings were watered to saturation with a water-soluble f e r t i l i z e r solution (100 mg/L N) containing micronutrients (Plant-Prod 20-20-20, Plant Products Co. Ltd., Bramalea, ON). Production of inoculum Twelve fungal symbionts were grown on modified Melin-Norkans (MMN) agar (Marx 1969) for 4-8 weeks u n t i l at least 50% of each Petri plate was covered by mycelium. Mycelial slurries (Danielson et a l . 1984b, Boyle et a l . 1987) were prepared by homogenizing mycelium and adhering agar from 10 Petri dishes for 15-20s i n a Waring blender and then adding d i s t i l l e d water to give a f i n a l volume of 300 mL. V i a b i l i t y of mycelial fragments was tested by culturing 5 mL aliquots on MMN agar. Inoculum for the control treatment was prepared by autoclaving mycelium and agar before preparing the slurry. Forest floor material from the three plantations was bulked, mixed thoroughly and passed through a 1 cm screen to remove large organic debris. It was stored at 2°C prior to use. Inoculation of seedlings Eight weeks after germination, a subsample of 60 seedlings was examined to ensure that there was no contamination by airborne spores. The Spencer-LeMaire books were subdivided into individual containers (using scissors and waterproof tape), and then the seedlings were inoculated with 4 mL of slurry or 4 cm of forest floor inoculum. The slurry was applied with a syringe equipped with a stainless steel needle (2-mm-wide aperture). The tip was inserted to the bottom of the plug and raised slowly while the slurry was ejected-in order to distribute the slurry evenly through the root zone. Forest floor inoculum was placed along one side of the seedling plug before the sides of the containers were taped. Soi l temperature treatment After inoculation, the containerized seedlings were transplanted into pots (4 L, 15 cm diameter, 17 cm high) f i l l e d with coarse gravel. The temperature of the gravel was maintained at either 6, 16 or 26°C by placing the pots in water baths. To minimize heat exchange between the gravel and air , the gravel surface of the pots was covered with styrofoara chips and the bath water level was maintained 2 cm above the gravel surface. The baths were located in a controlled environment chamber: 16-22°C day/night a i r temperature, 30-70% relative humidity, 18 h photoperiod, lig h t intensity of 400 /iiol/(m s) (400-700 nm wavelength) from cool white fluorescent and incandescent bulbs. Seedlings were watered as necessary to maintain the growing mix water potential above f i e l d capacity. Once a week, 10 mL of a water-soluble f e r t i l i z e r solution (100 mg/L N) containing micronutrients (Plant-Prod 20-20-20) was added to each seedling container ' and the pots were re-randomized within the water baths. Assessment of mycorrhiza formation and root morphology Eight weeks after inoculation, the seedlings were destructively sampled to assess the degree of mycorrhiza formation. A short root was considered mycorrhizal i f a Hartig net was present. Intact unwashed root plugs were scanned at 12-40x magnification and whole mounts of short roots were examined at lOOOx magnification as described by Danielson and Visser 17 (1984, 1990). This technique had several advantages for the dissertation research compared with methods which involve root washing, sectioning or staining, : (1) most short roots were found on the external surface of the plug and external hyphae which tend to be removed during root washing were easily detected, (2) early stages of infection (with only a portion of the short root occupied by a Hartig net) were more easily detected i n whole mounts than in sections and (3) characteristics used to c l a s s i f y morphological types of mycorrhizae (e.g., colour, hyphal ornaments) were not lost by washing and staining. Percent root colonization was recorded on a six-class scale: (1) 0% of short roots mycorrhizal, (2) 1-25% mycorrhizal, (3) 26-50% mycorrhizal, (4) 51-75% mycorrhizal, (5) 76-95% mycorrhizal, (6) > 95% mycorrhizal. Approximately 40 whole mounts of short roots were examined to c l a s s i f y each seedling. Accuracy of this subsampling system was checked by comparing the class ratings estimated from the subsample with those obtained from assessing a l l the short roots of 24 seedlings selected to represent infection levels from 0% to 100%. After mycorrhizal development was assessed, the root systems of each seedling were washed and cut into 5-cm segments. The number of short roots per seedling was estimated from (1) a randomly-selected subsample of these segments comprising approximately 1/3 of the root system and (2) the oven-dry weight ratio between the subsample and total root system. Short root number at the time of inoculation was estimated from a destructive harvest of 12 seedlings per temperature treatment. 18 Experimental design and s t a t i s t i c a l analysis The experiment was a completely randomized design with a f a c t o r i a l (3 x 14) arrangement of the treatments; there were 12 seedlings (six seedlings per pot) planted per treatment combination. Each seedling was an experimental unit. There were no significant differences (P > 0.25, randomization test) in root colonization between the two pots within each inoculation and treatment combination. Mycorrhiza class data showed l i t t l e or no variation within groups subjected to particular treatment combinations. The distribution of these data tended to be non-normal and heteroscedastic; thus i t did not meet the assumptions for parametric s t a t i s t i c a l analysis.(Eisenhart 1947). Therefore, s t a t i s t i c a l analysis of mycorrhizal class data was performed with various forms of the randomization test, one of the most powerful non-parametric s t a t i s t i c a l methods (Siegel 1956, Edgington, 1987). The randomization test for two samples (Siegel 1956), as programmed in FORTRAN to run on a VAX computer by Dr. Clarence Simmons of Forestry Canada, Victoria, B.C., was used to find the probabilities of observed differences between two means, under the null hypothesis that the data were from the same population and occurred in the observed groups purely by chance of the sampling. In the randomization test, a s t a t i s t i c , such as the difference between means of two groups of data, is compared with the same s t a t i s t i c derived from each of a l l possible permutations of their pooled data to the groups, as described by Siegel (1956). Applying the principles and broad definition of sampling randomization tests described by Edgington (1987), Dr. Simmons developed and programmed an extension of mean-based sampling randomization tests, going beyond using the simple difference of two means 19 as a test s t a t i s t i c to using orthogonal sets of linear contrasts on more than two means to obtain more complex linear combinations of means as test s t a t i s t i c s , enabling significance of single-degree-of-freedom main effects and interactions to be estimated. Where the sample sizes were too large to consider sampling a l l possible permutations, large with-replacement random samples of permutations were used instead, as described by Edgington (1987). This method called the "sample randomization test" by Edgington (1987) was programmed by Dr. Simmons in FORTRAN, using a library function to generate uniform distribution (pseudo-) random numbers in the process of deriving successive random permutations. The number of permutations or re-randomizations used in a particular sampling randomization test was generally 100,000. Following Edgington (1987), where the groups were subdivided by a cross-classification common to a l l , the re-randomization was done independently within each class of the cross-classification. Analysis of variance (ANOVA) was used to (1) test the effects of temperature and inocula on 8-week root morphology and (2) the effect of inoculum source ( f i e l d or nursery) on mycorrhiza formation. Mean mycorrhiza class data (for each inoculation and treatment combination) were used in the latt e r analysis; these data met the normal distribution and homogeneity of variance assumptions of ANOVA. Results 1. Root assessments There was no evidence of mycorrhiza formation by contaminant fungi in the eight-week-old seedlings prior to inoculation. On average, there were more than 200 short roots per seedling available for colonization by ECM fungi (Table 2.2). Temperature had a highly significant (P < 0.001, ANOVA) effect on short root production, accounting for approximately 40% of the total v a r i a b i l i t y i n short root data at 8 weeks. The mean number of short roots produced per seedling was reduced 80% and 20%, respectively i n the 6 and 26°C mixes, compared with the 16°C mix during the 8-week test period (Table 2.2). Inoculation treatment also influenced short root production (P < 0.05, ANOVA) but accounted for only 8% of the total v a r i a b i l i t y in 8-week short root data. There was no significant interaction between inoculation and temperature (P — 0.20) on short root production. Table 2.2. Effect of growing mix temperature on mean number of short roots per seedling Week Temperature °C 6 16 26 0 219 ( 60) 214 ( 46) 236 ( 61) 8 471 (293) 2311 (273) 1868 (591) NOTE: Standard deviations are shown in parentheses. 21 2. Interaction of temperature and inoculation treatments on mycorrhiza  formation Orthogonal contrasts conducted by randomization tests showed there were three significantly (P - 0.01) different patterns of ECM mycorrhiza formation i n response to s o i l temperature (Table 2.3). Percent root colonization for 8 of the 13 inocula, including the forest floor inoculum, was greatest at 16°C, decreasing at 6°C and 26°C (Fig. 2.1). In contrast, percent colonization decreased with temperature for L. bicolor 5313; and increased with temperature for the E-strain 947, T. terrestris (2088), T. terrestris (Laval) and L. laccata 101C inocula. Averaged across a l l inoculation treatments, the percentage of mycorrhizal short roots was lower at 6°C (P < 0.001) and 26°C (P - 0.13) compared with 16°C. Mean mycorrhiza classes for the 6, 16 and 26°C root-zone temperatures were 1.9, 3.0, and 2.8, respectively. 3. Effect of inoculum source on mycorrhiza formation ANOVA showed no effect (P - 0.50) of inoculum source ( f i e l d versus nursery) on percent root colonization as estimated by mean mycorrhiza class; nor was there an interaction between temperature and inoculum source (P - 0.87) on mean mycorrhiza class. Several inocula originating from cool forest s o i l environments (e.g., E-strain 947; L. bicolor 5268 ) successfully colonized a significant percentage of the short roots i n the 26°C peat:vermiculite mix; others (e.g., forest floor inoculum) formed sign i f i c a n t l y (P < 0.001) fewer mycorrhizae at 26°C compared to 16°C. 22 Table 2.3. Effect of inoculum and root-zone temperature on mean mycorrhiza class Growing mix temperature (°C) 6 16 26 Mean Inocula* s** Mycorrhiza class *** Control 1. .0 (0. 0) 1.0 (0.0) 1. 0 (0. 0) 1. .0 Ab N 1. .1 (0. 3)c 1.7 (0.7)c 1. ,3 (0. ,5)d 1. ,4d E 0188 N 1. .3 (0. 5)c 2.6 (1.5)c 2. .2 (1. 2)d 2. ,2c E 947 F 2, .8 (1. ,3)b 5.3 (0.9)a 6. .0 (0. ,0)a 4, ,5ab Ff F 3, ,4 (1. ,7)a 5.0 (l.l)ab 2. .7 (1. ,3)cd 3. ,5b He 5 F 1. .0 (0. 0)c 2.5 (1.6)c 1. .3 (0. .5) d 1. ,4d He 8 F 1, .0 (0. ,0)c 1.8 (1.0)c 1. ,5 (0. .5) d 1, .4d He 5247 F 1, .0 (0. ,0)c 2.3 ( l . l ) c 1. .8 (0. .8) d 1 .7cd He 125 F 2, .2 (0. ,7)b 2.7 (0.9)c 2. .3 (0, .5) d 2, .4c Lb 5313 N 2, .8 (1. ,3)b 2.7 (1.4)c 1. .2 (0. .4) d 2. .lc Lb 5268 F 4, .7 (0. ,5)a 5.4 (0.5)a 4. .3 (1. ,l)b 4, ,8a LI 101C N 1 .5 (0. ,5)c 3.2 (1.7)bc 3. .9 (1. ,0)bc 2 .8cb (continued on next page) 23 Table 2.3. (cont.) Tt 2088 N 1.0 (0.0) c 4.8 (0.8)b 5.9 (0.3)a 3.9b Tt Laval F 1.0 (0.0) c 1.6 (1.2) c 3.0 (2.3)bcd 1.9cd Mean 1.9 (1.2) 3.0 (1.6) 2.8 (1.8) NOTE: For individual s o i l temperatures, N - 12. Means i n columns with the same lett e r designation are not significantly different (P = 0.01, paired randomization tests). Standard deviation in parentheses. Abbreviations for inocula: Ab - A,, bvssoides. E = E-strain, Ff =» forest floor, He - H. crustuliniforme. Lb - L. bicolor. LI - L. laccata. Tt - T. terrestris S - source; either f i e l d (F) or nursery (N). Mean mycorrhiza classes: (1) 0% of short roots mycorrhizal, (2) 1-25% mycorrhizal, (3) 26-50% mycorrhizal, (4) 51-75% mycorrhizal, (5) 76-95% mycorrhizal, (6) > 95% mycorrhizal Figure 2.1. Effect of root-zone temperature on mycorrhiza formation by three inocula: T. t e r r e s t r i s 2088, Lj. bicolor 5313 and forest floor. 6 16 26 Temperature In the 6°C mix, most mycorrhizae were formed by three f i e l d sources (L* bicolor 5268, forest floor, and E-strain 947) and one nursery source (L. bicolor 5313). Both f i e l d and nursery sources (e.g., H. crustuliniforme 5247, T. terrestris 2088) f a i l e d to form mycorrhizae i n the 6°C mix. In the 16 and 26°C growing mixes, inocula that formed the most mycorrhizae were from both f i e l d (e.g., L. bicolor 5268) and nursery sources (e.g., T. terrestris 2088). Averaged across a l l growing mix temperatures, two nursery isolates (L. laccata 101C and T. terrestris 2088) and three f i e l d sources of inoculum (forest floor, L. bicolor 5268 and E-strain 947) colonized the greatest percentage of short roots eight weeks after inoculation. Only four inocula (E-strain 947, L. bicolor 5268, H. crustuliniforme 125 and forest floor) tolerated the whole range of test temperatures, with mean mycorrhiza classes greater than 2.0 i n 6, 16, and 26°C mixes. There were significant (P = 0.01) differences in mycorrhiza formation among isolates of L. bicolor and E-strain at a l l growing mix temperatures; and for H. crustuliniforme isolates at 6°C and T. terrestris at 16 and 26°C. Discussion Mycorrhiza formation occurred within eight weeks of inoculation at a l l three s o i l temperatures. Percent root colonization was greatest at 16°C for most inocula, confirming earlier observations that mycorrhiza formation is generally greatest at s o i l temperatures that are optimal for root growth (Parke 1983). The optimum temperature for spruce root growth is about 18°C (Heninger and White 1974, Ritchie and Dunlap 1980). High temperature limited percent colonization to a lesser degree than did cool temperature. Thirteen of the fourteen inocula formed some mycorrhizae at 26°C; but only eight formed any mycorrhiza at 6°C. The magnitude of the temperature effect was greater than appears from the percent colonization data because of the reduced production of short roots by seedlings i n the 6°C s o i l compared with that in the 16 or 26°C s o i l s . On the basis of total number of mycorrhizae formed per seedling, infection was severely reduced (by approx. 90%) at 6°C; and to a lesser degree (by 15%) at 26°C. The formation of mycorrhizae at 6 and 16°C eight weeks after inoculation is not consistent with the results of an e a r l i e r study conducted by Heninger and White (1974). They grew spruce seedlings inoculated with forest s o i l ECM fungi for eight weeks at s o i l temperatures between 15 and 31°C. No mycorrhiza formation was observed at s o i l temperatures below 19°C. However, there are reports of fungal growth in culture (Dennis 1985, Samson and Fortin 1986) and mycorrhiza formation in s o i l (Orlov 1957, Parke et a l . 1983b) at temperatures below 19°C. Orlov (1957, cited by Slankis 1974) observed mycorrhiza formation i n spruce at s o i l temperatures between 3 and 9°C. Vogt et a l . (1980) reported that the biomass of active mycorrhizae i n amabilis f i r stands was highest in f a l l and winter when s o i l temperatures averaged 1°C. In a review of s o i l effects on mycorrhizae, Slankis (1974) states that although mycorrhiza formation occurs i n cool s o i l s , i t is generally more rapid i n warm s o i l s . However, the rate of mycorrhiza formation at various s o i l temperatures is not well documented. Parke et a l . (1983b) reported moderate mycorrhiza formation i n Douglas-fir seedlings grown in 7.5°C soil s fourteen weeks after inoculation. Coutts and N i c o l l (1990a) observed mycorrhizal infection of Sitka spruce seed sown in 8-15°C s o i l twelve weeks after inoculation. Mycorrhiza formation was more rapid in this study compared to their results. Since the seedlings were non-mycorrhizal at the time of inoculation, the disparity between results of this and previous studies probably reflects differences in inoculation technique and, more important, in the method of assessing short roots. Eight-week-old seedlings were inoculated in this study; in the other studies (e.g., Heninger and White 1974, Parke et a l . 1983b, Coutts and Ni c o l l 1990a) seeds were inoculated. Heninger and White (1974) assessed short roots "visually" for the presence or absence of mantles; Coutts and Ni c o l l used low power (40x) magnification and did not check for the i n i t i a l stages of infection (e.g., hyphal penetration of the cortex and Hartig net formation) with high power magnification. Parke et a l . (1983b) did not describe their method of assessment but probably did not use high power magnification. Visual and low power methods have two important limitations. F i r s t , in this and other studies (e.g., Nylund and Unestam 1982) of the infection process i n spruce seedlings, formation of the Hartig net, which is the diagnostic c r i t e r i o n for mycorrhiza formation, preceded mantle formation. At low power magnification, short roots with a Hartig net but lacking a well-developed mantle w i l l l i k e l y be c l a s s i f i e d as non-mycorrhizal. The early stages of infection have been shown to influence host plant physiology (Nylund and Unestam 1982, Ekwebelam and Reid 1983, Daughtridge et a l . 1986); therefore, their detection is important to understanding the mycorrhizal symbiosis. Second, gross characteristics (i.e., lack of root hairs, distinctive colours, branching and swelling) commonly used to identify mycorrhizae "visually" or under low power magnification can be found on non-mycorrhizal root systems (Nylund and Unestam 1982, Harley and 28 Smith and references therein) and, therefore, they are not reli a b l e indicators of mycorrhiza formation. Infection of roots from mycelial inoculum involves three phases (1) the growth of hyphae from the inoculum, (2) primary infection of roots from these "free" hyphae, and (3) secondary infections as hyphae from established mycorrhizae spread to other parts of the root system. Soil temperature affects these processes through i t s influence on fungal growth and root colonization. In addition, s o i l temperature influences many aspects of host plant growth and physiology which influence mycorrhiza formation including (1) rate of root growth (Hermann 1977, Ritchie and Dunlap 1980), (2) l a t e r a l branching and the production of short roots (Wilcox and Ganmore-Neumann 1975), (3) the maturation rate of roots (Nightingale 1935, Smit-Spinks 1983) and (4) the composition and quantity of root exudates (Rovira 1969, Slankis 1974). Changes in the growth, branching and maturation of roots alter the a v a i l a b i l i t y of suitable short roots for colonization by ECM fungi; changes in root physiology influence fungal growth in the rhizosphere (Slankis 1974, Wilcox and Ganmore-Neumann 1975). It is d i f f i c u l t to separate the various effects of s o i l temperature on mycorrhiza formation. In the current study, spruce seedlings were grown for eight weeks in a warm growing medium (18°C) before inoculation to ensure that a v a i l a b i l i t y of suitable colonization sites did not i n i t i a l l y l i m i t mycorrhiza formation at any temperature. The poor mycorrhiza formation observed in the 6°C mix appeared to result from decreased v i a b i l i t y of the inoculum. Few hyphae were observed adhering to the roots of these seedlings and many isolates produced no mycorrhizae. Low s o i l temperatures also depress the production of root exudates which are an 29 important nutrient source for "free" hyphae (Garbaye and Wilhelm 1985) . The 26°C temperature may have limited the growth of hyphae directly or altered root composition and hence primary or secondary infection. A l l but one isolate formed mycorrhizae suggesting that the growth of "free" hyphae may have been less affected by high s o i l temperature than the primary and secondary phases of infection. The susceptibility of short roots to infection may have been reduced by rapid rates of maturation or by lower sucrose concentrations. Suberization and the development of secondary tissues are more rapid at high s o i l temperature (Barney 1951, Nightingale 1935), reducing the amount of time roots are susceptible to infection. High sucrose concentrations are positively correlated with the susceptibility of l o b l o l l y pine roots to infection by P. tinctorius (Marx et a l . 1977). Chalupa and Fraser (1968) compared sucrose concentrations of white spruce seedlings grown at s o i l temperatures ranging from 10 to 38°C with a constant a i r temperature of 21°C. They found that the level of root sucrose (% dry weight) was inversely proportional to root-zone temperature. Differences among isolates in mycorrhiza formation at the various s o i l temperatures were not related to the origin of the fungal isolate ( f i e l d or nursery). This conclusion must be considered with caution due to two important limitations of the study (1) the lack of two to three nursery and f i e l d isolates for each species of fungus, and (2) the differences in age and treatment of the various cultures used i n the study. The original intent of the study was to obtain at least two f i e l d and nursery isolates of E-strain, T. terrestris and L. laccata. three ECM fungi found i n the nursery and f i e l d . Isolates for a l l three species were d i f f i c u l t to obtain. I was most successful locating L. laccata isolates in the University of Alberta Microfungus collection. However, during the study, many of these isolates were re c l a s s i f i e d as L. bicolor. Not a l l the cultures were collected, isolated, cultured or stored in the same manner. Culture and storage conditions have been shown to affect the a b i l i t y of fungi to form mycorrhizae (Marx 1981, Hung and Molina 1986). It is possible that the physiological a c t i v i t y of isolates forming few mycorrhizae at a l l growing mix temperatures (e.g., H. crustuliniforme 8) had been impaired during culture or storage. Given the limitations of this study, the results are consistent with the results of in v i t r o studies of temperature response variation in ECM fungal Isolates cultured from sporocarp tissue (Dennis 1985, Samson and Fortin 1986, Cline et a l . 1987), i.e., the temperature response of isolates is not related to their geographic origin. Dennis (1985) reported differences as great as 10°C in the optimum temperature for mycelial growth; but there was no relationship between the origin of the fungus and optimum temperature. Samson and Fortin (1986) reported that the growth of 62 fungal isolates with similar collection, culture and storage histories was not related to the geographic origin of the isolate. 31 CHAPTER III EFFECTS OF COOL SOIL TEMPERATURE AND SLOW-RELEASE FERTILIZER ON THE PERSISTENCE OF ESTABLISHED MYCORRHIZAE Introduction Inoculant fungi may disappear i n the f i r s t year after outplanting (Chu-Chou 1979, Bledsoe et a l . 1982, Mason et a l . 1983, Thomas et a l . 1983, Castellano and Trappe 1985, Danielson and Visser 1989). Competition from indigenous ECM fungi and the i n a b i l i t y of inoculant fungi to adapt to the f i e l d environment are considered to be the major reasons for poor persistence. High s o i l temperature has been shown to reduce the persistence of established mycorrhizae. Parke et a l . (1983b) reported that high s o i l temperatures (35°C for one week) reduced the persistence of mycorrhizae formed from fungi indigenous to Oregon and northern California. In container nurseries, the a b i l i t y of established mycorrhizae to tolerate high temperatures could influence the success of a r t i f i c i a l inoculation programs. The a b i l i t y of established mycorrhizae to survive and colonize new roots at cool temperatures (< 10°C) is a more important consideration for a r t i f i c i a l inoculation of seedlings outplanted in the cool so i l s of northern B r i t i s h Columbia. If the inoculant fungus survives and colonizes new roots before the onset of warm, dry summer conditions, seedling stress during the f i r s t f i e l d season may be minimized. There have been no studies of the effect of cool s o i l temperatures on the persistence of established ECM fungi although a study conducted by R i f f l e and Tinus (1982) suggests i t is an important factor. They observed that Pj_ tinctorius (Pt), a fungus adapted to warm s o i l s , did not persist on the root systems of pine seedlings planted in South Dakotaand speculated that cool so i l s and a short growing season were responsible for i t s disappearance. The objectives of the research reported in this chapter were (1) to study the a b i l i t y of ECM fungi established In a nursery to persist i n cool s o i l s , (2) to determine i f the presence of indigenous forest s o i l ECM fungi altered the effects of s o i l temperature on persistence, and (3) to study the effect of nutrient a v a i l a b i l i t y at the time of transplanting on the persistence of established mycorrhizae i n a cool (12°C) s o i l . The third objective was included because the application of slow-release f e r t i l i z e r at the time of planting is one s i l v i c u l t u r a l option for improving early seedling growth on sites with low nutrient a v a i l a b i l i t y . Nutrient a v a i l a b i l i t y in boreal forests is strongly affected by s o i l temperature through i t s effect on the rate of decomposition (Nielsen and Humphries 1966, Van Cleve et a l . 1990). ECM fungi vary in their response to f e r t i l i z e r (Alexander and Fairley 1983, Hunt 1989). Two studies testing both a r t i f i c i a l Inoculation and f e r t i l i z e r at the time of planting (Grossnickle and Reid 1983, Marx et a l . 1985) were conducted on a mine reclamation site and an African savanna, respectively, and are not applicable to reforestation sites in northern B.C. It would be useful to know i f slow-release f e r t i l i z a t i o n at the time of planting influences the persistence of established mycorrhizae. Approach Persistence was sub-divided into two components (1) survival on roots within the container root plug and (2) degree of new root colonization by inoculant fungi. The studies were conducted in pots with the recognition that persistence could be influenced by confinement of the roots to a restricted s o i l volume. The duration of the studies was limited to twelve weeks to minimize these effects. Since mycorrhizal Infection is influenced by the a v a i l a b i l i t y of photosynthate (Harley and Smith 1983 and references therein), variation in ligh t intensity and the size of seedling shoots was minimized to reduce potential variation in photosynthetic efficiency and capacity. Non-mycorrhizal s o i l f l o r a also affect root infection by ECM fungi (Brown and Sinc l a i r 1981, Summerbell 1987, McAfee and Fortin 1988). Therefore, a s o i l slurry containing these microorganisms was added to the forest s o i l which was pasteurized to eliminate indigenous ECM fungal inoculum. Methods Production of inoculated tree seedlings Eight-week-old white spruce seedlings (seedlot 29144) were inoculated as described in Chapter II with forest floor inoculum and mycelial slurries of seven ECM fungi typical of the early stages of mycorrhizal succession: (1) E-strain 947, (2) Hebeloma crustuliniforme 5249 (3) Laccaria laccata 101D (4) H. crustuliniforme 125, (5) Laccaria bicolor 5268, (6) Amphinema  byssoides or (7) Thelephora terrestris 2088. Origins of the isolates are summarized in Table 2.1 in Chapter II. After inoculation, the seedlings were grown in a controlled environment for 14 weeks under three cultural regimes: (1) 6 weeks with an 18 h photoperiod, 20°C a i r temperature and 50-80% relative humidity, (2) followed by 4 weeks of a short photoperiod treatment (8 h) to induce terminal bud formation, and (3) 4 weeks of cool conditions (14 h photoperiod, 9-14°C a i r temperatures) to harden the seedlings for cold storage. Once a week, seedling blocks were rerandomized to minimize possible position effects within the growth chamber; and the seedlings were watered to saturation with a water-soluble f e r t i l i z e r solution (100 mg/L N) containing micronutrients (Plant-Prod 20-20-20, Plant Products Co. Ltd., Bramalea, ON). Every two weeks, the seedlings were watered to saturation with an insecticide (0.84 g/L Diazinon, Later Chemical Ltd., Richmond, B.C.) to minimize losses of fungal tissues. The seedlings were cold-stored for 10 weeks at 3-4°C before the s o i l temperature treatments. Following cold-storage, approximately one-third of the seedlings were culled to reduce between-seedling variation i n size; the largest and smallest seedlings were removed from each inoculation treatment. Native ECM fungi and s o i l temperature treatments Seedlings were removed from the Spencer-Lemaire containers and transplanted into pots (4 L, 15 cm in diameter) containing a forest s o i l mixture consisting of forest floor, mineral s o i l and per l i t e (1:1:2 by volume). The forest floor and mineral s o i l were collected from three vigorous spruce plantations established near Mackenzie, B.C. These soil s o were passed through a 1 cm screen to remove gravel and large organic debris before the perlite was added. The mean values of pH (1:4 soil:water suspension, Peech 1965), available phosphorus (Bray and Kurtz Nol, Bray and Kurtz 1945) and mineralizable nitrogen (anaerobic incubation, Waring and Bremner 1964) for this mixture were respectively 4.9, 35 /ig/g and 68 /ig/g. Five of the eight inoculation treatments were planted i n forest s o i l with and without indigenous ECM fungal propagules at two s o i l temperatures, 6 and 12°C. Because of space limitations i n the water baths, the remaining three inoculation treatments were planted only i n forest s o i l with ECM fungal propagules (Table 3.1). Soil without viable indigenous ECM fungal propagules was prepared by pasteurizing i t for 40 minutes at 65°C. An extract, prepared by passing a slurry of unpasteurized forest floor material and autoclaved s t e r i l e water through a 0.50 /im f i l t e r , was added to the cooled pasteurized s o i l to restore forest floor microbes. The extract comprised less than IX of the total s o i l volume. The ECM inoculum potential of pasteurized and unpasteurized forest s o i l mixture was assessed by transplanting 8-week-old non-inoculated control seedlings into each mixture. Three pots (6 seedlings/pot) were maintained at each s o i l temperature (6, 12°C). Mycorrhizal status of the seedlings was estimated 0, 5 and 12 weeks after transplanting by destruetive sampling. Water baths were used to maintain two s o i l temperatures (6, 12°C). Pots within each bath were randomized weekly to minimize possible position effects within the water baths. The pots were watered to f i e l d capacity as required but no f e r t i l i z e r was applied to the forest s o i l mixture during the experiment. F e r t i l i z e r treatment Seedlings inoculated with (1) forest floor, (2) E-strain 947 or (3) H. crustuliniforme 5249 were transplanted into pots f i l l e d with either u n f e r t i l i z e d or f e r t i l i z e d 12°C unpasteurized forest s o i l (1:1:2, by volume, forest floor, mineral s o i l and p e r l i t e ) . The forest s o i l was f e r t i l i z e d with a resin-coated slow-release f e r t i l i z e r , Osmocote 14-14-14 (Sierra Chemical Co., Milpitas, CA) with a three month release time. Each f e r t i l i z e d pot was f i l l e d with 1.25 kg of the forest s o i l mixture thoroughly mixed with 1.3 g of Osmocote (100 kg/ha N). Evaluation of seedling nutrition Twelve weeks after transplanting and f e r t i l i z e r application, newly-flushed foliage of seedlings in the f e r t i l i z e r experiment were analyzed for nitrogen (N) and phosphorus (P) concentrations i n the dry matter. Seedling foliage from each f e r t i l i z e r - i n o c u l a t i o n treatment was bulked and oven-dried (70°C, 48 h) for analysis. Dried needles were ground in a Wiley M i l l to pass through a 20-mesh screen; and digested using a wet oxidation with H2SO4, Se, salts and H2O2 (Parkinson and Allen 1975). N and P concentrations in the digest were determined by a colorimetric (auto analyser) analysis using salicylate/nitroprusside for N and ascorbic acid/molybdate-antimony for P (Technicon Industrial Systems 1977). Accuracy and precision of these analyses were checked by including 3 samples of Standard Reference Material 1575 (pine needles) from the National Bureau of Standards^" in each run of 40 samples. Personal communication with A. Gammell, MacMillan Bloedel Ltd, Nanaimo, B.C. Table 3.1. Fungal isolated and inoculum used to study the effects of s o i l temperature and the presence of native ECM fungal inoculum on persistence of inoculant fungi Forest s o i l with indigenous Forest s o i l without ECM fungi indigenous ECM fungi (not pasteurized) (pasteurized) Non-inoculated seedlings E-strain 947* H. crustuliniforme 5249 L. laccata 101D Forest floor inoculum H. crustuliniforme 125 L. bicolor 5268 A. byssoides Thelephora terrestris 2088 Non-inoculated seedlings E-strain 947 H. crustuliniforme 5249 L. laccata 101D Forest floor inoculum H. crustuliniforme 125 NOTE: Non-inoculated seedlings were used to test the inoculum potential of indigenous ECM fungi. Included i n the f e r t i l i z e r experiment. 38 Mycorrhizal assessment Mycorrhizal infection was estimated on a six-class scale using low„ and high power magnification (as described i n Chapter II). The mycorrhizal status of f i f t e e n seedlings, selected randomly from each inoculation and s o i l temperature treatment combination after the largest and smallest seedlings were culled, was evaluated at the time of transplanting. A l l remaining seedlings were assessed 12 weeks after transplanting. The mycorrhizal status of roots within the original container root plug and new roots formed outside the plug were evaluated separately at 12 weeks. Mycorrhizae formed by indigenous forest s o i l ECM fungi were c l a s s i f i e d using characteristics (1) of the mycorrhizae (e.g., morphology, colour, abundance of extramatrical (EM) hyphae, presence of cystidia or mycelial strands), (2) of the EM hyphae (e.g., diameter, presence of ornamentation, c e l l wall pigmentation, clamps) and (3) of the mantle structure in plan view (e.g, shape, size and orientation of hyphae), which are rel a t i v e l y stable for a particular fungal genus over a range of host plants (Trappe 1967, Zak 1973, Haug and Oberwinkler 1987). Mantle structure was described using the textura system (Eckblad 1968, Alexander 1981, Haug and Oberwinkler 1987). Morphological types of indigenous mycorrhizae were grouped into several taxonomic types based on characteristics of fungal genera, for example Hebeloma-like. as recommended by Godbout and Fortin (1985) and Danielson and Pruden (1989). Descriptions of mycorrhizae formed by the pure cultures of inoculant fungi and forest s o i l ECM fungi are contained in Appendix B. The mycorrhizal types which occurred on non-inoculated seedlings transplanted into unpasteurized forest s o i l served as a basis for identifying indigenous ECM types. Morphological types formed by forest 39 s o i l fungi were generally quite distinct from those of the inoculant fungi. Evaluation of seedling growth Root c o l l a r caliper at the time of transplanting (0 week) was measured for each seedling. Root biomass, l a t e r a l root length, and number of short roots were measured at 0 and 12 weeks. The root system of each seedling was carefully extracted and cut lengthwise into two halves: one to assess the degree and type of mycorrhiza infection, and the other to estimate root length and short root number. Roots in the second sample were gently washed to remove the growing medium, cut into 3-5 cm long segments, thoroughly mixed, and a random subsample of fragments (approximately one-quarter of the total root system) was transferred to a Petri dish placed over a 1 cm grid. The number of short roots (sensu Sutton 1980) was counted in this subsample; the length of the long l a t e r a l roots was estimated using a modification (Tennant 1975) of a line intersect method (Newman 1966). Root biomass of each seedling was calculated by summing the oven-dry weights (70°C, 48 h) of the various subsamples. The total root length and number of short roots per seedling were estimated from dry weight ratio between the length subsample and whole root system. 40 Experimental Design and S t a t i s t i c a l Analysis Three completely randomized designs were used to test the various treatments on persistence: (1) a 2 x 2 x 5 f a c t o r i a l with 12 seedlings per treatment combination to study the effects of s o i l temperature (6, 12°C) i n the presence or absence of native ECM fungal inoculum on the persistence of 5 sources of inocula. (2) a 2 x 8 f a c t o r i a l with 12 seedlings per treatment combination to study the effects of s o i l temperature (6, 12°C) on the persistence of 8 sources of inocula. (3) a 2 x 3 f a c t o r i a l with 8 seedlings per treatment combination design to study the effects of f e r t i l i z e r (none, 100 kg/ha N) on the persistence of 3 sources of inocula. Seedlings in a l l experiments were grouped, four to a pot. Since variation between pots had an insignificant (P > 0.30) effect on mycorrhiza class or seedling root growth, individual seedlings were considered to be an experimental unit. The distribution of mycorrhiza class data did not meet the assumptions of parametric tests (Eisenhart 1947). Therefore, s t a t i s t i c a l analysis was conducted using non-parametric randomization tests as described in Chapter II. To compensate for differences in mycorrhizal status at the beginning of the experiment, persistence on new roots was compared to persistence on roots within the original plug using a paired-sample randomization test (Siegel 1956) . The effects of s o i l temperature, presence of native ECM fungal inoculum and slow-release f e r t i l i z e r on seedling root growth were tested by 41 least squares analysis of covariance (ANCOVA) using i n i t i a l root coll a r caliper as the covariate; or by least squares analysis of variance (ANOVA) when caliper interacted significantly with treatments, invalidating the "homogeneity of slope" assumption of ANCOVA (Hicks 1973). Analyses were performed on log-transformed values for the short root data to meet the homogeneity of variance assumption of ANOVA and ANCOVA. A l l tests were conducted using a microcomputer s t a t i s t i c a l package, SYSTAT (Wilkinson 1988). Results 1. Inoculum potential of forest s o i l mixtures Root systems of 8-week-old non-inoculated seedlings transplanted into the unpasteurized s o i l mixture were colonized rapidly by forest s o i l ECM fungi. At five weeks, 66% of these seedlings had some mycorrhizae; at twelve weeks, more than 95% of the seedlings had been infected by forest s o i l mycorrhizal fungi. At 12 weeks, mean mycorrhiza formation for individual seedlings exceeded 37% and 50%, respectively, in the 6 and 12°C s o i l s . At five weeks, no mycorrhizae were found in 8-week-old seedlings transplanted into pasteurized 6 and 12°C s o i l s . However, at twelve weeks, a ligh t infection of T. terrestris was observed at both temperatures on approximately 20% of the seedlings. T. terrestris inoculum may have survived pasteurization of the forest s o i l ; i t is more l i k e l y , however, that this infection resulted from air-borne spores. The growth chambers were located outdoors adjacent to a conifer nursery. 42 2. I n i t i a l mycorrhizal status of seedlings Mean mycorrhiza class for the various inoculation treatments ranged from 4.2 to 6.0 at the time of transplanting. Mycorrhiza formation was lower (P < 0.001) i n the A. bvssoides and T. terrestris treatments (mean mycorrhiza classes of 4.3 and 4.7, respectively) compared with the other isolates (mean classes 5.9 to 6.0). T. terrestris mycorrhizae were identified on 15% of the seedlings inoculated with A. bvssoides. This infection was weak with (1) fewer than 25% of the short roots of these seedlings infected, (2) extramatrical (EM) hyphae, mycelial strands and cystidia uncommon and (3) mantle formation discontinuous. Some seedlings inoculated with forest floor were also colonized by T. terrestris; 15% had 50% or more of the short roots colonized weakly by T. ter r e s t r i s . It was not clear whether these infections originated from forest s o i l propagules or from air-borne spores in the nursery. Seedlings inoculated with T. terrestris 2088 may also have been contaminated by air-borne spores of T. te r r e s t r i s . However, most mycorrhizae observed on seedlings inoculated with T. terrestris 2088 were well-developed (i.e., mantles covered most of the infected short roots, numerous cystidia and mycelial strands), strongly suggesting that they developed from the inoculant fungus and not from spores. Mycorrhizae formed from air-borne spores in seedlings inoculated with forest floor inoculum or A . bvssoides were not well-developed (i.e., exhibited weak mantle development, few mycelial strands). 43 3. Seedling root growth and morphology Lateral roots egressed horizontally from the top to bottom of the root plug. Root biomass, la t e r a l root length, and number of short roots per seedling measured at 12 weeks increased (P < 0.01) with s o i l temperature (Table 3.2). Pasteurization of the forest s o i l mixture had no effect (P > 0.20) on these root parameters. Seedlings assigned to each temperature treatment were not significantly different (P > 0.25) at the time of transplanting. Slow-release f e r t i l i z e r increased root biomass (P < 0.001) and l a t e r a l root length (P - 0.02) at 12 weeks; but had no effect (P - 0.22) on the number of short roots per seedling (Table 3.3). Table 3.2. Effect of s o i l temperature on mean seedling root biomass (oven-dry weight), root length and number of short roots for seedlings at 0 and 12 weeks Root parameter Duration of temperature treatment 0 weeks 12 weeks 6°C 12°C biomass (mg) length (cm) short roots/seedling 90 ( 26) 256 (122) 863 (392) 190 ( 14) 465 (197) 1394 260 (036) 606 (306) 1772 NOTE: Standard deviations are shown in parentheses. Data were averaged across 8 inocula; N = 96. Short root data for the 12 week measurement were log-transformed to meet the assumptions of ANOVA; means are shown in the original scale. 44 4. Survival of inoculant fungi within the original root mass Survival of inoculant fungi was high on roots within the container root mass with mean mycorrhiza class ranging from 4.7 to 6.0. Neither the presence of native inoculum (P > 0.50) nor s o i l temperature (P > 0.40) had an effect on survival within the original root mass. With one exception, mycorrhizae appeared turgid and healthy. E-strain mycorrhizae, particularly those i n the 6°C s o i l , were frequently shrivelled and dark-brown or black. Table 3.3. Effect of slow-release f e r t i l i z e r (NPK) at 12°C s o i l temperature on root parameters at 0 and 12 weeks Root Duration of f e r t i l i z e r treatment parameters 0 weeks 12 weeks -NPK +NPK biomass (mg) 90 (22) 231 (51) 388 (84) length (cm) 233 (122) 550 (224) 752 (335) short roots/seedling 887 (352) 1808 2122 NOTE: Data averaged across 3 inocula; N - 24. Short root data for the 12 week measurement were log-transformed before ANOVA; means are shown in original units (i.e., back-transformed means of logarithms). Except for these transformed data, standard deviations are shown i n parentheses. It is inappropriate to back-transform standard deviations (Steel and Torrie 1980, p. 236). 45 5. Effects of temperature and indigenous inoculum on the colonization of  new short roots Infection of new short roots formed outside the original root mass by inoculant fungi was good with more than 75% of short roots infected (Table 3.4) even though the potential for mycorrhiza formation by indigenous ECM fungi was significant. Non-inoculated seedlings transplanted into unpasteurized forest s o i l were rapidly infected by indigenous ECM fungi. EM hyphae of the inoculant fungi extended along l a t e r a l roots as they egressed from the container plugs, colonizing newly-initiated short roots. The intensity of new root colonization was highly correlated with the degree of infection of the original root mass (P - 0.85, P < 0.001). At the time of transplanting, percent root colonization by A. bvssoides and T. terrestris was lower compared with other fungal symbionts. To compensate for this difference, persistence of the various test fungi on newly-formed roots was compared to that on roots within the container plug, i.e., essentially using i n i t i a l infection as a covariate. In unpasteurized s o i l with a l l eight inocula, percent colonization of new roots was lower in the 12°C s o i l than in the 6°C s o i l (Table 3.4). At 6 C, only one fungal isolate, H. c rustulmiforme 5249, exhibited reduced (P - 0.03) persistence on new roots compared to old roots; at 12°C, three isolates, L. laccata 101D (P - 0.001), H. crustuliniforme 5249 (P - 0.06), and A. bvssoides (P - 0.06) exhibited reduced persistence on new roots. Indigenous ECM fungi colonized short roots not infected by L. laccata or A. bvssoides. In contrast, most short roots not colonized by H. crustuliniforme 5249 remained non-mycorrhizal. Table 3.4. Mean mycorrhiza class for roots within original container plug (old roots)- and for new roots formed outside the container plug Old roots New roots pasteurized unpasteurized 6°C 12°C 6°C 12°C 6°C 12°C Inocula Mean mycorrhiza class E 6. ,0 6.0 6. .0 6. ,0 5. ,9 6. ,0 He 5249 5. .9 5.8 5, ,7 6, .0 5. .4 5. ,3 He 125 6. ,0 6.0 6. .0 6, .0 6. .0 6. .0 LI 6, ,0 6.0 6, .0 6 .0 6, .0 4. .3 Ff 6, .0 6.0 6. .0 6, .0 6. .0 6. ,0 Lb 6, .0 6.0 6 .0 6, .0 Ab 4, .4 4.2 4, .3 3. .8 Tt 4, .3 5.0 4 .2 4, .8 NOTE: N - 15 and 12, respectively for old and new roots. Abbreviations for inocula: Ab - A_,_ bvssoides. E - E-strain, Ff - forest floor, He - H. crustuliniforme. Lb — L. bicolor. LI •» L. laccata. Tt - T. terrestris 5 f * Mean mycorrhiza classes: (1) 0% of short roots mycorrhizal, (2) 1-25% mycorrhizal, (3) 26-50% mycorrhizal, (4) 51-75% mycorrhizal, (5) 76-95% mycorrhizal, (6) > 95% mycorrhizal. For the five inocula in both soi l s and temperatures, the presence of indigenous fungi reduced new root colonization (P — 0.001) with significant interactions between the presence of indigenous native fungi, s o i l temperature and inoculation treatments on new root colonization. Soil temperature had no effect on new root colonization in the absence of indigenous ECM fungi, but did affect new root colonization in the presence of indigenous inoculum. The presence of indigenous inoculum reduced (P - 0.04) new root colonization by H. crustuliniforme 5249 and L. laccata 101D, but had no effect on new root colonization by other inocula. The presence of indigenous ECM fungal inoculum reduced new root infection by H. crustuliniforme 5249 at both s o i l temperatures, but reduced new root infection by L. laccata 101D only at 12°C. 6. Effect of nutrient addition (Osmocote) on persistence At the time of transplanting, mean mycorrhiza class for roots within the container plug averaged 6.0, 5.9, and 6.0 respectively for the E-strain, H. crustuliniforme and forest floor seedlings. Slow-release f e r t i l i z e r had no effect (P > 0.40) on survival of inoculant fungi within the original root plug but did affect colonization of new roots formed outside the plug (Table 3.5). In the unfertilized forest s o i l , colonization of new root growth was greater than 75% for a l l inoculant fungi. F e r t i l i z a t i o n had no effect on new root colonization by forest floor, but reduced (P - 0.05) that by E-strain by approximately 20% While new root colonization by H. crustuliniforme 5249 was lower than that i n old roots, nutrient addition had no effect (P > 0.05) on new root colonization. Short roots not infected with E-strain were colonized by forest floor fungi; those not infected by H. crustuliniforme 5249 remained non-mycorrhizal. 48 Table 3.5. Degree of short root colonization by inoculant fungi at the time of slow-release f e r t i l i z e r (Osmocote 14-14-14 NPK) application and at 12 weeks after application Roots at the time of f e r t i l i z e r application New roots formed after f e r t i l i z a t i o n -NPK +NPK Inocula Mean mycorrhiza class E He 5249 Ff 6.0 5.9 6.0 6.0 5.3 6.0 4.6 5.6 6.0 NOTE: N - 16 at the time of f e r t i l i z e r application; N - 8 for both f e r t i l i z e r treatments at 12 weeks. Abbreviations for inocula: E «• E-strain, Ff - forest floor, He = H. crustuliniforme. Mean mycorrhiza classes: (1) 0% of short roots mycorrhizal, (2) 1-25% mycorrhizal, (3) 26-50% mycorrhizal, (4) 51-75% mycorrhizal, (5) 76-95% mycorrhizal, (6) > 95% mycorrhizal. 7. Foliar nutrition Slow-release f e r t i l i z a t i o n appeared to increase the levels of f o l i a r N of seedlings inoculated with forest floor; but had l i t t l e effect on f o l i a r N and P of the other seedlings (Table 3.6). Levels of f o l i a r P (% dry weight) decreased s l i g h t l y in a l l inoculation treatments probably due to growth dilution. F e r t i l i z e d seedlings had more than twice the P content (mg/seedling) i n new foliage than did unfe r t i l i z e d seedlings. Table 3.6. Effect of slow-release f e r t i l i z e r (NPK) and inoculation treatments on N and P concentrations (%. oven-dry weight) in new foliage at 12 weeks Inoculation -NPK +NPK treatment N% P% N% P% Forest floor 1.03 0.21 1.73 0.19 E-strain 1.67 0.22 1.80 0.19 H. crustuliniforme 0.93 0.20 0.90 0.15 NOTE: N - l ; each sample consists of pooled foliage from 8 seedlings. 50 Discussion In general, spread of inoculant fungi to new roots produced outside the original container plug was considered good (> 50% infection) i n the experimental conditions (cool, acidic soils) of the study. This result suggests mycorrhizae established in the nursery have the potential to significantly influence mycorrhiza development, seedling nutrition, growth and physiology at least i n the f i r s t growing season after planting. A similar conclusion was reached by Dahlberg (1990) for lodgepole and Scots pine seedlings outplanted in Sweden and by Fleming et a l . (1984) for s i l v e r birch seedlings outplanted i n Great Britain. In the study conducted by Fleming et a l . (1984) a short period (8 weeks) of mycorrhizal inoculation in the nursery influenced mycorrhiza formation in the f i e l d for at least 45 months. Soi l temperature and the presence of indigenous ECM fungi interacted to influence the infection by inoculant fungi of short roots i n i t i a t e d outside the root plug. When native ECM fungi inoculum was present, inoculant fungi colonized a greater percentage of new roots i n the 6°C s o i l than in the 12°C s o i l . Inoculant fungi may persist and influence seedling growth and physiology for a longer period on routine reforestation sites in boreal forest regions compared with sites i n warm forest regions where most mycorrhizal research has been conducted. In the absence of native ECM inoculum, s o i l temperature had no effect on the percentage of new roots colonized by inoculant fungi. Infection of old and new roots by inoculant fungi exceeded 95% i n 6 and 12°C pasteurized s o i l s . Several factors may be responsible for the reduced persistence of the test fungi i n unpasteurized 12°C s o i l . Extramatrical (EM) hyphae of the inoculant fungi were observed spreading along l a t e r a l roots as they 51 egressed from the container root plug, colonizing short roots near established mycorrhizae. Indigenous mycorrhizae when present were found near the tips of elongating l a t e r a l roots. This pattern of infection by inoculant and indigenous fungi was also observed by McAfee and Fortin (1986) on jack pine seedlings two months after planting in various f i e l d environments. In the 6°C s o i l , short roots tended to emerge into forest s o i l containing extramatrical hyphae of the inoculant fungi. In the 12°C s o i l , the more rapid rate of root elongation increased the probability that short root apices would contact indigenous sources of inoculum before being colonized by EM hyphae of the inoculant fungi. Warming of the s o i l from 6 to 12°C appeared to increase the rate of root extension more than the rate of hyphal extension. Short root production and root elongation was 70% greater in the 12°C s o i l . Second, cool s o i l temperatures may have slowed primary infection from "free" indigenous propagules (hyphal fragments, germinating spores) more than secondary infection from hyphae attached to established mycorrhizae. Established mycorrhizae have access to host plant photosynthate for mycelial respiration and growth. Whereas established infections of T. terrestris 2088 and A. bvssoides survived and colonized new roots i n 6°C so i l s , these isolates did not form mycorrhizae from "free" mycelial inoculum at 6°C (see Chapter II). Significant differences in persistence were evident for the various fungal isolates. Averaged across a l l treatments (temperature, +/- native inoculum), established L. bicolor. T. ter r e s t r i s . E-strain, H. crustuliniforme 125 inocula were the most successful colonizers of roots outside the plug i n the study conditions, i.e., cool, acidic, wet forest s o i l s . L. laccata. H. crustuliniforme 5249, and A. bvssoides inocula were 52 the least successful. In f i e l d studies, Laccaria species have been shown to persist and compete successfully i n a variety of s o i l types during the f i r s t growing season (McAfee and Fortin 1986, Wilson et a l . 1987, Danielson and Visser 1989). L. bicolor competed successfully with native ECM fungi i n acidic (pH 3.7-6.1) disturbed forest so i l s of the boreal forest zone (McAfee and Fortin 1986). Long-term persistence of Laccaria spp. is more variable and may depend on s o i l type. Danielson and Visser (1989) reported that L. proxima persisted only one growing season i n neutral (pH 6.3-7.5) o i l sands amended with peat in a cool, continental climate. In contrast, Wilson et a l . (1987) found that L. proxima persisted for at least four growing seasons in a poorly-drained acidic peat s o i l . Shaw e_t a l . (1987b) also reported good persistence (> 50% of short roots) of L. laccata for two years on Sitka spruce seedlings planted in variety of organic and mineral s o i l microsites In a cool, moist maritime climate. Long-term persistence of Laccaria spp. also may depend on the overwinter survival of EM hyphae. Coutts and Ni c o l l (1990a) monitored mycelial growth of established T. terrest r i s and L. proxima infections on potted Sitka spruce seedlings for a summer and winter. They found that EM hyphae of L. proxima disappeared during the winter as s o i l temperatures dropped below 6°C, decreasing the likelihood that short roots emerging the following spring would contact L. proxima inoculum. T. terrestris is one of the most common fungi in forest nurseries throughout the world. Although i t is adapted to the high f e r t i l i t y and moisture conditions of nurseries (Marx et a l . 1984, Hunt 1989), T. terrestris established in a nursery can successfully persist after outplanting (Cruz 1974, Ruehle 1982, Thomas et a l . 1983, Danielson and Visser 1989). Cruz (1974) found that T. terrestris persisted on slash pine seedlings planted i n poorly-drained, acidic s o i l s , whereas, Pisolithus  tinctorius was replaced by native fungi. Extramatrical hyphae of T. terrestris have been shown to overwinter successfully at s o i l temperatures below 6°C (Coutts and N i c o l l 1990a), increasing the probability that short roots formed the following year would emerge into forest s o i l containing T. terres t r i s inoculum. E-strain has been shown to survive for several years after outplanting, particularly i n dry, neutral to moderately alkaline so i l s (Danielson and Pruden 1989, Danielson and Visser 1989) or i n burnt, clear-cut so i l s (Mikola 1965). whether E-strain w i l l persist for several years in cool, wet, acidic s o i l s has not been determined. Its a b i l i t y to survive and compete at cool temperatures may be the deciding factor. E-strain is known to tolerate a wide range of s o i l pH values (Mikola 1965) and poor s o i l drainage (Levisohn 1954). The high degree of new root colonization (> 75% of short roots) for both isolates of H. crustuliniforme i n the acidic s o i l s of the study was not expected. Previous studies have suggested that the persistence of Hebeloma spp. would be low (Lamb 1979, Chu-Chou and Grace 1981, Mason et a l . 1983), especially in acidic s o i l s . Mason et a l . (1983) observed that native Hebeloma fungi colonized birch seedlings planted in mineral so i l s but not those planted i n peat s o i l s ; and that H. sacchariolens established on nursery birch seedlings did not persist in an acid peat s o i l . In vitro studies (Hung and Trappe 1983, Dennis 1985) indicate Hebeloma spp. are adapted to neutral to alkaline s o i l s . Optimum growth in culture occurs at pH 7 (Hung and Trappe 1983), with some growth even at pH 9 (Dennis 1985). 54 Danielson and Pruden (1989) reported that Hebeloma-like native s o i l fungi persisted for a long period on urban spruce growing in dry, neutral to moderately alkaline s o i l s . Data on f i e l d persistence of established A. bvssoides infections is not available i n the mycorrhiza literature. However, the common occurrence of this species in coniferous forests suggests established infections may compete successfully with native ECM fungi. Based on fruit-body distribution, Eriksson and Ryvarden (1973, cited i n Danielson et a l . 1984d) consider A. bvssoides to be a characteristic species of European coniferous forests. In North America, A. bvssoides has been collected from mature stands of jack pine (Danielson 1984b) and white spruce (Danielson et a l . 1984d). The optimum s o i l pH, moisture, and temperature conditions for persistence of established A. bvssoides infections is not known. The poor persistence of A. bvssoides in the forest s o i l mixture used in this study suggests i t s a b i l i t y to compete with native ECM fungi may be low in cool, wet, acid s o i l s . However, the A. bvssoides isolate used in the study originated from a container nursery environment; and may have been better adapted to a more f e r t i l e , less acidic growing medium. More isolates need to be tested to determine the ecological characteristics of this species. The three inocula sources varied in their response to nutrient addition at the time of transplanting. F e r t i l i z a t i o n reduced colonization of roots outside the plug by E-strain; but not colonization by H. crustuliniforme or by the forest s o i l inoculum. High rates of NPK application have been shown to inhibit mycorrhiza formation i n nursery stock (Crowley et a l . 1986, Gagnon et a l . 1987, Hunt 1989). Inhibition may be due to a build-up of f e r t i l i z e r salts in the growing medium (Crowley et a l . 1986) or to a reduction in root sucrose concentrations (Marx et a l . 1977). E-strain is known to be sensitive to high rates of soluble (Danielson and Visser 1988) and slow-release (Gagnon et a l . 1987, Hunt 1989) NPK f e r t i l i z e r in container growing medium. However, the application rate used in this study was not high and should not have reduced mycorrhiza formation by E-strain; the amount of slow-release f e r t i l i z e r applied was less than half the rate found to be inhibitory to mycorrhiza formation by other researchers, e.g., Crowley et a l . (1986), Gagnon et a l . (1987) and Hunt (1989). Shaw et a l . (1987a) reported that E-strain could successfully infect conifer roots at application rates much higher than those used in this study. Also levels of f o l i a r N and P (1.90% and 0.22%, respectively) were not excessively high. E-strain can successfully colonize roots of seedlings with similar levels of N and P (Danielson and Visser 1988). Possibly, fertilizer-induced changes in root growth, host physiology (e.g., composition or quantity of root exudates, hormones or root content of sucrose) or in the population of rhizosphere microorganisms decreased the a b i l i t y of E-strain hyphae to contact and colonize short roots before they were occupied by indigenous inoculum. Alternatively, E-strain may be more sensitive to an increase in f e r t i l i z e r salts or to a lowering of root sucrose concentrations i n cool moist soi l s than in warm nursery mixes. Danielson and Visser (1989) noted that competition from indigenous ECM fungi is the most frequent explanation for lack of persistence by inoculant fungi. Active replacement of inoculant by indigenous fungi has been observed in the f i e l d . However, in their study of f i e l d persistence of nine ECM fungi on jack pine seedlings planted on a mine reclamation site, they observed that replacement of inoculant fungi by indigenous fungi was often passive, i.e., the inoculant fungus infecting a short root died and was subsequently replaced by an indigenous fungus with no apparent 56 physical contact between the two fungi. Lack of persistence with non-interactive replacement is more l i k e l y due to poor adaptation of the inoculant fungus to the host plant or environment. Danielson and Visser (1989) hypothesized that ill-adapted mycorrhizae lose vigour because they do not function effectively as sinks for host plant carbohydrate. Neither active nor passive replacement of inoculant fungi was observed during this study. Twelve weeks may be too short a time for active or passive replacement to occur. In addition, an interruption i n root growth may be necessary before one fungus is able to replace another (Marks and Foster 1967). Root growth was continuous in the controlled environment, possibly because s o i l moisture was not limiting. The unhealthy appearance of many E-strain mycorrhizae suggests this fungus may be replaced in a longer-term study. Alternatively, the lack of replacement In the study may r e f l e c t the "free" nature of the indigenous inoculum, i.e., hyphal fragments or dormant propagules. In f i e l d environments, the infecting hyphae of indigenous ECM fungi may be connected to a carbohydrate reserve ( l i v i n g plant root) which supports faster growth and greater competitiveness with inoculant fungi. Except i n the H. crustuliniforme treatment, short roots not infected by inoculant fungi were colonized by native ECM fungi. Most short roots not infected by H. crustuliniforme remained non-mycorrhizal, suggesting that this fungus may inhibit colonization of the root system by other fungi. Other researchers have reported similar inhibitory effects i n species of Laccaria. Hebeloma and Rhizopogon. Gagnon et a l . (1987) noted that L. bicolor or H. cylindrosporum infections on container-grown pine seedlings appeared to inhibit root colonization by fungi indigenous to container nurseries (e.g., T. t e r r e s t r i s ) . McAfee and Fortin (1986) 57 reported that Rhizopogon rubescens prevented infection by ECM fungi native to boreal forests. They hypothesized that Rj. rubescens might secrete chemicals which inhibit root colonization by competing ECM fungi. Cultures of Rhizopogon have been shown to secrete chemicals that inhibit other ECM fungi^". The i n i t i a l mycorrhizal status of the seedlings and the pattern of l a t e r a l root egress from the container plug may have contributed to the high persistence of inoculant fungi i n this study. Inoculum density is a major factor determining the a b i l i t y of fungal Isolates to form mycorrhizae in a nursery environment (Garbaye 1983, E g l i and Kalin 1985); and may have a significant influence on f i e l d persistence of inoculant fungi. Colonization of roots outside the plug was positively correlated with the percentage of container plug roots infected by the inoculant fungi prior to transplanting. Analysis of data presented by Danielson and Visser (1989) for nine ECM fungi showed that percent colonization at the time of planting was also positively correlated (P - 0.006) with the percentage of new roots infected by inoculant fungi one year after planting. Egress of l a t e r a l roots from the entire root plug, as occurred in this study, improves f i e l d persistence of inoculant fungi (Ruehle 1983, 1985). Ruehle concluded that persistence of Pj. tinctorius was greater on outplanted bare-root l o b l o l l y pine seedlings than on container-grown seedlings because la t e r a l roots egressed horizontally from the entire root system of the bare-root stock. In contrast, l a t e r a l roots elongated v e r t i c a l l y from the bottom of the container root plugs. P_j_ tinctorius did not colonize these v e r t i c a l laterals, apparently due to inhibitory ^ Castellano 1987, unpublished data cited in Castellano and Molina 1989. 58 biological factors (e.g., native ECM symbionts, microbes or pathogenic fungi). Pj. tinctorius was able to successfully colonize v e r t i c a l l y egressing roots in s t e r i l i z e d s o i l . The possible effects of inoculum potential and la t e r a l root egression on f i e l d persistence should be considered when interpreting the results of outplanting t r i a l s . Although Bledsoe et a l . (1982) cited competition as the major reason for poor f i e l d persistence of L. laccata and H. crustuliniforme inoculum on the root systems of outplanted container-grown Douglas-fir seedlings, three results of their study suggest that inoculum density and la t e r a l root egression patterns affected persistence: (1) fewer than 25% of the roots formed outside the plug were colonized by native forest s o i l ECM fungi five months after planting, (2) new la t e r a l roots egressed v e r t i c a l l y from the bottom of the root plugs, and (3) the inoculant fungi had not colonized the root plug extensively prior to outplanting. Inoculum distribution within the plug was not described. It is possible that low infection levels in the bottom of the container plugs reduced the potential for colonization of v e r t i c a l l y egressing l a t e r a l roots. Summary Fungal symbionts established i n a warm container nursery environment persisted within the original root mass and colonized new roots i n i t i a t e d outside the container plug when white spruce seedlings were tranplanted into cool so i l s (6, 12°C). Even the common indigenous nursery fungus, T. ter r e s t r i s . considered to be poorly adapted to f i e l d environments, rapidly colonized new roots. Soil temperature and the presence of indigenous ECM fungi interacted to influence new root colonization by inoculant fungi. 59 Indigenous f o r e s t f u n g i (from " f r ee" i n o c u l a p ropagu les ) were poor r o o t c o l o n i z e r s a t 6°C, and t h e i r presence reduced pe rcen t r o o t c o l o n i z a t i o n by i n o c u l a n t f u n g i o n l y a t 12°C. F a c t o r s wh ich c o n t r i b u t e d to s u c c e s s f u l c o l o n i z a t i o n o f new r o o t s by i n o c u l a n t f u n g i , i n c l u d e d (1) degree o f r o o t c o l o n i z a t i o n by i n o c u l a n t f u n g i i n the n u r s e r y , (2) the p a t t e r n o f new r o o t growth , and (3) the r e l a t i v e weakness o f e c t o m y c o r r h i z a l p ropagu les (spores and h y p h a l fragments) compared w i t h l i v e e c t o m y c o r r h i z a l a t t achments . The n e g a t i v e e f f e c t o f NPK f e r t i l i z e r a t the t ime o f t r a n s p l a n t i n g on new r o o t c o l o n i z a t i o n by E - s t r a i n f u n g i suggests t h a t i n t e r a c t i o n s between i n o c u l a n t f u n g i and s i l v i c u l t u r a l p r a c t i c e s need f u r t h e r i n v e s t i g a t i o n . 60 CHAPTER IV EFFECT OF VARIOUS MYCORRHIZAL FUNGI ON THE ACCLIMATION OF WHITE SPRUCE SEEDLINGS TO LOW SOIL TEMPERATURES Introduction In cold s o i l s , planted seedlings are subject to water stress even when s o i l water content is high. Low root-zone temperatures reduce the permeability of root c e l l membranes and increase the viscosity of water resulting i n high plant resistance to water flow and low water absorption (Kramer 1940, Kaufmann 1975, Running and Reid 1980, Teskey et a l . 1984, Orlander and Due 1986). In 7°C s o i l , for example, water absorption by Scots pine is approximately 30% of that in 25°C s o i l (Orlander and Due 1986) . If seedlings transpire more water than the amount absorbed under these conditions, they may develop shoot water de f i c i t s despite adequate s o i l moisture (Goldstein et a l . 1985, Lopushinsky and Kaufmann 1984). Cold soi l s also decrease root and shoot growth of newly planted stock (Barney 1951, Lopushinsky and Kaufmann 1984). Decreased metabolic ac t i v i t y or turgor would influence the growth of root tissues. In addition, carbon assimilation may be reduced in cool soi l s (Babalola et a l . 1968, Lawrence and Oechel 1983b, Delucia 1986), limiting the a v a i l a b i l i t y of current photosynthate for new shoot and root growth. Any reduction in root growth after planting increases the susceptibility of seedlings to summer drought (Lopushinsky and Kaufmann 1984). When water uptake is reduced by high root resistances, the development of severe water stress is prevented in the short-term by delayed flushing of new shoot tissue (Blake 1983) and by stomatal closure (Lopushinsky and Kaufmann 1984). The delay in flushing is important because newly-flushed needles of spruce seedlings transpire at 4-5 times the rate of mature needles (Blake 1983). Cold-stored white spruce seedlings, however, may not be able to regulate stomata of mature needles immediately after transplanting. Grossnickle and Blake (1985) observed that the stomata of white spruce seedlings, i n contrast to those of jack pine seedlings, did not close f u l l y at night for two to three weeks after they were removed from cold storage. Consequently, the white spruce seedlings were unable to control water loss even when photosynthesis was not occurring. Resistance to water flow, or i t s inverse hydraulic conductance, is affected by many plant characteristics including (1) total volume or surface area of the root system (Carlson 1986), (2) total plant size and shoot/root ratio (Levy et a l . 1983, Koide 1985), (3) amount of new root growth or the proportion of unsuberized to suberized roots (Kramer and Bullock 1966, Sands et a l . 1982), (4) nitrogen and phosphorus nutrition (Safir et a l . 1972, Graham and Syvertsen 1984, Radin and Eidenbock 1984), and (5) presence of root disease (Marschner 1986). Ectomycorrhizal infection influences a l l of these plant characteristics (Harley and Smith 1983 and references therein) suggesting that i t would alter root resistance to water flow as has been shown for vesicular-arbuscular (VA) infections i n crop plants. These effects have been attributed to improved phosphorus nutrition (Nelsen and Safir 1982, Graham and Syvertsen 1984, Koide 1985), changes in plant root/shoot ratios (Andersen et a l . 1988) or in the balance of plant growth regulators (Levy and Krikun 1980, Allen et a l . 1981). VA infections also have been shown to influence stomatal conductance (Levy and Krikun 1980), possibly by altering plant nutrient status or the balance of plant growth regulators (e.g., 62 cytokiniris or abscisic acid) that affect stomatal opening and closing. If cold-stored spruce seedlings do not quickly acclimate.to the environment of the planting site, transplant stress may develop (Grossnickle and Blake 1985). The objectives of the work reported in this chapter were (1) to describe the effects of inoculation on seedling response to low s o i l temperature during the transplant stress period, i.e., the f i r s t 6 weeks after planting, and (2) to test the hypothesis that inoculation with specific fungi could speed seedling acclimation. Five variables were chosen to compare the acclimation of cold-stored inoculated and non-inoculated seedlings: flushing date, amount of new root growth, resistance to water flow, pre-dawn stomatal conductance, and the rate of net photosynthesis. Resistance to water uptake was measured using a whole plant rather than a root pressurization technique. Root pressurization measurements are subject to artifacts (Passioura 1988) and may underestimate the resistance of seedlings (Grossnickle and Russell 1990). These parameters were measured for three weeks after transplanting, the time required for cold-stored spruce and pine seedlings to acclimate physiologically to cool so i l s (Grossnickle and Blake 1985). Two s o i l temperatures (6, 12°C) were chosen to bracket the s o i l temperature (7-8°C) below which root resistance of lodgepole pine (Running and Reid 1980) and Engelmann spruce (Kaufmann 1975) rises exponentially, and the s o i l temperature (10°C) at which root growth of spruce sharply declines (Tyron and Chapin 1983). Methods 3 . E i g h t - w e e k - o l d seed l ings sown i n 40 cm Spencer-LeMaire Rootra iners were i n o c u l a t e d , grown and c o l d - s t o r e d as d e s c r i b e d i n Chapter I I . They were then t r a n s p l a n t e d i n t o pots c o n t a i n i n g a mixture o f f o r e s t f l o o r , m i n e r a l s o i l and p e r l i t e (1:1:2 by volume) maintained at e i t h e r 6°C or 1 2 ° C . The surface o f the s o i l was covered w i th a 2 cm l a y e r o f white Styrofoam ch ips i n order to minimize heat exchange between the s o i l and a i r . A p r e l i m i n a r y study showed that s o i l temperature v a r i e d v e r t i c a l l y by 1 . 5 ° C and h o r i z o n t a l l y by l e s s than 1 . 0 ° C . S ix i n o c u l a t i o n treatments were i n c l u d e d i n t h i s experiment: (1) 4 3 cm o f f o r e s t f l o o r from vigorous spruce p l a n t a t i o n s , (2) 4 mL o f s t e r i l i z e d agar s l u r r y ( c o n t r o l ) , or 4 mL of a m y c e l i a l s l u r r y prepared from pure c u l t u r e s o f (3) Hebeloma c r u s t u l i n i f o r m e 5249, (4) L a c c a r i a b i c o l o r 5268, (5) E - s t r a i n 947, and (6) Thelephora t e r r e s t r i s ( L a v a l ) . A l l Inocula were c o l l e c t e d from f o r e s t s i t e s (Table 2 . 1 ) . Seedl ings were not f e r t i l i z e d a f t e r t r a n s p l a n t i n g but were watered wi th tap water o f the appropr ia te temperature (6 or 1 2 ° C ) to m a i n t a i n s o i l moisture p o t e n t i a l at -0.01 MPa, i . e . , the s o i l s were both c o o l and mois t . The three week study was conducted i n a c o n t r o l l e d environment chamber: 30 to 50% r e l a t i v e humid i ty , r e s p e c t i v e day /n ight a i r temperatures o f 2 0 / 1 2 ° C , and an 18-h photoper iod wi th a photosynthet i c photon f l u x dens i ty o f 400 2 /imol/(m s) from a combination o f incandescent and c o o l white f l u o r e s c e n t l i g h t s . Two bu lked samples of root and shoot t i s s u e (8 seed l ings per sample) from each i n o c u l a t i o n treatment were analyzed for n i t r o g e n (N) and phosphorus (P) at the time o f t r a n s p l a n t i n g . A f t e r oven-dry ing ( 7 0 ° C , 48 h ) , m i l l i n g and wet o x i d a t i o n with H 2 S O 4 , Se, s a l t s and H 2 O 2 (Parkinson and 64 Allen 1975), tissues were analyzed for N and P by colorimetric analysis using salicylate/nitroprusside for N and ascorbic acid/ molybdate-antimony for P (Technicon Industrial Systems 1977). Stem diameter, shoot biomass, root biomass and morphology (lateral root length and short root number), pre-dawn stomatal conductance, net photosynthesis and resistance to water flow were measured 2, 9 and 21 days after transplanting on 8 seedlings per temperature and inoculation treatment. Individual seedlings were carefully removed from the pots to minimize disturbance to the remaining seedlings; cavities were f i l l e d with forest s o i l mixture. Disturbance to the remainder of the seedlings in a pot was negligible due to the coarse friable nature of the forest s o i l mixture and to the lack of significant root elongation during the experiment. Measurements of biomass and root morphology at day 2 were assumed to be representative of these parameters at the time of transplanting (day 0). Flushing was recorded 9, 16 and 21 days after transplanting. A seedling was considered flushed i f the terminal bud had broken through the bud scales. The root system of each seedling was cut lengthwise with one half examined for mycorrhizal infection. Mycorrhiza formation by inoculant and nursery contaminant fungi was assessed at low and high power as described in Chapter III. The second half was carefully washed and cut into 2-3 cm long segments. No attempt was made to remove a l l extramatrical hyphae from mycorrhizae. Approximately a third of these segments were selected randomly to estimate short root numbers and root length. Root length was estimated using Newman's line intersect method (1966) as modified by Tennant (1975). Total seedling root length and short root number were derived from the dry weight (70°C for 48 h) ratio between sample segments 65 and the whole root system. Stomatal conductance, net photosynthesis and internal needle CO2 were measured using a portable gas exchange system (LI-6200, Li-Cor Ltd.). Measurements were conducted 4 to 6 hours into the li g h t period. A preliminary study showed that net photosynthesis and stomatal conductance of individual seedlings were not affected (P > 0.25) by the time of measurement during this period. A l l measurements were made on mature needles; any newly flushed needles were removed carefully with tweezers prior to measurement. Incorporation of newly flushed needles causes errors in physiological measurements (e.g., Lippu and Puttonen 1989) since their photosynthetic rates are low in comparison to those of mature needles (Troeng and Linder 1982). Net photosynthetic rates were calculated on the basis of needle dry weight and surface area which was estimated from needle length and displaced volume (Brand 1987). Photosynthetic nitrogen-use efficiency or PNUE, the rate of net photosynthesis per unit of shoot nitrogen, was derived from net photosynthesis and shoot f o l i a r N data. Growth, conductance and net photosynthesis data were tested by least-squares analysis of variance (ANOVA) using SYSTAT (Wilkinson 1988). Data were analyzed using a completely randomized three-way f a c t o r i a l design with fixed treatments: s o i l temperature (2 levels), inoculation (6 types) and time (3 levels) with 4 pots (each containing 6 seedlings) nested within the s o i l temperature and inoculation treatments. When pot-to-pot v a r i a b i l i t y was insignificant (P < 0.25, Bancroft 1964) compared to seedling-to-seedling v a r i a b i l i t y , these two sources of v a r i a b i l i t y were combined as the error term to test main effects and interactions. Otherwise pot-to-pot v a r i a b i l i t y was used as the error term. Data were transformed when necessary to meet the normality and homogeneity of variance assumptions of ANOVA (Eisenhart 1947). Linear contrasts were used to compare inoculation treatments. The error rate for multiple comparisons was controlled using the Bonferroni procedure, i.e., for an overall rate of p, k comparisons were conducted at p/k (Wilkinson 1988). Correlation analysis was used to examine relationships between seedling physiology and seedling morphology and nutrition. Root resistance to water uptake was estimated by measuring "soil-to-xylem" resistance (RSX) to water flow. Root resistance i s considered to be the major resistance along this pathway unless so i l s are very dry (Passioura 1982), with s o i l water potentials less than -0.1 MPa (Gardner and Ehlig 1962). The root resistance of lodgepole pine seedlings is 67% and 93%, respectively, of total plant resistance at 7°C and 0°C (Running and Reid 1980). RSX was measured indirectly using a modification of Elfving et al.'s (1972) model of plant water relations in which, needle water potential (Vneedle) I s considered a function of s o i l water potential (Ysoil)• resistance to water flow through the soil-plant-air-continuum (RSPAC) and transpirational flux density (TFD): Vneedle - Vsoil - RSPAC*(TFD) Assuming (1) that experimental seedlings had a negligible capacity for water storage, (2) that V Xyl em approximated U n e e ( j i e and (3) that the water potential of the s o i l was zero, this model was reduced to: -RSX - (yxylem>/TFD The f i r s t two assumptions are reasonable given the small size of the seedlings used i n this study. The third was met by maintaining s o i l water content above f i e l d capacity. Three seedlings per inoculation-temperature treatment combination were measured to determine pre-dawn Uxylem using a pressure chamber and a 6 7 hand-held lens (Ritchie and Hinckley 1975). Five seedlings were measured to determine mid-day (.4—hours into the light period) Uxylem a n c^ transpirational flux density (TFD) using a portable gas exchange system (LI-6200). RSX for each treatment combination was calculated from the slope of the regression of U X y i e m on log-transformed values of TFD. This transformation was necessary to meet the requirement for residual homogeneity of variance i n least squares regression analysis (Steel and 2 Torrie 1980, p. 248). R values for these regressions ranged from 0.60 to 0. 90. Equality of regression slopes (RSX) was examined by t-tests (Neter and Wasserman 1974). Results 1. Mycorrhizal status Sixty percent of the non-inoculated control seedlings were non-mycorrhizal at the time of transplanting; the remainder had a weak infection of T. ter r e s t r i s . i.e., less than 25% of the short roots exhibiting a Hartig net, negligible mantle development and rare occurrence of cystidia, external hyphae and mycelial strands. Colonization by inoculant fungi was successful with a l l plants showing some infection. More than sixty percent of the short roots of seedlings inoculated with H. crustuliniforme (5249), E-strain (947) and L. bicolor (5268) were infected by the test fungus; other short roots were predominantly non-mycorrhizal. Hebeloma mycorrhizae produced abundant extramatrical (EM) hyphae; E-strain mycorrhizae produced few EM hyphae. Three-quarters of the seedlings inoculated with T. terrestris had at least 50% of the short roots colonized by this fungus; other short roots remained non-mycorrhizal or were colonized by E-strain fungi. These 68 Thelephora mycorrhizae were well-developed (i.e., mantles covered more than 75% of the short roots, cystidia and mycelial strands were common) indicating that infection resulted from the inoculum and not from air-borne spores of Thelephora. Eighty-five percent of the seedlings inoculated with forest floor were colonized primarily by forest floor fungi; 15% of these seedlings were infected by forest floor fungi and by T. ter r e s t r i s . 2. Seedling size and morphology at the time of transplanting At the time of transplanting, seedling morphology (Table 4.1) and nutrition (Tables 4.2) differed by inoculation treatments (P < 0.001). Seedlings assigned to each temperature treatment were not significantly different (P > 0.60) at the time of transplanting. Root length and short root number were least for seedlings inoculated with L. bicolor: and greatest for those inoculated with T. ter r e s t r i s . Root biomass ranged from a low of 68 mg for seedlings inoculated with L. bicolor to a high of 105 mg for those inoculated with T. terrestris. Comparison of f o l i a r N and P concentrations with published f o l i a r analyses of spruce seedlings (e.g., Leyton 1948, Ingestad 1959, Swan 1962, Beaton et a l . 1965, Swan 1971, Benzian and Smith 1973, Morrison 1974, Farr et a l . 1977, Ballard and Carter 1986) suggested that nitrogen was very deficient i n seedlings inoculated with E-strain, H. crustuliniforme and T. terrestris (Table 4.2); and that P levels were adequate for spruce seedling growth i n a l l inoculation treatments. Table 4.1. 69 Effect of inoculation treatment on seedling morphology at the time of transplanting Inocula Shoot/ # short Root # short root roots/ length roots/cm ratio seedling (cm) of root Control 2. .7 (0. .7) 936 (373) 347 (130) 2. .8 (0. • 8) E 2. ,0 (0. .4) 695 (263) 170 ( 82) 4. ,4 (1. 3) Ff 2. .9 (0. .8) 1010 (396) 290 (118) 3. .6 (0. .7) He 2. .7 (0. .7) 958 (320) 240 ( 76) 4, .2 (1. .2) Lb 3. ,0 (0. 8) 491 (130) 159 ( 64) 3. .5 (1. .5) Tt 2. ,3 (0. • 6) 1061 (480) 328 (113) 3. .2 (0. .9) NOTE: N — 16; standard deviations are shown i n parentheses. Short roots were not included in the estimate of root length. Abbreviations for inocula: E - E-strain, Ff — forest floor, He = H. crustuliniforme. Lb «- L. bicolor. Tt - T. ter r e s t r i s . Table 4.2. Effect of inoculation treatment on seedling N and P concentrations (X oven-dry weight) at the time of transplanting Inocula* NX Shoot PX NX Root P% Control 1. .2(0.06) 0. 25(0.01) 1.4(0.24) 0, .26(0.04) E 0. ,9(0.03) 0. 27(0.02) 1.9(0.11) 0. .42(0.09) Ff 1. .4(0.05) 0. 36(0.01) 1.6(0.03) 0. .35(0.01) He 0. .9(0.09) 0. 24(0.05) 1.4(0.06) 0. ,25(0.01) Lb 1, .1(0.06) 0. 37(0.04) 1.3(0.19) 0, .29(0.06) Tt 0. .8(0.07) 0. 23(0.02) 1.2(0.11) 0. ,24(0.01) NOTE: N - 2; each sample consisted of pooled root or shoot tissues from 8 seedlings. One standard deviation in parentheses. Abbreviations for inocula: E - E-strain, Ff - forest floor, He =• H. crustuliniforme. Lb - L. bicolor. Tt - T. te r r e s t r i s . 71 3. Resistance to water flow from s o i l to xylem (RSX) RSX decreased over time in both s o i l temperatures (Figure 4.1). Two days after transplanting, resistance to water flow was greater (P < 0.01) in the 6°C s o i l ; at 9 and 21 days, there was no appreciable difference in RSX (P > 0.11) between the 6 and 12°C soils. Xylem pressure potential increased (became less negative) from day 2 to 21 in the 6°C s o i l (Table 4.3) even though transpiration rates more than doubled, increasing from 0.3 to 0.8 fig H20/(cm2s) . There were significant (P = 0.05) differences in RSX among inoculation treatments 2 and 9 days at both s o i l temperatures; at 21 days, these differences were significant only in the 6°C s o i l (Table 4.4). RSX with several exceptions declined in a l l inoculation treatments and temperature combinations. In the 6°C s o i l , RSX of seedlings inoculated with E-strain or L. bicolor did not decrease significantly from 2 to 21 days; in the 12°C s o i l , RSX of non-inoculated seedlings was consistently low from 2 to 21 days. Table 4.3. Range in mean mid-day mean xylem pressure potentials (-MPa) of the various inocula at 2, 9 and 21 days Soil temperature 2 9 21 6°C 12°C 1.1-1.5 0.5-1.0 0.8-1.0 0.7-0.9 0.5-0.8 0.5-0.6 72 Figure 4.1. Effect of s o i l temperature on RSX [MPa/log (/ig H20/cm2s)] at 2, 9 and 21 days.- Ver t i c a l bars represent one standard error of the estimate. Table 4.4. Summary of soil-to-xylem resistance (RSX) estimates at 2, 9 and 21 days Inoculation 6°C s o i l 12°C s o i l 2 days after transplanting Control 0 113 (0.011) 0.016 (0 008) E-strain 0 041 (0.013) 0.063 (0 009) Forest Floor 0 107 (0.013) 0.039 (0 013) H. crustuliniforme 0 112 (0.038) 0.057 (0 018) L. bicolor 0 073 (0.029) 0.027 (0 007) T. terrestris 0 046 (0.022) 0.074 (0 027) 9 days after transplanting Control 0 060 (0.004) 0.015 (0 014) E-strain 0 068 (0.013) 0.033 (0 011) Forest Floor 0 051 (0.009) 0.065 (0 015) H. crustuliniforme 0 069 (0.009) 0.049 (0 018) L. bicolor 0 034 (0.008) 0.021 (0 009) T. terrestris 0 046 (0.006) 0.034 (0 010) 21 days after transplanting Control 0 011 (0.007) 0.016 (0 009) E-strain 0 053 (0.004) 0.012 (0 009) Forest Floor 0 016 (0.006) 0.011 (0 005) H. crustuliniforme 0 032 (0.008) 0.006 (0 004) L. bicolor 0 060 (0.014) 0.020 (0 010) T. terrestris 0 008 (0.006) 0.009 (0 005) NOTE: Units for RSX are MPa/log (/ig K^ O/cm s); standard error of regression estimate (N — 8) in parentheses. At 21 days, RSX for seedlings inoculated with E - s t r a i n , H. crustuliniforme and L. b i c o l o r were influenced by s o i l temperature (respective P values of 0.05, 0.10, and 0.05); suggesting that i n o c u l a t i o n with these fungi slowed the rate of seedling acclimation to the 6°C s o i l . Differences among i n o c u l a t i o n treatments i n RSX values were not r e l a t e d to the presence or absence of root pathogens; symptoms of root disease (e.g.,lesions, necrosis) were not evident i n any of the in o c u l a t i o n treatments. Nor were they c o r r e l a t e d (P > 0.30) with (1) root growth (length, dry weight and number of short roots) a f t e r transplanting, (2) with seedling s i z e and shoot/root r a t i o or (3) shoot N and P n u t r i t i o n . RSX values at 21 days were r e l a t e d to root biomass, root form and n u t r i t i o n at the time of transplanting (Table 4.5). However, there were no s i g n i f i c a n t c o r r e l a t i o n s (P > 0.10) between these parameters and RSX estimates at 2 and 9 days. RSX at 21 days was in v e r s e l y proportional to l a t e r a l root length, number of short roots per seedling, root biomass and root length per u n i t root dry weight. Figure 4.2 shows RSX at 21 days as a function of i n i t i a l root biomass. These r e l a t i o n s h i p s were strongest i n the 6°C s o i l (Table 4.5). Seedlings inoculated with E - s t r a i n or L. b i c o l o r . with the highest RSX at 21 days, had approximately 50% fewer short roots and 50% les s l a t e r a l root length per seedling at the time of transplanting (Table 4.1) than non-inoculated c o n t r o l seedlings or those inoculated with f o r e s t s o i l , T. t e r r e s t r i s or H. crustuliniforme. Root %P and RSX were weakly and p o s i t i v e l y c o r r e l a t e d (P = 0.07) due to the coincidence of high values of RSX and root %P (Table 4.2) i n E-s t r a i n seedlings. There was no as s o c i a t i o n (P > 0.35) between RSX and root P when E - s t r a i n data were excluded from the c o r r e l a t i o n a n a l y s i s . 75 Figure 4.2. Relationship between seedling resistance to water flow (RSX) at 21 days and i n i t i a l root biomass; units for RSX 2 are MPa/log (/xg H^ O/cm s). 0.08 j 0.07 -0.06 -0.05 -RSX 0.04 -0.03 -0.02 -0.01 -0 — 40 50 60 70 80 90 100 I n i t i a l r o o t biomass (mg) 110 120 76 Table 4.5. Pearson correlation coefficients (r) between 21-day RSX and root morphology and nutrition at the time of transplanting Variable S o i l temperature 6°C 12°C 6 & 12°C # short roots per seedling -0. .93 (0.01) -0. ,70 (0. 12) -0. 65 (0. ,02) root length (cm) per seedling -0. ,94 (0.01) -0. .07 (0. 91) -0. .60 (0. ,04) # short roots/ cm of root 0. .41 (0.43) -0. .32 (0. 53) 0. .32 (0. .31) length/unit root biomass (cm/g) -0, .90 (0.01) 0. .10 (0. .85) -0. .56 (0. ,06) root biomass -0 .79 (0.06) -0 .65 (0. .17) -0. .65 (0, .02) root %P 0, .61 (0.20) -0 .12 (0, .83) 0. .54 (0, .07) NOTE: Probability values for the Pearson correlation coefficients are shown in parentheses. N - 6 for individual s o i l temperatures. 77 4. Root and Shoot Growth after transplanting New root.growth was evident at the 9 day sample. Inoculation treatments accounted for 40% of the v a r i a b i l i t y i n f i n a l (21 day) root biomass data; whereas s o i l temperature treatments accounted for only 8%. Inoculation effects were due i n large part to differences i n root biomass at the time of planting. When covariance analysis was used to compensate for i n i t i a l differences i n seedling size, inoculation accounted for only 9% of the v a r i a b i l i t y in the f i n a l root biomass data. Increases i n root biomass during the test period ranged from less than 10% for some inocula (e.g., E-strain and forest soil) to greater than 35% for others (e.g., H. crustuliniforme). Mean root dry weight increased by 10 and 33%, respectively, in the 6 and 12°C s o i l s . Terminal buds began flushing after day 9. Soi l temperature had no effect on the incidence of flushing at 16 or 21 days. Sixteen days after transplanting, a greater (P < 0.01) percentage (69%) of the non-inoculated seedlings had flushed compared to inoculated seedlings (25% averaged across the 5 inocula). Seedlings inoculated with forest s o i l flushed ear l i e r than those inoculated with other inocula (Table 4.6). 5. Pre-dawn Stomatal Conductance Analysis of variance showed no effect of inoculation (P - 0.04) on pre-dawn conductance (Table 4.7). Values of pre-dawn stomatal conductance values were very low (averaging less than 0.04 cm/s) indicating that stomata were f u l l y closing at night. Two days after transplanting, pre-dawn conductance was lower (P - 0.05) in the 6°C s o i l (0.02 cm/s) than in 12°C s o i l (0.04 cm/s). So i l temperature had no effect on pre-dawn stomatal conductance at 9 and 21 days. 78 Table 4.6. Effect of inoculation treatment on terminal bud break Inoculation X of seedlings flushed treatment 16 days 21 days Non-inoculated 69 100 E-strain 19 94 Forest s o i l 56 100 H. crustuliniforme 6 88 L. bicolor 19 88 T. terrestris 25 94 NOTE: N - 16 for each inoculation treatment. 79 Table 4.7. ANOVA of pre-dawn stomatal data Source of variation df Mean-square F-ratio Prob. Soi l temperature (ST) 1 2.45 7.0 0.01 Inocula (I) 5 0.61 1.7 0.14 Time (T) 2 1.23 3.5 0.03 ST x I 5 0.24 0.7 0.64 ST x T 2 1.69 4.8 0.01 I x T 10 0.20 0.6 0.83 ST x T x I 10 0.51 1.5 0.18 Error 72 0.35 80 6. Rate of Net Photosynthesis (Pn) Data for each measurement time were analyzed separately by two-way analysis of variance because pot-to-pot variation was significant at 9 and 21 days but not at 2 days. Parallel results were obtained for Pn calculated on a per unit shoot weight and unit leaf area basis. Therefore only the results for Pn on a leaf area basis are presented. Inoculation treatments had more influence on net photosynthesis (Pn) than did s o i l temperature (Table 4.8). S o i l temperature had the greatest effect (P - 0.06) at 9 days with net photosynthetic rate reduced by 13% in the 6°C s o i l . In comparison, Pn varied by 40 to 60% across inoculation treatments 2 and 21 days after transplanting. Two days after transplanting, there was a significant interaction between inoculation and temperature treatments on Pn. In the 6°C s o i l , Pn of control seedlings and those inoculated with either L. bicolor or forest floor exceeded that of seedlings inoculated with other fungi (Table 4.9); in the 12°C s o i l , the Pn of seedlings inoculated with T. terrestris was lower (P < 0.01) compared with a l l other seedlings. At 21 days, there was no interaction between temperature and inoculation treatments, therefore only the mean values for inoculation treatment are presented (Table 4.10). Regardless of s o i l temperature, Pn was higher (P •= 0.01) i n control, forest floor and L. bicolor treatments compared to E-strain, H. crustuliniforme and T. terrestris treatments. Photosynthetic nitrogen-use efficiency (PNUE) showed no effect of s o i l temperature (P > 0.21) or inoculation treatment (P > 0.12). 81 Table 4 . 8 . ANOVA of log-transformed Pn (pmoles CO2/111 s) data Source of variation df Mean-square F-ratio Prob. Two days after transplanting So i l temp (ST) 1 0.17 3.0 0.09 Inocula (I) 5 0.49 8.7 0.00 ST x I 5 0.18 3.1 0.02 Error 48 0.06 Nine days after transplanting Soil temp (ST) 1 Inocula (I) 5 ST x I 5 Pots (ST x I) 36 Error 12 Twenty-one days 0.48 3.8 0.06 0.14 1.1 0.37 0.07 0.6 0.71 0.13 0.06 after transplanting So i l temp (ST) 1 Inocula (I) 5 ST x I 5 Pots (ST x I) 36 Error 12 0.09 1.2 0.29 0.45 6.0 0.00 0.12 1.6 0.18 0.07 82 Table. 4 .9 . Interaction of s o i l temperature and inoculation on mean values of net photosynthetic rate 2 days after transplanting Inoculation Net PS (umoles C02/m2 s) treatment 6°C s o i l 12°C s o i l Forest Floor 1.3 1.1 Control 1.1 1.1 L. bicolor 1.0 1.1 T. terrest r i s 0.7 0.7 E-strain 0.7 1.1 H. crustuliniforme 0.6 0.9 NOTE: Log-transformed Pn data were analyzed; values shown are in the original units; N - 5. 83 o Table 4.10. Comparison of net photosynthesis (/zmoles C02/m s) 21 days after transplanting with shoot N (% oven-dry weight), seedling N (mg/seedling) and the ratio of shoot N to root N at the time of transplanting Inocula Pn Shoot Seedling Shoot N/ %N mg N root N Ff 1. ,4 1. ,4 (0. 05) 4. ,7 (0. 4) 2. .4 (0. .1) Control 1. .3 1. .2 (0. .05) 3. .7 (0. .1) 2. .2 (0. 3) Lb 1. ,2 1. .1 (0. .06) 3. .8 (0. 3) 2. ,7 (0. 2) Tt 1, ,0 0. .8 (0. .07) 3, .1 (0. .1) 1. .5 (0. 3) E 0. .9 0, .9 (0, .03) 3. .6 (0. .3) 1. .1 (0. .2) He 0, ,8 0. ,9 (0. .09) 3. .2 (0. 2) 1. .7 (0. 1) NOTE: Log-transformed Pn data (N - 10) were analyzed; values shown are i n the original units. N - 2 for nutrient parameters; standard deviations shown in parentheses. Abbreviations for inocula: Ff =• forest floor, Lb = L. bicolor. Tt = T. te r r e s t r i s . E - E-strain and He - H. crustuliniforme. 84 Pn at 21 days correlated positively with i n i t i a l seedling N nutrition (Table 4.10) including shoot N (3! oven-dry weight) (r - 0.78, N - 12, P = 0.003), total seedling N (r - 0.66, N - 12, P - 0.02) and the ratio of shoot N to total seedling N (r - 0.70, N - 12, P - 0.01); and to a lesser degree with shoot XP (r - 0.50, N - 12, P - 0.10) and the ratio of shoot to total seedling P (r - 0.57, N - 12, P - 0.05). Inoculation treatments significantly influenced (P - 0.01) a l l of these nutrient parameters. Except at day 2, there was no correlation (P > 0.20) between Pn and xylem pressure potential (XPP). At day 2, net photosynthetic rate was inversely related (P - 0.01) to XPP, i.e., seedlings with high rates of Pn tended to have lower values (more negative) of XPP. 85 Discussion Inoculation with E-strain, H. crustuliniforme. T, terrestris and L. bicolor delayed terminal bud break. The delay was not greater than 6 days, and the significance for seedling acclimation to cool s o i l s was not clear. Shoot nutrition may have influenced the rate of flushing. Levels of shoot nitrogen were highest in the two treatments (control, forest floor) with the most rapid rate of bud break. Late-season nitrogen f e r t i l i z e r applications accelerate bud break of Sitka spruce seedlings (Benzian et a l . 1974). The lack of an appreciable difference (only 0.1%)'in shoot nitrogen between certain treatments (e.g., L. bicolor and control) with significantly different rates of flushing, suggests, however, that non-nutritional factors also influenced bud break. Spring shoot growth may be stimulated by a plant growth substance exported from roots (Atkin et a l . 1973, Lavender et a l . 1973). In cultures, ECM fungi have been shown to synthesize some of these substances, for example auxins and cytokinins (Ek et a l . 1983, Harley and Smith 1983 and references therein), which are thought to influence the timing of bud break in trees (Kramer and Kozlowski 1979). Garbaye (1986) found that some ectomycorrhizal fungi (e.g., H. crustuliniforme. L. laccata) promoted earlier bud break in oak and beech seedlings than others (e.g., T. t e r r e s t r i s ) . Mycorrhizal-induced variation in time of bud break was attributed to differences i n the a b i l i t y of ECM fungi to synthesize plant growth regulators; however, Garbaye did not compare the nutritional status of seedlings infected with different fungi. 86 Averaged across inoculation treatments, seedling resistance to water flow, RSX, decreased from 2 to 21 days at both s o i l temperatures.. This result is i n agreement with other long-term studies of s o i l temperature effects on cold-stored white spruce seedlings (Grossnickle and Blake 1985, Grossnickle 1987). In contrast to these studies, however, seedling acclimation with respect to resistance to water flow was more rapid; occurring at 9 days compared to 14 days (Grossnickle and Blake 1985) or more than 21 days (Grossnickle 1987). Averaged across a l l inocula, resistance to water flow decreased as root biomass increased over time (N -6, r - -0.86, P - 0.03), suggesting that new root growth contributed to the decrease in resistance from 2 to 21 days observed at both s o i l temperatures. Previous studies have shown positive correlations between new root growth and the inverse of resistance, hydraulic conductivity of spruce root systems (Colombo and Asselstine 1989). The hydraulic conductivity of new unsuberized roots may be 3-to 4-fold greater than that of suberized roots (Sands et a l . 1982, Carlson 1986). Therefore a rel a t i v e l y small increase in root biomass can have a significant impact on root system conductivity. The s t a t i s t i c a l l y insignificant 10% increase in root biomass in the 6°C s o i l would be sufficient to increase whole root system conductance by 30 to 40%. For comparison with published RSX values, 21-day RSX values were estimated without log transforming the transpiration data. The values of RSX calculated in this manner (0.14 and 0.06 MPa/[ug H20/cm2s], respectively in the 6 and 12°C soils) were considerably lower than the 21-day values of RSX for non-flooded 10°C s o i l (1.02 MPa/[ug H20/cm2s]) reported by Grossnickle (1987) and the 18-day value of RSX (0.48 MPa/[ug H20/cm2s]) for 10°C s o i l calculated from a regression of RSX on time 87 developed by Grossnickle and Blake (1985). In this study, transpiration rates increased and plant moisture status improved from day 2 to 21. In contrast, during the three weeks of the study conducted by Grossnickle and Blake, transpiration rates did not show any detectable increase over time. RSX values in the present study were more similar to those measured by Running and Reid (1980) for cold-stored lodgepole pine exposed for two days to cool temperatures. Estimates of RSX obtained from their regression of o RSX on root temperature were 0.37 and 0.12 MPa/(ug R^ O/m s), respectively, for 6 and 12°C s o i l temperatures. A comparison of methods for these various studies suggests that l i f t i n g date and cold-storage conditions (i.e., duration and temperature) contribute to the resistance to water uptake after transplanting. Cold-storage conditions increased in severity i n the same order as RSX values: this study ( f a l l - l i f t e d , 2 months, 4°C), Grossnickle and Blake (spring-l i f t e d , 2 months, -2°C) and Grossnickle ( f a l l - l i f t e d , at least 6 months, -2°C). The lodgepole pine seedlings used by Running and Reid (1980) were only cold-stored 2 months at 2°C. Cold-storing (-5°C) white spruce seedlings for more than 18 weeks reduces their root growth capacity at low s o i l temperatures (Camm and Harper [1991]). New root growth potential, an indicator of overall seedling vigor (Lavender 1988), was higher in this study with mean root biomass increasing by 9 mg and 28 mg, respectively, in the 6 and 12°C s o i l s . In the same time period, Grossnickle and coworkers found root biomass increased by only 4-6 mg in 10°C s o i l even though their seedlings were much larger; 6 mm in stem diameter compared to 1.2 mm. 88 There were significant differences among inoculation treatments in resistance to water flow i n both soi l s at 2 and 9 days.; but only i n the 6°C s o i l at 21 days. Differences at 21 days correlated with root size, length and short root number at the time of transplanting. These root parameters varied significantly with inoculation treatment but not s o i l temperature treatment. Although inoculation treatments significantly influenced new root growth during the 21 day test period, differences among inoculation treatments in RSX at 21 days were not correlated to the amount of new root growth (dry weight, short roots). Again the i n i t i a l inoculation effects on roots outweighed the effects of inocula on incremental changes in roots. Seedlings inoculated with E-strain and L. bicolor which had the smallest root biomass, length and short root number at the time of transplanting exhibited negligible decreases in resistance to water flow over time. This result emphasizes the importance of root size and morphology at the time of planting. In a review of seedling characteristics which correlate with f i e l d performance, Lavender (1988) notes that growth of conifer seedling after planting is often more closely related to root mass or volume at the time of planting than to root growth capacity, especially when new root growth is limited by droughty or cool s o i l s . The growth of outplanted white and black spruce seedlings has been shown to increase with seedling size at the time of planting (Dobbs 1976, Sutherland and Day 1988). In controlled environment studies, ectomycorrhizal infection has been found to reduce the hydraulic conductance of whole root systems of radiata pine (Sands and Theodorou 1978) and Douglas-fir seedlings (Coleman et a l . 1987, 1990). Coleman et a l . (1990) concluded that the smaller size (length, dry weight) of mycorrhizal root systems compared to non-89 mycorrhizal control seedlings was the major factor responsible for the reduction i n root conductivity. Sands and Theodorou (1978) also attributed at least part of the mycorrhizal effect to the smaller size of mycorrhizal seedlings compared with non-mycorrhizal seedlings. In a later study, Sands et a l . (1982) concluded that mycorrhizal infection had no effect on hydraulic conductivity per unit root length. Based on previous studies an inverse relationship between root phosphorus and resistance was expected. VA-induced decreases in root resistance have been attributed, directly or indirectly, to improved root phosphorus nutrition (Graham and Syvertsen 1984, Koide 1985). In greenhouse-grown Douglas-fir seedlings, Coleman et a l . (1990) found that root phosphorus and conductance were positively correlated. Nitrogen and phosphorus deficiencies i n crop plants have been shown to increase root resistance per unit root dry weight or length (Radin and Boyer 1982, Radin and Eidenbock 1984). The mechanism involved is not known; although phosphorus deficiencies are known to alter membrane permeability (Ratnayake et a l . 1978). In this study, root phosphorus and 21-day RSX were weakly and positively correlated (P - 0.07), the inverse of published studies; seedling resistance increased with root %P. The coincidence of high values of RSX and root phosphorus in E-strain seedlings accounted for this unexpected relationship. RSX was not correlated with root phosphorus when E-strain data were excluded from the analysis. 90 Levels of root phosphorus for a l l inoculation treatments in the present study were 2 to 3-fold greater than, those reported (< 0.10%.) by Coleman et a i . (1990). If plants are not deficient in phosphorus, root resistance may be independent of this nutrient. Two days after transplanting, resistance to water flow was lower i n non-inoculated seedlings in 12°C s o i l , but higher in the 6°C s o i l , compared with average inoculated seedlings. This suggested that inoculation, on average, decreased the temperature sen s i t i v i t y of seedling root systems. Root resistance increases rapidly below a threshold s o i l temperature of approximately 7°C for spruce and pine seedlings (Kaufmann 1975, Running and Reid 1980). The sharp increase in resistance has been attributed to a phase transition in root c e l l membrane li p i d s (Kaufmann 1975, Running and Reid 1980). It is possible that fungal membrane l i p i d s have a different threshold temperature for phase transition or that inoculation alters the balance of plant growth substances affecting root conductivity. Exogenously applied ABA has been shown to increase root hydraulic conductance and reduce the effect of cool s o i l temperatures on root resistance (Fiscus 1981, Markhart 1984). Pn values were 10 to 20% of the maximum values reported for spruce seedlings (e.g., Brix 1979, Beadle et a l . 1981, Binder et a l . 1987). The low values r e f l e c t two factors: (1) Pn was measured at non-saturating light intensities, and (2) surface area rather than projected leaf area was used in the calculations. On a dry weight basis, the mean photosynthetic rate of 4.4 mg C02/g/h was about 50% of the maximum photosynthetic rate reported by Brix (1979). Inoculation treatments had a much greater effect on the rate of net photosynthesis than did s o i l temperature. A decrease i n s o i l temperature from 12 to 6°C reduced the rate of net photosynthesis at 9 days by 13%. Similar or smaller temperature differentials have been shown to reduce the net photosynthetic rate by 15 to 20% in short-term (Delucia 1986) and long-term studies (Llppu and Puttonen 1989). There was no significant effect of s o i l temperature at day 2 or 21. At 21 days, there was a 46% difference in the rate of net photosynthesis among the six inoculation treatments; compared to a 6% difference between the two s o i l temperatures. In both s o i l temperatures, inoculation with T. te r r e s t r i s . E-strain or H. crustuliniforme reduced the net photosynthetic rate of seedlings both on an area and dry weight basis compared with non-inoculated control seedlings or those inoculated with forest s o i l or L. bicolor. In previous research, mycorrhizal inoculation has been shown to have no effect (Dosskey et a l . 1990) or a positive effect on net photosynthesis per unit needle area (Parke et a l . 1983a, Reid et a l . 1983, Dosskey et a l . 1990). Positive effects have been attributed to (1) enhancement of the nutrient or water status of seedlings (Benecke and Gobi 1974, Parke et a l . 1983a), (2) changes in the balance of plant growth regulators (Slankis 1973) or (3) increased demand for carbohydrates to produce fungal tissues such as extramatrical hyphae (Reid et a l . 1983, Dosskey et a l . 1990). Reid et a l . (1983) reported that mycorrhizal infection of pine seedling root systems increased respiration of ^ C02 about 4-fold. Dosskey et a l . (1990) observed that some mycorrhizal fungi increased net photosynthetic rate in Douglas-fir seedlings, but others had no effect. They hypothesized that fungi which formed mycorrhiza with abundant external hyphae (e.g., Rhizopogon vinicolor) were more l i k e l y to create a carbon demand and 92 stimulate net photosynthetic rate than those species with weak extramatrical development (e.g., L. laccata). According to the "carbon demand" hypothesis, the increased photosynthetic sink created by mycorrhizal fungi stimulates the rate of net photosynthesis. A substantial portion of current photosynthate (as much as 38%) may be incorporated into ectomycorrhizae (Tranquillini 1964). Carbon demand of the various mycorrhizal root systems was not measured. However, several observations suggest that i t was unlikely that differences i n carbon demand were responsible for the inoculation-induced changes in net photosynthetic rate. F i r s t , s o i l temperature had a negligible effect on net photosynthetic rate, even though new root growth (dry weight) was 3-fold greater i n the 12°C s o i l . Carbon lost through root respiration also increases with s o i l temperature. Graphical data presented by Lawrence and Oechel (1983a) shows about a 60% increase in root respiration as s o i l temperature rises from 6 to 12°C. Second, the rate of net photosynthesis was not associated with the amount of extramatrical (EM) hyphae produced by different fungi. Compared with non-inoculated controls, the rate of net photosynthesis was depressed both in seedlings with mycorrhizae producing abundant amounts of EM hyphae (e.g., H. crustuliniforme) and in those with mycorrhizae producing few EM hyphae (e.g., E-strain). The results also suggest inoculation effects were independent of seedling water status. Net photosynthesis and stomatal conductance of many plant species is relatively constant over a wide range of xylem pressure potentials (Hsiao 1973); decreasing significantly for a variety of conifer species only at potentials in the range of -1.5 to -2.0 MPa (Turner and Jarvis 1975, Running 1976, Beadle et a l . 1981). The levels of xylem pressure potential measured in this study were not indicative of severe moisture stress. The lowest value of xylem pressure potential for any seedling measured in this experiment was -1.7__MPa two days after transplanting; mean values for the inoculation treatments were greater than -1.5 MPa at a l l sample dates for both s o i l temperatures. Nitrogen deficiency increases the sen s i t i v i t y of plants to water de f i c i t s (Marschner 1986). Consequently, the rate of net photosynthesis of seedlings used i n this study may have been more sensitive to a moderate plant moisture stress than those used in other studies (e.g., Beadle et a l . 1981). If this were the case, values of xylem pressure potential and net photosynthesis should decrease in p a r a l l e l . Except for 2-day data, however, these data were not correlated. At this time, seedlings with low (the most negative) values of xylem pressure potential tended to have the highest photosynthetic rates. If plant moisture stress negatively affected net photosynthetic rates, the opposite trend would have occurred. Net photosynthetic rate correlated positively with shoot nitrogen and phosphorus (% dry weight). Levels of shoot nitrogen and phosphorus varied significantly with inoculation treatment; but not with s o i l temperature suggesting that inoculation effects on net photosynthetic rate involved changes in the nutritional status of seedlings. A high percentage (>80%) of the nitrogen in conifer foliage is contained in proteins used to maintain photosynthesis (Small 1972, Chapin and Kedrowski 1983). Numerous studies have shown positive correlations between f o l i a r nitrogen and the rate of net photosynthesis in conifers (Brix 1981, Smolander and Oker-Blom 1989) with the net photosynthetic rate of Douglas-fir increasing with f o l i a r nitrogen, N, to an optimum of 1.74% (Brix 1981). Levels of shoot nitrogen were below this optimum, ranging from 0.08 to 1.4 %. Reid et a l . (1983) demonstrated that mycorrhizal infection of pine seedlings increased 94 net photosynthesis per unit leaf area for nine months after inoculation. Mycorrhizal seedlings had consistently higher f o l i a r nitrogen and phosphorus concentrations suggesting enhancement of net photosynthetic rate was due to nutrition. However, they were not able to s t a t i s t i c a l l y test difference i n f o l i a r nutrition between mycorrhizal and non-mycorrhizal seedlings. Net photosynthetic rate correlated positively with the proportion of total seedling nitrogen in shoot tissue. Inoculation treatments affected nutrient distribution as well as nutrient content. The low proportion of total nitrogen contained i n the shoot tissues of E-strain seedlings may account for their low rate of net photosynthesis compared with seedlings in other inoculation treatments (e.g., control, forest floor and L. bicolor seedlings) with a similar total nitrogen content. Black (1986) reported that mycorrhizal infection by L. laccata increased the proportion of nitrogen and phosphorus retained i n the root tissues of nursery-grown Douglas-fir seedlings. The presence of sporocarps at the time of sampling for nutrient analysis suggested to him that L. laccata had sequestered nutrients and diverted them to reproductive tissues instead of releasing them to the host plant. Unfortunately, I did not look for reproductive structures in this study. The cold storage effect on pre-dawn stomatal conductance reported by Grossnickle and Blake (1985) (i.e., cold-stored seedlings were not able to f u l l y close their stomata at night) was not observed i n this study, possibly because cold storage duration and temperature were less stressful. It would be useful to know i f inoculation affected pre-dawn stomatal conductance when seedlings are stored for longer periods at freezing temperatures. Summary The various inocula influenced the rate at which white spruce seedlings acclimated to low s o i l temperatures with respect to resistance to water flow and net photosynthetic rate, but had no effect on pre-dawn stomatal conductance. Differences among inoculation treatments were related to root size (dry weight, short root number) and nutritional status (shoot N and P) of the seedlings at the time of transplanting. CHAPTER V EFFECT OF VARIOUS MYCORRHIZAL FUNGI ON THE GROWTH AND NUTRIENT STATUS OF WHITE SPRUCE SEEDLINGS TRANSPLANTED INTO COOL SOILS Introduction The a b i l i t y to improve the growth and nutrition of spruce seedlings planted i n cold so i l s was the f i n a l c r i t e r i o n used to assess the potential efficacy of ectomycorrhizal (ECM) fungi. Several studies (e.g., Marx 1977, Last et a l . 1984, Wilson et a l . 1987) have reported that a r t i f i c i a l inoculation of nursery seedlings with specific ECM fungi improves f i e l d performance. However, not a l l studies (e.g., Bledsoe et a l . 1982, Shaw et a l . 1987b) have shown that mycorrhizal inoculation benefits seedling growth or nutrition. Although rarely reported, ECM fungal inoculation may even depress the growth of conifer seedlings (e.g., Bledsoe et a l . 1982, Sands and Theodorou 1978). Negative growth effects have not been given much attention in the mycorrhizal research literature. Perhaps this reflects the common view that the ECM fungus-host plant relationship is a symbiosis in the narrowest sense of the term, i.e., mutualistic relationship between two l i v i n g organisms in which both partners benefit. The host plant benefits from improved nutrient uptake and the fungus benefits from host-derived carbohydrates. The lack of attention to negative host.plant response may also r e f l e c t the practical objective of much ECM research, i.e., to improve reforestation success. Funding for this objective i s not based on negative growth results. 97 "Most research on inoculation with ectomycorrhizal fungi has been based on two premises. F i r s t , any ectomycorrhizal association on roots of tree seedlings Is far better than none......Second, some species of ectomycorrhizal fungi on . certain sites are more beneficial to trees than other fungal species that naturally occur on such sites." (Marx and Kenney 1982, p. 131) Wilcox (1983) recommends examining the mycorrhizal-host plant relationship using the broader (and original) definition of a symbiotic relationship. In addition to mutualistic partnerships, this definition encompasses (1) relationships in which one partner benefits but the other neither benefits or loses (commensalism), and (2) those in which one partner benefits at the expense of the other (parasitism). Environmental conditions l i k e l y play a important role in determining the type of symbiotic relationship which develops between ECM fungi and host plants (Wilcox 1983, Harley and Smith 1983). Cool s o i l temperatures may s h i f t the balance of the mycorrhizal symbiosis in favor of the fungi. Research with VA-infected seedlings has shown that cool temperatures eliminate the benefits normally derived from mycorrhizal infection (Moawad 1978, Smith and Roncadori 1986). The growth of VAM plants may even be reduced compared to that of non-mycorrhizal plants at low temperatures (Furlan and Fortin 1973, Hayman 1974, Schenck and Schroder 1974). The lack of host plant response to infection at sub-optimum temperatures may be due to the i n a b i l i t y of established fungi to absorb or transport phosphorus while s t i l l u t i l i z i n g host carbon. Smith and Roncadori (1986) reported that mycorrhizae increased the phosphorus and copper uptake of cotton plants at s o i l temperatures of 24°C and 36°C; but mycorrhizae had no effect on nutrient uptake at a s o i l temperature of 18°C. Comparable research has not been conducted with ectomycorrhizal conifer seedlings. 98 The objectives of the research reported in this chapter were to study the effect of ECM inoculation on the relative growth and nutrition of spruce seedlings grown in cool forest so i l s to determine: (1) i f the presence of "any" ECM fungus is better than "none" when seedlings are planted into cool s o i l s , (2) i f some ECM fungi are more effective than others in cool s o i l s , (3) i f there are interactions between temperature and inoculation effects on seedling growth and physiology. (4) i f Inoculation-induced changes in whole seedling response to cold so i l s are related to the uptake, distribution or use of nutrients. Approach Both root and shoot growth and nutrition were measured so that changes in dry matter, nutrient distribution and nutrient use could be described. Soil temperature (Davidson 1969) and mycorrhizal inoculation (Harley and Smith 1983, Black 1986) have been shown to alter dry matter and/or nutrient distribution between root and shoot. Bowen (1973) suggested that mycorrhizal plants use limited nutrients more e f f i c i e n t l y than non-mycorrhizal plants. Nutrient analyses were limited to four nutrients, nitrogen (N), phosphorus (P), iron (Fe) and calcium (Ca). P was analysed because mycorrhizal benefits are often attributed to improved P uptake; N and Fe because these nutrients are often seriously deficient in young spruce plantations (Ballard 1985). Ca was included in the study because i t s uptake is related to seedling transpiration (Barber 1984). Compared to N 99 and P, the level of Ca in newly flushed foliage is more dependent on the mass flow of water and less dependent on redistribution from older plant tissues (Marschner 1986). Since the experiment was conducted in containers, growth and nutrition of the seedlings were measured several times during the experiment. Periodic sampling is particularly important for container experiments because of the limited amount of available nutrients and water (Parke 1985). Seedlings inoculated with different ECM fungi may deplete the limited resources of containers at different rates. Mycorrhizal treatments which result in rapid depletion of water or nutrients may not show a benefit to the host plant unless growth is measured before these resources are depleted. Periodic sampling also allowed for the calculation of growth analysis indices (Hunt 1982) which are more independent of i n i t i a l seedling size and nutritional status than are absolute measures of growth. Most studies of mycorrhizal efficacy are confounded by differences in i n i t i a l seedling size. Three approaches were used to compensate for size differences: (1) after cold-storage, the largest and smallest seedlings were culled to minimize the differences, (2) the i n i t i a l caliper of a l l seedlings was measured and used in covariance analysis to adjust treatment means for differences In i n i t i a l size, and (3) the relative rather than absolute growth rates of seedlings were calculated. An alternative approach would have been to produce seedlings with equivalent dry weights, nutrient contents and patterns of nutrient and dry matter distribution using different levels of nutrients for each inoculation treatment. In practice this is d i f f i c u l t to achieve because different aspects of the host growth and physiology respond differently to mycorrhizal infection and f e r t i l i z a t i o n (Pacovsky et a l . 1986) with mycorrhizal and f e r t i l i z e r treatments interacting to influence seedling growth and nutrition. Growth analysis techniques (Hunt 1982) were used to examine whole plant response to the stress of cold s o i l s . Relative growth rate was used as a whole plant index of physiological vigor (Margolis and Brand 1990). Three components of relative growth rate (root efficiency, nutrient-use efficiency and biomass allocation) were used to compare the effect of inoculation treatments on acquisition and use of nutrients. Root efficiency was estimated by calculating the rate of mineral uptake per unit root weight per unit time, termed the specific absorption rate by Welbank (1962). Root dry weight was assumed to be a good index of the absorption capacity of roots. Hackett (1969) studied relationships between dry weight, volume, surface area and length of barley root systems. He found that these relationships were relatively independent of nutrient regime, plant age and variety; consequently, for comparative purposes, nutrient uptake could be expressed on the basis of any of these root dimensions. In a previous study (Chapter IV), root dry weight, length and number of short roots per seedlings were highly correlated (P < 0.001). 101 On the premise that ECM fungi predominantly infect short roots, the efficiency of mycorrhizal and non-mycorrhizal root systems has been compared on the basis of nutrient uptake per short root (Alexander and Fairley 1986, Hdgberg 1989). Since many ECM fungi (e.g., E-strain) infect long l a t e r a l roots (Robertson 1954), nutrient uptake per unit root weight was considered a better index of root efficiency than uptake per short root. Ideally, nutrient uptake should be based on the surface area of roots, including fungal structures such as external hyphae. However, measurement of surface area was not feasible. Methods Inoculation treatments included: (1) a non-inoculated control ( s t e r i l i z e d agar medium), (2) forest floor inoculum, and pure cultures of (3) Hebeloma crustuliniforme 5249, (4) Laccaria bicolor 5268, (5) Amphinema  bvssoides 0288, (6) Thelephora terrestris 2088, (7) E-strain 947. Except for the Amphinema and Thelephora isolates, the test inocula were obtained from northern B.C. or Alberta forest sites (as described i n Table 2.1). T. terrestris 2088 and A. bvssoides 0288 were isolated In 1988 from surface-s t e r i l i z e d ectomycorrhizae of containerized spruce seedlings grown respectively at the Canadian Forest Products nursery in Saanichton, B.C. and at the Balco Canfor Reforestation Centre in Kamloops, B.C. Seedlings were inoculated, grown, cold-stored and culled as described previously (Chapters II, III, and IV). Mean seedling caliper, after culling, was 1.2 mm. Twenty-four seedlings, selected to represent the f u l l range of caliper values, were destructively sampled to develop regressions of shoot and root dry weight on i n i t i a l caliper. The remaining seedlings were transplanted into pots (4 l i t r e s , 15 cm diameter) containing a mixture 102 of unpasteurized forest floor, mineral s o i l and per l i t e (1:1:2 by volume). Each pot held 6 seedlings from one inoculation treatment; 8 pots were f i l l e d for each inoculation treatment; and 4 pots were randomly assigned to each of 2 water baths (i.e., s o i l temperature treatments, 6 and 12°C). The forest s o i l mixture contained propagules of indigenous ECM fungi and non-mycorrhizal microflora. The mean values of pH (1:4 soil:water suspension, Peech 1965), available phosphorus (Bray and Kurtz No 1, Bray and Kurtz 1945) and mineralizable nitrogen (anaerobic incubation, Waring and Bremner 1964) for this mixture were respectively 4.9, 35 /ig/g and 68 Mg/g-After transplanting, the seedlings were grown for 12 weeks with an 18 h photoperiod, relative humidity of 30 to 50%, a i r temperature of 20°C day/12°C, and light intensity of 400 /raol/(m2s) in the 400-700 nm wavelength. No f e r t i l i z e r was applied during the experiment. An insecticide (0.84 g/L Diazinon, Later Chemical Ltd., Richmond, B.C.) was applied every 2 weeks to minimize insect grazing on fungal mycelium. The pots were re-randomized once a week to minimize the effects of position within the water baths and to a lesser extent within the growth chamber. A s o i l water retention curve was developed to estimate s o i l water tension from the weight of a pot and i t s forest s o i l mixture. The weight of container-grown seedlings at various water tensions was estimated from a water retention curve developed for the plug medium (peat:vermiculite) and the fresh/dry weight ratio of seedlings at the time of planting. For the f i r s t 5 weeks, the pots were watered as necessary to maintain a high s o i l moisture potential (above -0.03 MPa). During the 5 to 10 week period, watering was reduced i n frequency with the objective of gradually drying the 6 and 12°C soil s to a s o i l moisture potential less than -0.06 MPa to 103 compare the response of the various inoculation treatments to a mild s o i l water d e f i c i t as would occur i n the f i e l d . Between 10 and 12 weeks the pots were watered to maintain a high s o i l water content. Measurements of seedling growth and physiology were made at 5, 10 and 12 weeks on 8 seedlings per treatment combination. Seedlings were carefully extracted to minimize root loss and disturbance to the remaining seedlings. The loose nature of the forest s o i l : p e r l i t e mixture f a c i l i t a t e d seedling extraction. Root growth was slow and roots of neighbouring seedlings did not intermingle or reach the bottom of the containers u n t i l the f i n a l sample in the 12°C s o i l . Growth measurements included: root, mature shoot and new foliage biomass, la t e r a l root length, short root number and caliper. Total root length and short root number were estimated from dry weight ratios between subsamples and the total root system as described in Chapter IV. At the same time, roots within the original container plug and new roots formed outside the plug were assessed for mycorrhizal infection as described In Chapter III. Shoot and root tissues from 8 seedlings per treatment combination were bulked separately for chemical analysis of nitrogen (N), phosphorus (P) and calcium (Ca) at 0, 5, and 12 weeks. Only shoot tissues were analyzed for "active" iron (Fe) concentrations. New and mature shoot tissue were separated for chemical analysis; the mature shoot sample included stem tissue. Wet oxidation of milled, oven- dried (70°C, 48 h) tissue with H2SO4, Se, salts and H2O2 (Parkinson and Allen 1975), was followed by colorimetric analysis for N (salicylate/nitroprusside) and P (ascorbic acid/molybdate-antimony) and atomic absorption spectrophotometry for Ca (Technicon Industrial Systems 1977). Active Fe was extracted with 1 M HCl (Oserkosky 1933, cited in Ballard and Carter 1986) and analyzed by 104 atomic absorption spectrophotometry. It was impossible to remove a l l external hyphae from mycorrhizal root systems. Therefore, root biomass and nutrient data include undetermined amounts of fungal mycelium, especial ly in the H. crustuliniforme and A. bvssoides treatments. Percent cal iper growth was calculated from measurements of i n i t i a l (Ci) and f i n a l cal iper (Cf) using the equation: [100X x ( C f - C i ) / C i ] . The allometric constant (k) for shoot and root growth was calculated by dividing shoot re lat ive growth rate (RGR) by root RGR (Huxley 1932). I n i t i a l shoot and root dry weights were estimated from the regressions of dry weight on cal iper conducted at the time of transplanting. I n i t i a l shoot/root rat io was used as a covariate in the analysis of the shoot/root allometric constant. Physiological measurements ( i . e . , net photosynthesis, transpiration and xylem pressure potential) are described in Chapter IV. Two indices of potential resource-use eff iciency were derived from the nutr i t iona l and physiological data: (1) instantaneous nitrogen- and phosphorus-use eff ic iency (respectively, INUE and IPUE) defined as the rat io of photosynthetic capacity to shoot N or P content, and (2) water-use eff ic iency (WUE) defined as the rat io of carbon assimilated (net photosynthetic rate) to water evaporated (transpiration rate) . Relative growth rate, RGR, was subdivided into an algebraical ly equivalent set of four indices (1) re lat ive root weight or RWR, representing the proportion of tota l seedling biomass al located to the nutrient absorbing system, (2) specif ic absorption rate (A), representing the rate of nutrient uptake per unit root dry weight or root eff ic iency, (3) speci f ic u t i l i z a t i o n rate or NUE, representing the rate of dry weight increment per unit of absorbed nutrient and (4) the re lat ive increase in 105 nutrient content (R). The equation for this relationship (Hunt 1982) for nitrogen i s presented below: RGR - (RWR x A x NUE) / (R) or 1 dW - R W x _ ! d N x l d W W dt W RW dt N dt 1 dN N dt where RW, W, N and t are respectively root dry weight, seedling dry weight, seedling nitrogen content, and time. Mean values of these parameters were used to calculate the growth indices as shown in Table 5.1. The calculation of these indices assumes a linear relationship exists between root weight and nutrient content; and between root and shoot dry weights (Welbank 1962, Hunt 1973, 1982). Plots of the data showed these assumptions were met. 106 Table 5.1. Equations used to calculate growth analysis indices Index Equation relative growth rate (RGR) [g growth/(g seedling/week) (lnW 2-lnW 1)/(t 2-ti) root weight ratio (RWR) (g root/g seedling) t(RW1/W1)+(RW2/W2)]/2 specific absorption [g N/(g root/week] for N (AN) [(N 2-N 1)/(t 2-t 1)][(lnRW 2-lnRW 1)/(RW 2-RW 1)] nitrogen-use efficiency [g growth/(g N/week)] (NUE) [(W 2-W 1)/(t 2-t 1)][(lnN 2-lnN 1)/(N 2-N 1) Relative increase in N (RN) [g N/(g N seedling/week] ( l n N 2 - l n N 1 ) / ( t 2 - t 1 ) NOTE: RW, W, N and t are respectively root dry weight, seedling dry weight, seedling nitrogen content, and time. Subscripts indicate the time of measurement (i.e., 0, 5, 12 weeks). 107 The experiment was a completely randomized design with a f a c t o r i a l arrangement of the 7 inoculation treatments and.2 s o i l temperatures; 24 seedlings per treatment combination. Data were tested by least squares analysis of variance (ANOVA) or analysis of covariance (ANCOVA), i f the assumption of p a r a l l e l slopes was met (Hicks 1973), using a microcomputer s t a t i s t i c a l program, SYSTAT (Wilkinson 1988). The ANOVA model consisted of three fixed treatments ( s o i l temperature, inoculation and time treatments) with the 4 pots nested within the s o i l temperature and inoculation treatments. The mean square for between-pot error was tested for significance against the residual error. If i t were insignificant (P > 0.25, Bancroft 1964), these two errors were pooled, with a l l treatments and interactions tested against the pooled error term. Otherwise, the between-pot error was used to test main effects and interactions. Some parameters were log-transformed to meet the assumptions of normality and homoscedasticity for ANOVA and ANCOVA (Eisenhart 1947). V a r i a b i l i t y of these parameters was proportional to the value of their mean. Tabulated means of these parameters when shown in the original units are geometric means of the original data. Data which were log-transformed are identified in the tables. Preplanned and post-hoc tests of means were conducted using linear contrasts (SYSTAT, Wilkinson 1988). A preplanned linear contrast of the control treatment (no inoculation) versus the 6 inoculation treatments was conducted on the entire data set when interactions between s o i l temperature and inoculation treatments were not s t a t i s t i c a l l y significant. Otherwise this contrast was conducted separately for each s o i l temperature. A similar comparison of mycorrhizal and non-mycorrhizal (at the time of transplanting) seedlings was also conducted excluding control seedlings with T. terrestris contamination and inoculated seedlings with poor (less than 50% of short roots colonized) nursery infection by inoculant fungi. This reduced the total sample size from 336 to 314 seedlings; with the sample for non-inoculated seedlings decreasing from 48 to 36. Relationships among nutrition, growth and physiological parameters were examined by linear correlation analysis using either individual seedling data (growth versus physiological parameters) or mean values for the 14 treatment combinations (nutrition versus growth and physiological parameters). Cause and effect relationships cannot not be inferred from correlation analysis but the degree of association between two parameters can be described. Furthermore, lack of significant correlation negates a cause and effect relationship between two parameters. Results 1. Mycorrhizal status Mycorrhizal infection of new roots formed outside the container plugs was rapid i n the 6 and 12°C so i l s (Table 5.2). Most new roots (> 75%) of seedlings inoculated with E-strain, T. te r r e s t r i s . H. crustuliniforme. L. bicolor were infected by the inoculant fungi. New roots of seedlings inoculated with A. bvssoides were infected (> 30% of short roots) with other ECM fungi, mainly those native to the forest s o i l during. The degree of infection on new roots by A. bvssoides correlated positively (r = 0.80, P < 0.001) with the degree of A. bvssoides infection on roots within the original root mass. Table 5.2. 109 Mycorrhizal infection of new roots formed outside the container plug i n 6°C and 12°C s o i l Inocula Percent mycorrhizal infection 6°C 12°C 5wk lOwk 12wk 5wk lOwk 12i Control 16 82 88 42 88 88 Forest s o i l 88 88 • 88 88 88 88 E-strain 88 84 84 88 80 81 L. bicolor 88 88 88 88 88 88 * H. crust. 77 77 85 85 77 77 A. bvssoides 70 57 63 60 50 52 T. terrestris 69 78 82 88 88 88 NOTE: A l l control seedlings showed some new root infection by 10 weeks; new roots of a l l inoculated seedlings were infected by 5 weeks. N = 8 seedlings for each time-inoculation-temperature combination. Percent mycorrhizal infection for each seedling was estimated on a six-class scale: 0, 1-25, 26-50, 51-75, 76-95, >95; the midpoint of each class was used to calculate average infection per seedling. H. crust. - H. crustuliniforme 110 Infection of non-inoculated seedlings by indigenous fungi was rapid. A l l non-inoculated seedlings were infected by mycorrhizal fungi 10 weeks, after transplanting. Mycorrhizae formed from forest s o i l ECM dominated i n the new root system of 80% of the non-inoculated seedlings; Thelephora-like mycorrhizae dominated in the other 20%. These latt e r mycorrhizae probably resulted from contamination by air-borne spores of T. terrestris prior to transplanting. At the time of transplanting, approximately 35% of the control seedlings showed some mycorrhiza formation by T. t e r r e s t r i s . 2. Phenology Seedlings began to flush 10 to 14 days after transplanting. About 65% , 40% and 50%, respectively, of new foliage, root and caliper growth was completed 5 weeks after transplanting. Shoot elongation ceased and terminal buds formed between 5 and 10 weeks. Early i n the 5 week drying cycle, I realized i t was impossible to decrease the s o i l water potential of the 6°C s o i l to -0.06 MPa, a level which would significantly reduce root growth (Day and MacGillivray 1975). The lowest potential reached was -0.02 MPa which was above f i e l d capacity -0.03 MPa. Poor seedling growth and low transpiration rates combined with condensation of water from the surrounding air (15-20°C) on the cool s o i l surface slowed net water loss from the pots. Therefore, I abandoned the idea of comparing the response of the various inoculation treatments to a drying cycle and watered both soil s to maintain s o i l water tension above f i e l d capacity. I l l 3. Total Seedling Biomass Seedling biomass at the time of transplanting was influenced by inoculation treatment (P - 0.01) but not by s o i l temperature (P > 0.60). Mean values for the seven inoculation treatments ranged from 0.20 to 0.24 g/seedling. After transplanting, seedling biomass was strongly influenced by inoculation treatment, time and s o i l temperature (Table 5.3). There was also a significant but weak interaction between s o i l temperature and time. Seedling biomass was greater i n the 12°C s o i l at 10 (0.67 g at 12°C versus 0.73 g at 6°C) and 12 weeks (0.69 g versus 0.73 g); however, at 5 weeks s o i l temperature had no effect on total seedling biomass (0.64 versus 0.63 g). Inoculation treatments accounted for a much greater percentage (22%) of the variation in biomass data than did time (3.5%), s o i l temperature (<1%) or the time-temperature interaction (<1%). There were no significant interactions between the three main effects (i.e., inoculation, temperature and time). Therefore, mean values for inocula, averaged across time and temperature, are presented i n Table 5.4. Forest s o i l , L. bicolor or T. terrestris inoculation increased seedling biomass compared with non-inoculated seedlings; in contrast, E-strain or H. crustuliniforme inoculation decreased total biomass, and A_,_ bvssoides inoculation had no effect. 112 Table 5.3. Analyses of covariance for total, shoot and root biomass Source of variation df Mean F-ratio prob. square Total seedling biomass Time of sample (T) 2 0. .49 17. ,9 <0, ,001 Soil temperature (ST) 1 0. .15 5. 6 0, ,02 Inocula (I) 6 1. ,00 36. ,9 <0. ,001 T x ST 2 0. 08 2. 9 0. ,05 T x I 12 0. ,03 1. ,1 0. ,36 ST x I 6 0. ,04 1. ,4 0, ,20 T x ST x I 12 0. ,03 1. ,1 0, ,34 i n i t i a l caliper 1 11. ,26 414, ,8 <0, ,001 Error 291 0. ,04 Mature shoot biomass (MSB) Time of sample (T) 2 0. .71 17. .8 <0, ,001 Soil temperature (ST) 1 1, .36 34, .2 <0, .001 Inocula (I) 6 0. .70 17, .5 <0, ,001 T x ST 2 0, .16 3, .9 0, .02 T x I 12 0. .03 0, ,7 0 .73 ST x I 6 0, .01 0, .1 ' 0, .99 T x ST x I 12 0. .05 1. .3 0, .22 i n i t i a l caliper 1 16, .02 403, .5 <0 .001 Error 293 0 .04 113 Table 5.3. (continued) New foliage biomass (NFB) Time of sample (T) 2 10 .24 43.9 <0.001 Soil temperature (ST) 1 3 .92 16.8 <0.001 Inocula (I) 6 8 .26 35.4 <0.001 T x ST 2 0 .29 1.3 0.29 T x I 12 0 .16 0.7 0.78 ST x I 6 0 .34 1.5 0.19 T x ST x I 12 0 .36 1.5 0.11 i n i t i a l caliper 1 7 .29 31.3 <0.001 Error 293 0 .23 Root biomass Time of sample (T) 2 0. 120 69.4 <0.001 Soil temperature (ST) 1 0. 180 104.3 <0.001 Inocula (I) 6 0. 014 8.4 <0.001 T x ST 2 0. 016 9.2 <0.001 T x I 12 0. 003 1.6 0.10 ST x I 6 0. 007 4.2 <0.001 T x ST x I 12 0. 002 1.3 0.22 i n i t i a l caliper 1 0. 342 198.1 <0.001 Error 291 0. 002 114 4. Mature shoot biomass --. Mature shoot biomass (MSB) varied with time (P <0.001), s o i l temperature (P <0.00T), inoculation treatment (P <0.00T) and to a lesser degree with a second-order interaction between temperature and time (P — 0.02) (Table 5.3). MSB increased during the f i r s t five weeks after transplanting in both s o i l temperatures but showed different trends with time for the 5 to 12 week period. In the 6°C s o i l , MSB decreased gradually from 5 to 12 weeks; in the 12°C s o i l , MSB was essentially constant from 5 to 10 weeks and then decreased from 10 to 12 weeks (Fig. 5.1). The decrease coincided with an increase i n root biomass, suggesting carbohydrate stored in mature shoot tissue was allocated to new root growth. Mean MSB, averaged across time and s o i l temperature treatments (Table 5.4), ranged from 0.40 g in the forest s o i l treatment to 0.29 g in the H. crustuliniforme and A. bvssoides treatments. 5. New foliage biomass and caliper growth New foliage biomass (NFB) increased with time (P < 0.001) and s o i l temperature (P < 0.001) and varied with inoculation treatment (P < 0.001) (Table 5.3). Inoculation with forest floor and L. bicolor increased NFB (P < 0.001) compared with control seedlings or those inoculated with other fungi; inoculation with E-strain or H. crustuliniforme reduced NFB (P < 0.001) relative to other treatments (Table 5.4); and inoculation with A. bvssoides and T. terrestris had no effect on NFB (P > 0.10). Figure 5.1. Mean biomass of mature shoot, new foliage and tissues at 0, 5, 10 and 12 weeks at 6 and 12°C temperatures. 116 Table 5.4. Total seedling, mature shoot (MS) and new foliage (NF) biomass (oven-dry weight) averaged across time and temperature compared with seedling nitrogen status at the time of transplanting Inocula Biomass (g) I n i t i a l N Total MS NF mg/plant shoot % Control 0.68 0. .33 0. .12 3, .8 1, .2 Forest floor 0.86 0. ,40 0. ,22 4, ,9 1. ,4 E-strain 0.65 0. .36 0. ,08 3. .8 0, ,9 L. bicolor 0.75 0. .34 0. ,21 4. .0 1. ,1 * H. crust. 0.54 0. .29 0. ,08 3. ,4 0, ,9 A. bvssoides 0.64 0. .29 0. ,14 3, ,9 1, ,1 T. terrestris 0.73 0. .36 0, ,14 3, .3 0. ,8 NOTE: Biomass data were log-transformed for analysis of covariance. A l l biomass values shown are adjusted means using i n i t i a l caliper as a covariate; biomass data were averaged across time and s o i l temperature (N = 48). Caliper data analyzed using i n i t i a l caliper as a covariate; adjusted means (N =• 16) are shown. Nitrogen nutrition data (N = 2) were averaged across temperature. N content calculated as mg/seedling; shoot N as % oven-dry weight. Each sample consisted of tissue pooled from 8 seedlings. H. crust. - H. crustuliniforme 117 Analysis of covariance showed no significant (P > 0.21) interaction between s o i l temperature and inoculation treatments on percent caliper growth. Averaged across inocula, caliper growth increased (P < 0.001) from 54% i n the 6°C s o i l to 72% in the 12°C s o i l . Stem growth was similar for non-inoculated (56%) and inoculated seedlings (64%, averaged across 6 inocula). However, there was considerable difference in caliper growth among inoculation treatments. Inoculation with forest s o i l (79%), L. bicolor (82%) or T. terrestris (71%) increased stem growth compared with non-inoculated seedlings (56%). Inoculation with H. crustuliniforme reduced stem growth (46%); and inoculation with E-strain or A. bvssoides had no effect on stem growth (53%). 6. Root biomass Root biomass varied (P < 0.001) with time, s o i l temperature and inocula (Table 5.3). Soil temperature effects interacted with those of time and inoculation. In both s o i l s , root biomass increased during the 10 weeks following transplanting. From 10 to 12 weeks the rate of root growth remained unchanged in the 6°C s o i l , whereas i t accelerated in the 12°C s o i l (Fig. 5.1). Mean root biomass data averaged across time are shown in Table 5.5. In contrast to other seedlings, root growth of seedlings infected with E-strain or H. crustuliniforme did not respond to increasing s o i l temperature. 7. Root morphology Both s o i l temperature and inocula influenced the number of short roots per seedling (Table 5.6). There was a significant interaction between these main effects with a different ranking of inoculation treatments for the two s o i l temperatures (Table 5.7). In the 6°C s o i l , seedlings inoculated with H. crustuliniforme or A. bvssoides produced the greatest number of short roots; in the 12°C s o i l , control seedlings and those inoculated with forest floor and A. bvssoides produced the greatest number. Table 5 .5 . Interaction of s o i l temperature and inocula on mean root biomass (g oven-dry weight/seedling) Inocula S o i l temperature 6°C 12°C Control 0. ,19 0, ,25 Forest floor 0. .18 0. ,25 E-strain 0. .20 0, ,21 L. bicolor 0. ,16 0. ,23 H. crustuliniforme 0, ,16 0, ,17 A. bvssoides 0. ,18 0. ,21 T. terrestris 0. ,18 0. ,24 NOTE: Biomass data were log-transformed for analysis of covariance; adjusted means are shown i n the original units. Means are averaged across time (N - 24). 119 Table 5 .6 . Analysis of (co)variance for root morphology data Source of variation df Mean- F-ratio Prob. square Number of short roots/seedling Time of sample (T) 2 3.35 27. 2 <0. 001 Soil temperature (ST) 1 3.39 27. 6 <0. 001 Inocula (I) 6 3.03 24. .6 <0. ,001 T x ST 2 0.05 0. ,4 0. .19 T x I 12 0.05 0. ,5 0. .94 ST x I 6 0.26 2. .1 0. .05 T x ST x I 12 0.14 1. ,2 0. .31 caliper 1 9.92 80. ,5 <0. .001 Error 289 0.12 Short roots per unit root weight Time of sample (T) 2 61. .5 9. .7 <0. .001 Soi l temperature (ST) 1 4. ,6 0. .7 0. .39 Inocula (I) 6 230. .6 36. .2 <0. .001 T x ST 2 3. ,5 0. .6 0, .58 T x I 12 3. ,9 0. .6 0. .83 ST x I 6 8. .0 1. ,3 0. .28 T x ST x I 12 5. .8 0. ,9 0, .54 Error 290 6. .4 120 I n c o n t r a s t to o t h e r t rea tments ( i n c l u d i n g the c o n t r o l ) , s h o r t r o o t f o r m a t i o n i n the H . c r u s t u l i n i f o r m e t rea tment showed no response to i n c r e a s i n g s o i l tempera ture . The number o f s h o r t r o o t s pe r u n i t r o o t we igh t was s t r o n g l y i n f l u e n c e d by i n o c u l a t i o n bu t not by s o i l temperature t rea tments (Table 5 .6) . On average , i n o c u l a t i o n decreased (P < 0.001) the r a t i o o f s h o r t r o o t s pe r u n i t o f r o o t b iomass . T h i s e f f e c t was due to low s h o r t r o o t r a t i o s i n s e e d l i n g s i n f e c t e d w i t h e i t h e r T. t e r r e s t r i s or L . b i c o l o r (Table 5.7) . 8. Drv mat te r a l l o c a t i o n S o i l temperature (P < 0.001) and i n o c u l a t i o n t rea tments (P < 0.001) i n f l u e n c e d s h o o t - t o - r o o t a l l o m e t r y ; bu t the re was no i n t e r a c t i o n between these two f a c t o r s (P > 0 . 2 0 ) . Mean v a l u e s f o r the a l l o m e t r i c c o e f f i c i e n t (k) and s h o o t / r o o t r a t i o s are shown i n Tab le 5 . 8 . A l l o m e t r i c da t a p a r a l l e l e d the s e e d l i n g biomass d a t a . Shoot growth r e l a t i v e to r o o t growth was h i g h e s t f o r the f o r e s t s o i l and L . b i c o l o r t rea tments and lowes t f o r the H . c r u s t u l i n i f o r m e and E - s t r a i n t r ea tmen t s . New r o o t and shoot growth c o r r e l a t e d p o s i t i v e l y ( r - 0 .86 , P - 0.012) i n the 12°C but no t the 6°C s o i l . A t 6°C, the r a t i o o f c u r r e n t f o l i a g e produced pe r u n i t o f new r o o t biomass was t w i c e t h a t a t 12°C; t h i s r e s u l t e d i n h i g h e r s h o o t - t o - r o o t r a t i o s and a l l o m e t r i c c o e f f i c i e n t s i n the 6°C s o i l . 121 Effect of inocula on root morphology # of short roots per seedling per mg root 6°C 12°C 6 and 12°C Non-inoculated 1652 2253 9. .9 (2. .6) Forest floor 1525 2080 9, .3 (3. 2) E-strain 1249 1541 7. ,4 (1. 9) L. bicolor 854 1188 5. .8 (1. 6) H. crustuliniforme 1702 1588 10. .8 (3. • 0) A. bvssoides 1845 2018 10. .6 (3, .4) T. terrestris 934 1200 5, .6 (1 • 6) NOTE: Means are averaged across time. Adjusted means (N = 24) are shown for short root number; these data were log-transformed before analysis of covariance. Data for the ratio of short roots to root biomass were not transformed or adjusted by covariance; standard deviations for these means (N = 48) are shown in parentheses. Table 5.7. Inocula Table 5.8. 122 Effect of s o i l temperature and inoculation treatment on the allometric coefficient (k) and shoot/root ratios K Shoot/root ratio 0-5 wk 0-12 wk 0 wk 12 wk Inocula Non-inoculated 1.1 Forest floor 1.8 • E-strain 1.2 L. bicolor 2.2 H. crustuliniforme 1.0 A. bvssoides 1.2 T. terrestris 1.6 Soil temperature 6°C 1.7 12°C 1.1 0.8 2.7 (0.2) 2.2 (0.6) 1.2 2.6 (0.2) 3.1 (0.9) 0.6 2.4 (0.3) 1.9 (0.7) 1.1 2.4 (0.2) 2.7 (0.8) 0.7 2.5 (0.2) 2.2 (0.5) 0.8 2.6 (0.2) 2.2 (0.5) 0.8 2.5 (0.2) 2.1 (0.8) 1.0 2.5 (0.2) 2.8 (0.7) 0.7 2.5 (0.2) 1.9 (0.5) NOTE: Analysis of covariance for the shoot-to-root allometric constant (K) was conducted on log-transformed data using shoot/root ratio at the time of transplanting as a covariate. Adjusted means are shown i n the original scale. Standard deviations for shoot/root data are shown in parentheses. N = 16 and 56, respectively for the inoculation and s o i l temperature data. 123 9. Effects of inoculation and mycorrhizal infection on seedling growth  and morphology The hypothesis that "any" mycorrhizal symbiont is better than "none" at the time of transplanting was tested on the whole data set (inoculation versus no inoculation) and a subset (mycorrhizal infection versus none) which excluded non-inoculated seedlings exhibiting any mycorrhizal development at the time of transplanting and inoculated seedlings with less than 50% root colonization by the inoculant fungus. The probabilities associated with these comparisons for seedling growth and morphology are shown Table 5.9. Neither inoculation nor mycorrhizal infection had a significant impact on shoot growth or dry matter distribution. Mycorrhizal infection had no s t a t i s t i c a l l y significant effect on root biomass. In contrast, the root biomass of non-inoculated seedlings was greater than that of the average inoculated seedling. Contamination by T. terrestris appears to have increased the mean root weight of the control (non-inoculated) seedlings. Both inoculation and mycorrhizal infection altered the form of the root system; by decreasing the number of short roots per seedling and per unit root dry weight. 124 Table 5 . 9 . Probabilities that inoculation or mycorrhizal infection present at the time of transplanting influenced the growth and morphology of transplanted seedlings Parameter Inoculation Mycorrhizal infection total seedling biomass 0.96 0.68 old shoot biomass 0.65 0.81 new foliage biomass 0.28 0.50 caliper growth 0.20 0.48 root biomass at 12 weeks 6°C s o i l 0.05 0.16 12°C s o i l 0.02 0.10 short roots/seedling 0.001 <0.001 short roots/mg root <0.001 0.03 biomass distribution 0-5 weeks 0.24 0.34 0-12 weeks 0.91 0.92 NOTE: Probabilities were determined from linear contrasts. Unless otherwise indicated, data were averaged across time and s o i l temperature. 125 10. Effect of s o i l temperature on nutrient concentrations Complete shoot and root N, P, Ca, and Fe concentration data are presented i n Appendix C. Shoot nutrient concentrations grouped by s o i l temperature and time are summarized in Table 5.10. Shoot N, P, Ca and Fe concentrations showed no effect of s o i l temperature at the time of transplanting (0 weeks, P > 0.30); but 12 weeks later, Ca, N and P concentrations were higher (P < 0.01) i n the 12°C s o i l ; this effect was weaker for Fe (P - 0.10). Root N, P, and Ca concentrations were not influenced by s o i l temperature. Mature shoot data were compared to published f o l i a r analyses for spruce seedlings and trees (e.g., Leyton 1948, Ingestad 1959, Swan 1962, Beaton et a l . 1965, Swan 1971, Benzian and Smith 1973, Morrison 1974, Farr et a l . 1977, Ballard and Carter 1986) to determine which nutrients might limit spruce seedling growth. At the time of transplanting, levels of shoot P, Ca and Fe concentrations were within the range considered adequate for spruce; but N levels were low, suggesting this nutrient was mildly to severely deficient. At 12 weeks, after terminal bud formation, levels of P and Ca in newly-flushed needles were well above 0.15%, the level associated with deficiencies i n spruce. Foliar N levels were indicative of a slight deficiency and very severe deficiency, respectively, i n the 12°C (1.10% N) and 6°C soil s (0.64% N). At 5 weeks, newly-flushed needles showed visual symptoms of nitrogen deficiency (e.g., needle chlorosis and reddish-purple tips) as described by Morrison (1974) in both s o i l s . These symptoms persisted in the 6°C s o i l during the 12-week test duration, but disappeared in the 12°C s o i l between 5 and 10 weeks. 126 Table 5.10. Effect of s o i l temperature on shoot concentrations of N, P, Ca and Fe (oven-dry weight basis) Week Mature shoot tissue New foliage 6°C 12°C 6°C 12°C Ca% 0 0. .46 ( .03) 0. .49 (• .06) 5 0. .30 ( .03) 0, .36 (• .04) 0. .11 (.04) 0, .17 ( .01) 12 0, .31 ( .03) 0. .47 ( .03) 0. .24 (.02) 0. .46 ( .05) - - ... N% 0 1. .02 (• .21) 1. .07 (• .21) 5 0. .50 ( • 05) 0. .55 ( • 07) 0. .79 (.04) 0, .74 ( .05) 12 0. .49 ( .08) 0. .78 ( .10) 0. .64 (.12) 1. .10 ( • 27) - • P% 0 0. .28 ( • 07) 0. .29 ( .05) 5 0. .14 ( .02) 0. .17 ( .04) 0. .21 (.02) 0. .18 ( .01) 12 0. .14 ( .03) 0. .23 (• .04) 0. .17 (.03) 0. .23 ( .02) Fe /ig/g 0 61 (14) 65 ( 6) 5 38 (11) 36 ( 4) 23 (2) 22 (2) 12 38 ( 9) 52 (18) 26 (9) 35 (4) NOTE: N = 7 for N, P, and Ca; N = 5 for Fe. Values averaged across inoculation treatments. Standard deviations in parentheses. 127 Active Fe in new foliage averaged 26 Mg/g f ° r the 6°C s o i l , suggesting Fe uptake may also have limited shoot growth. Concentrations below 30 Mg/g i n current f a l l foliage indicate a possible Fe deficiency (Ballard and Carter 1986). 11. Effect of inoculation treatments on nutrient concentrations Inoculation treatment influenced i n i t i a l shoot and root N, P, Ca, and Fe concentrations. The lack of replicate analyses within each temperature and inoculation treatment combination at 5 and 12 weeks meant that (1) interactions between s o i l temperature and inoculation treatments could not be examined s t a t i s t i c a l l y and (2) the effect of inocula had to be averaged across the 2 s o i l temperatures. Averaged across both temperatures, neither mature or new shoot nutrient concentrations showed any significant effect of inoculation at 5 or 12 weeks. However, comparison of 12-week new foliage %P data (Table 5.11) with published analyses indicated that, in the 6°C s o i l , P as well as N may have limited the growth of seedlings inoculated with either E-strain or H. crustuliniforme. Table 5.11. Concentration (% oven-dry weight) and content (mg) of N and P in new foliage biomass at 12 weeks Inocula Temp N% P% mg N mg P Control 6 0. .65 0. .17 0. .82 0. .21 12 0. .87 0. .22 1. .50 0. .38 Forest floor 6 0. .62 0. ,15 1. .51 0. ,37 12 1. .03 0. .21 3. .33 0, ,68 E-strain 6 0. .51 0. .13 0. .56 0, ,14 12 1. .67 0. .22 1. .57 0. ,21 L. bicolor 6 0, .66 0. .20 1. .49 0. .45 12 0. .93 0. .26 2. .64 0. ,74 H. crustuliniforme 6 0, .47 0. .14 0. .62 0. .18 12 0, .93 0. .20 0. .94 0. .20 A. bvssoides 6 0, .82 0. .19 1. .14 0. .26 12 1. .04 0. .23 2. .22 0. ,49 T. terres t r i s 6 0, .75 0, .18 1. .16 0. .28 12 1. .23 0. .25 1. .93 0, ,39 Mean 6 0. .64 0, .17 1. .04 0. .27 12 1, .10 0. .23 2. .02 0. .44 NOTE: Each sample includes pooled tissues of 8 seedlings per inoculation-temperature combination. 129 Averaged across both temperatures, root N and P varied significantly (P < 0.01) among inoculation treatments at 5 weeks. Inoculation (averaged across 6 inocula) increased root N and P concentrations. Root N of inoculated seedlings ranged from 1.28% to 1.93% in the 6°C s o i l and from 1.34% to 1.67% i n the 12°C s o i l s ; equivalent values for control seedlings were 1.13% and 1.25%. The level of root P for inoculated seedlings ranged from 0.19 to 0.30% i n the two s o i l s ; root P of control seedlings was 0.18% in both s o i l s . Root N levels were higher than those reported by Farr et a l . (1977) for naturally regenerated Sitka spruce seedlings and by Benzian and Smith (1973) for nursery Sitka spruce seedlings; root P concentrations were similar to their published values. 12. Nutrient Uptake and distribution The uptake of N, P and Ca after transplanting was independent (P > 0.40) of seedling nutrition at the time of transplanting. Total seedling N and P uptake data during the 12-week experiment are presented i n Table 5.12. These data suggest that inoculation, on average increased N uptake in the 12°C s o i l ; but had no effect on N and P uptake in the 6°C s o i l . There was considerable difference in uptake among the various inoculation treatments. Regardless of s o i l temperature, N and P uptake was greatest for seedlings inoculated with T. terrestris and least for those inoculated with H. crustuliniforme. T. terrestris seedlings absorbed 150% and 60% more N, respectively at 6 and 12°C, than did non-inoculated seedlings. Nutrient uptake was inversely related (P < 0.05) to the number of short roots per seedling and to the number of short roots per unit root weight. Table 5.12. 130 I n i t i a l seedling N and P content and N and P uptake between 0 and 12 weeks I n i t i a l content N P Uptake 6°C N Uptake 12°C N P Inocula . rug/seedling. Control Forest floor E-strain L. bicolor H. crust. A. bvssoides T. terrestris 3.8 4.9 3.8 4.0 3.4 3.9 3.3 0.78 1.19 0.99 1.16 0.81 0.90 0.88 1.8 1.2 1.0 2.3 0.5 2.0 2.9 0.27 0.22 0.15 0.31 0.05 0.27 0.45 2.6 4.6 3.9 5.4 1.9 3.6 6.5 0.62 0.78 0.38 1.12 0.19 0.50 1.16 Mean of inoculated seedlings 3.9 0.99 1.7 0.24 4.3 0.69 H. crust. =- H . crustuliniforme. 131 The distribution of N, P, and Ca between shoot and root tissues was strongly influenced by inoculation at 0 and 5 weeks (P < 0.02); but inoculation effects were weak by the 12-week sample (P values ranged from 0.11 for calcium to 0.05 for the nitrogen). Soil temperature had no effect on nutrient distribution at any sample time (P > 0.25). Nitrogen distribution data are shown in Table 5.13; P and Ca distribution data followed similar trends. The proportion of nutrient i n shoot tissues was greatest for seedlings inoculated with forest s o i l and L. bicolor: and least for seedlings inoculated with E-strain or H. crustuliniforme: paralleling the effects of inoculation treatments on total seedling growth. The distribution of N and Ca was similar (P > 0.25) for Ca and N in non-inoculated and inoculated seedlings (averaged across 6 inocula); but the proportion of P (2.3) in shoot tissues of inoculated seedlings, on average was higher (P — 0.02) compared with that of non-inoculated seedlings (2.1). Table 5.13. Effect of inocula on the ratio of shoot N to root N N shoot/root 5 weeks 12 weeks mg/mg Control 1.1 (0.06) 0.9 (0.09) Forest floor 1.6 (0.03) 1.6 (0.20) E-strain 0.9 (0.02) 0.9 (0.15) L. bicolor 1.3 (0.23) 1.2 (0.09) H. crustuliniforme 0.9 (0.03) 0.9 (0.21) A. bvssoides 1.1 (0.15) 1.1 (0.33) T. terrest r i s 1.2 (0.26) 1.0 (0.15) NOTE: N = 2; standard deviations in parentheses. 133 13. Relationship of Nutrition to Growth There were significant positive correlations between mean values of seedling biomass and nutrition (Table 5.14). However the various growth parameters correlated with different aspects of seedling nutrition. Final new foliage biomass (12 week data) correlated strongly with i n i t i a l shoot nutrient concentrations. To a lesser extent, so did the relative growth of shoot to roots (allometric constant). In contrast, caliper growth and 12-week root biomass were positively related to nutrient uptake but not to i n i t i a l shoot nutrition. New foliage biomass was not correlated with nutrient uptake even though both of these parameters increased, 20% and 140% respectively, with s o i l temperature. A possible explanation for the lack of correlation was the timing of the temperature effects. Most new foliage growth occurred in the 0-5 week period; whereas nutrient uptake was most strongly influenced by s o i l temperature i n the 5-12 week period. When data within each s o i l temperature were analyzed separately, mean allometric constant data correlated positively with the N and P content of shoot tissues at the time of transplanting (r>0.88, N=>7, P< 0.01); and to a lesser extent with total seedling N and P content (P > 0.03). There were no significant correlations with total N or P uptake nor with root efficiency after transplanting. 134 Table 5.14. Pearson c o r r e l a t i o n c o e f f i c i e n t s (r) r e l a t i n g s e e d l i n g i n i t i a l shoot n u t r i t i o n and n u t r i e n t uptake to c a l i p e r growth (0-12 weeks), s h o o t - t o - r o o t a l l o m e t r y (k ) , 12-week new f o l i a g e and root biomass N u t r i t i o n C a l i p e r Root New f o l i a g e A l l o m e t r y parameter growth biomass biomass (k) Shoot/0 week NX 0.43(0. ,13) 0.26(0.36) 0. .76(0, ,002) 0. .60(0.02) PX 0.64(0. ,01) 0.17(0.56) 0. ,85(0. ,001) 0. ,68(0.01) CaX 0.35(0. .23) 0.27(0.36) 0. .63(0. ,02) 0, ,39(0.17) Fe Mg/g 0.09(0. ,80) 0.17(0.56) 0. ,06(0. ,83) 0. ,18(0.53) Uptake/0- 5 weeks N 0.64(0. .01) 0.43(0.13) 0. .34(0. .24) 0, .13(0.67) P 0.63(0. ,01) 0.56(0.04) 0. ,24(0, 41) 0. .07(0.81) Ca 0.39(0. .16) 0.72(0.004) 0. .20(0. .50) 0, .43(0.12) Uptake/5- 12 weeks N 0.71(0, .01) 0.87(<0.001) 0. ,34(0, .24) 0. .43(0.13) P 0.81(0, .001) 0.94(<0.001) 0, .59(0. .15) 0, .21(0.47) Ca 0.59(0, .02) 0.74(0.002) 0. .41(0, .14) 0. .41(0.15) Fe 0.70(0 .01) 0.72(0.009) 0, .35(0 .26) 0, .27(0.40) NOTE: N = 14 except for Fe data (N = 12); p r o b a b i l i t y va lues i n parentheses; c o n c e n t r a t i o n data are based on oven-dry weight; uptake u n i t i s mg/seedl ing f o r N, P and Ca and mg/shoot f o r Fe . 1 3 5 14. Growth A n a l y s i s R e l a t i v e growth rate (RGR) was expressed as 4 in d i c e s (1) r e l a t i v e root r a t i o (RWR) representing the proportion of biomass a l l o c a t e d to the n u t r i e n t absorbing organ, (2) s p e c i f i c absorption rate (A) representing the rate of n u t r i e n t uptake per u n i t root weight, (3) r e l a t i v e r a t e of n u t r i e n t accumulation (R) and (4) nutrient-use e f f i c i e n c y (NUE), representing the amount of biomass produced per u n i t of absorbed n u t r i e n t . These components were c a l c u l a t e d f o r N, P and Ca. N, P and Ca in d i c e s showed p a r a l l e l trends across temperature and i n o c u l a t i o n treatments, and therefore, only the N i n d i c e s are presented i n d e t a i l . Nitrogen-use e f f i c i e n c y (NUE) accounted f o r the greatest amount of v a r i a b i l i t y (>80%) i n the r e l a t i v e growth rate data. Neither NUE or RGR was r e l a t e d to i n i t i a l s e e d l i n g biomass (P > 0.40). However, RGR c a l c u l a t e d f o r the 0-5 week pe r i o d c o r r e l a t e d p o s i t i v e l y (r = 0.74, N = 14, P < 0.01) with t o t a l s e e d l i n g N content and shoot N concentration at the time of t r a n s p l a n t i n g . RGR (5-12 weeks) was not r e l a t e d (P > 0.70) to the n u t r i t i o n a l status of seedlings at e i t h e r 5 or 12 weeks. Root weight r a t i o d i d not account f o r a s i g n i f i c a n t (P > 0.50) prop o r t i o n of the v a r i a b i l i t y i n the RGR data, i n d i c a t i n g the pro p o r t i o n of biomass a l l o c a t e d to the absorbing system was not an important determinant of RGR. RGR (0-5 weeks) d i d not vary (P > 0.30) with root e f f i c i e n c y ( s p e c i f i c absorption r a t e or A) but RGR (5-12 weeks) d i d vary s i g n i f i c a n t l y (P < 0.05) with root e f f i c i e n c y . 136 15. Effect of s o i l temperature on growth indices So i l temperature had no effect (P > 0.25) on relative growth rate (RGR) or i t s components in the 0-5 week period. During the 5-12 week period, absorption of N per unit root weight, AN, (P < 0.001), root weight ratio, RWR, (P - 0.01) and growth per unit of nitrogen, NUE, (P - 0.04) were greater i n the 12°C s o i l (Table 5.15). Results for P uptake paralleled those for N. Absorption of calcium (ACa and RCa ) was higher (P -0.03) i n the 12°C compared to the 6°C s o i l during the 0-5 week period; this difference increased in the 5-12 week period (P = 0.003). Table 5.15. Effect of s o i l temperature on growth analysis indices in the 5 to 12 week period Index 6°C 12°C RGR 0. .02 (0, ,01) 0, ,03 (0. ,01) RWR 0. .26 (0. ,03) 0. ,31 (0. ,02) AN 0, .50 (0, ,10) 1, ,80 (0, ,20) RN 0. ,02 (0. ,01) 0. ,06 (0. ,01) NUE 2, .30 (0. .70) 3, .50 (1. .30) NOTE: Standard deviations in parentheses. Units for RGR, RWR, AN, RN and NUE are respectively: [g growth/(g seedling/week)], (g root/g seedling), [mg N/(g root/week)], [g N/(N seedling/week)], and [g growth/(g N/week)]. 137 16. Effect of Inoculation on Growth Analysis Indices For the f i r s t 5 weeks after transplanting, RGR was influenced by inoculation treatment (P < 0.001, ANOVA). Averaged across both s o i l temperatures, RGR was greatest for seedlings inoculated with forest s o i l and least for those inoculated with H. crustuliniforme (Table 5.16). In the 5-12 week period, inoculation treatment, averaged across both s o i l temperatures, had no effect (P » 0.88) on RGR. However, the data suggest that there was an interaction between the effects of s o i l temperature and inocula on RGR. Unfortunately there were not enough replicates to test the s t a t i s t i c a l significance of these trends. Ranking of inocula differed for the two s o i l temperatures. In the 6°C s o i l , RGR of the L. bicolor and A. bvssoides treatments was lowest; in the 12°C s o i l , RGR of the E-strain treatment was lowest. RGR of seedlings infected with L. bicolor. T. terrestris or A. byssoides exhibited the greatest increase with s o i l temperature. RWR showed an effect of inoculation treatment (P < 0.04) for both time periods (Table 5.17). In seedlings inoculated with forest s o i l and L. bicolor. a smaller proportion of total biomass (P < 0.001) was allocated to the absorbing system than i n other treatments. 138 Table 5.16. Relative growth rate, RGR, [(g growth/(g seedling/week)] by inoculation treatment for the 0-5 and 5-12 week periods Inocula 0-5 weeks 5-12 weeks 6 & 12°C 6°C 12°C 6 & 12°C Control 0. .15 (0.01) 0. ,024 0 .031 o. 027 (0.005) Forest s o i l 0. .18 (<0.01) 0. ,016 0 .033 0. 024 (0.012) E-strain 0. .15 (<0.01) 0. .017 0 .009 0. 013 (0.006) L. bicolor 0. .13 (0.01) 0. ,012 0 .052 0. 032 (0.028) * H. crust. 0. .09 (0.01) 0. ,020 0 .034 0. 027 (0.009) A. bvssoides 0. .13 (0.01) 0. ,013 0 .028 0. 021 (0.011) T. terrest r i s 0. .13 (<0.01) 0. ,016 0 .037 0. 027 (0.015) NOTE: Standard deviations in parentheses; N = 2 when data averaged across s o i l temperature. H. crust. — H. crustuliniforme Table 5.17. 139 Effect of inoculation treatment on relative root ratio, RWR, (g root/g seedling) 0-5 weeks 5-12 weeks Control 0. .30 (0. .01) 0. 30 (0. ,03) Forest s o i l 0. .25 (0. .01) 0. ,24 (0, ,04) E-strain 0. .30 (0. 01) 0. ,30 (0, .03) L. bicolor 0. .24 (0. .01) 0. ,25 (0, .03) H. crustuliniforme 0. .30 (0. 01) 0. ,30 (0. .02) A. bvssoides 0, .29 (0. .02) 0. ,30 (0, .03) T. terrestris 0. .29 (0. .03) 0, ,28 (0 .05) NOTE: N = 2; standard deviations in parentheses. 140 During the 0-5 week period, specific absorption of nitrogen, AN, (P -0.008) and relative increase in nitrogen, RN, (P - 0.004) varied with inoculation treatments. AN (Table 5.18) and RN (Table 5.19) were greatest for seedlings inoculated with L. bicolor and T. terr e s t r i s ; and least for control seedlings and those inoculated with H. crustuliniforme (Table 5.18, 5.19). AP and RP showed less response to inoculation treatments (P = 0.03 and 0.05, respectively) for the 0-5 week period; ACa and showed no effect of inoculation (P > 0.60). During the 5-12 week period, AN and RN showed no effect of inoculation treatment but the data suggest there was an interaction between s o i l temperature and inoculation effects on AN and RN. In the 6°C s o i l , non-inoculated seedlings had higher rates of N absorption (AN and RN) than did inoculated seedlings (averaged across 6 inocula); i n the 12°C s o i l this pattern was reversed. AN and RN more than doubled in the warmer s o i l for a l l the inoculated seedlings; i n contrast values for non-inoculated (control) seedlings were similar for both s o i l temperatures. Inoculation with E-strain or H. crustuliniforme appeared to reduce N absorption efficiency in the 6°C s o i l ; but not i n the 12°C s o i l . Neither AN nor RN correlated with the number of short roots per unit root dry weight (P < 0.02). Table 5.18. 141 Specific absorption rate for nitrogen, AN, for the 0-5 and 5-12 week periods Inocula 0-5 weeks 5-12 weeks 6 & 12°C 6°C 12°C 6 & 12°C Control 0.9 ( 0.2) 1.0 1.1 1. .1 (0.1) Forest s o i l 1.5 ( 0.9) 0.7 1.8 1. .2 (0.6) E-strain 1.5 ( 0.1) 0.1 1.9 1. ,0 (0.9) L. bicolor 3.2 ( 0.1) 0.4 2.5 1. .4 (1.0) * H. crust. 0.7 (<0.01) 0.1 1.2 0, .6 (0.5) A. bvssoides 1.9 ( 0.2) 0.5 1.6 1. .0 (0.6) T. terrestris 3.1 ( 0.1) 0.7 2.5 1. .6 (0.5) NOTE: N — 2 when data averaged across temperature; standard deviations in parentheses. AN units are [mg N/(g root/week)]. H. crust.™ H. crustuliniforme. Table 5.19. Effect of inocula on the relative increase in nitrogen, RN, [g N/(g N seedling/week] for the 0-5 and 5-12 week periods Inocula 0-5 weeks 5-12 weeks 6 & 12°C 6°C 12°C 6 & 12°C Control 0. .03 ( o. .01) 0. .040 0. .048 0. .04 ( .01) Forest s o i l 0. .04 ( o. .02) 0. .021 0. .054 0. .04 (. .02) E-strain 0. .05 « o . .01) 0. .002 0. .065 0. .03 ( .05) L. bicolor 0. .08 (<o. .01) 0. .010 0. .068 0. .04 (• .04) * H. crust. 0. .02 « o . .01) 0. .005 0. .044 0. .03 ( .03) A. bvssoides 0. .06 (<0. .01) 0. .016 0, .056 0. .04 ( .03) T. terrestris 0. .10 (<0. .01) 0. .022 0, .080 0. .05 ( .04) NOTE: N = 2 when data averaged across temperature; standard deviations in parentheses. H. crust.*- H. crustuliniforme. 143 Nitrogen-use efficiency, calculated on the basis of absorbed nitrogen, for the 0-5 week period correlated weakly (P - 0.07) with i n i t i a l seedling nutrition. Therefore, a second estimate of nitrogen-use efficiency (growth per unit of i n i t i a l N) which did not vary with i n i t i a l N content ( P > 0.25) was calculated. Inoculation treatment influenced (P < 0.001) both estimates of NUE in the f i r s t 5 weeks after transplanting. NUE was greatest i n the control and forest s o i l treatments and least in the H. crustuliniforme treatment (Table 5.20). In the 5-12 week period, NUE did not vary significantly (P = 0.75) with inoculation treatment. With the exception of the E-strain treatment, NUE increased with s o i l temperature. The poor NUE of E-strain seedlings in 12°C s o i l was associated with a low relative growth rate even though root efficiency (AN and RN) indices were similar to those of other inoculation treatments. NUE values for the 5-12 week period were not correlated (P > 0.80) with seedling nitrogen (content or concentration) at 5 weeks. 144 Table 5.20. Effect of inocula on nitrogen-use efficiency estimated on the basis of absorbed N (NUEa) and on the basis of i n i t i a l N content (NUEi) for the 0-5 week period; and on the basis of absorbed N for the 5-12 week period Inocula 0-5 weeks (6,12°C) NUEa 5-12 weeks NUEa NUEi 6°C 12°C 6,12°C Control 16. .0(1.2) 40(3. 5) 3. .2 4. .2 3. .7(0. 7) Forest s o i l 16. .8(1.0) 37(0. 1) 2. .3 3. .6 2. .9(1. .0) E-strain 15. .2(0.1) 39(0. .7) 2. .4 0. .9 1. .7(1. 1) L. bicolor 12. .9(1.4) 33(2. 9) 1. .4 5. .2 3. .3(2. 7) * H. crust. 9, .7(1.6) 25(3. .4) 2. .9 4. .1 3. .5(0. 8) A. bvssoides 12. .4(0.8) 32(1. .8) 1. .6 3. .0 2. .3(1. 0) T. terres t r i s 14. .9(0.3) 40(0. .6) 2. .0 3. .8 2. .9(1. 3) NOTE: N •= 2 when data averaged across temperature; standard deviations in parentheses. Unit for NUEa are [g growth/(g absorbed N/week^]; units for NUEi are [g growth/(g i n i t i a l N/week)]. H. crust.- H. crustuliniforme. 145 17. Rate of Net Photosynthesis (Pn) Values of Pn were 50 - 75% lower compared to values reported by other researchers (e.g., Beadle et a l . 1981, Binder et a l . 1987), probably because needle surface area rather than projected needle area was used to calculate Pn. Values calculated on an oven-dry needle weight basis, were comparable to those reported by Brix (1979) for white spruce seedlings grown in a growth chamber; the maximum value of Pn reported was 9.1 mg 002/g/h dry weight of needles. On a dry weight basis, mean values of Pn in the 6°C s o i l ranged from a low of 1.5 at 5 weeks to a high of 2.9 mg 002/g/h at 10 weeks; and from 3.0 at 5 weeks to 7.3 mg C02/g/h at 12 weeks in the 12°C s o i l . Data collected at 5, 10, and 12 weeks were analyzed separately to meet the homogeneity of variance assumption of ANOVA (Table 5.21). At 5 weeks, there was a significant interaction between s o i l temperature and inoculation treatments on the rate of Pn. Averaged across a l l inocula, Pn increased with s o i l temperature. At 5 weeks, however, Pn showed no response (P > 0.20) to temperature i n the control and L. bicolor treatments (Table 5.22). The Pn of seedlings inoculated with forest s o i l exhibited the greatest response to s o i l temperature; increasing from 0.5 to 1.4 /xmol C02/m2s i n the 12°C s o i l . At 10 and 12 weeks, interactions between s o i l temperature and inoculation treatments were not significant (P > 0.07). 1 4 6 Table 5.21. Analysis of variance for Pn data at 5, 10 and 12 weeks Source of variation df Mean F-ratio prob. square 5 weeks Soil temperature (ST) 1 4.574 54. .0 <0. ,001 Inoculation (I) 6 0.429 5. ,1 <0. ,001 ST x I 6 0.270 3. ,2 0. ,009 Error 56 0/095 10 weeks Soil temperature (ST) 1 1.906 20. .4 <0. ,001 Inoculation (I) 6 0.223 2. .4 0. .04 ST x I 6 0.192 2. .1 0. .07 Error 56 0.093 12 weeks Soil temperature (ST) 1 28.568 171. .2 <0. .001 Inoculation (I) 6 0.061 0, .4 0, .90 ST x I 6 0.276 1. .7 0, .15 Error 56 0.167 NOTE: Pn data were log-transformed for ANOVA. 147 Table 5.22. Effect of inoculation treatments on the rate of Pn (/xmol CC>2/m s) and WUE (/imol C02/mmol H 2 O ) Pn 5 wk Pn 10 wk WUE Inocula 6°C 12°C 6/12°C 6/12°C Control Forest s o i l E-strain L. bicolor H. crust. A. bvssoides T. terrestris 0.51 0.49 0.43 0.84 0.39 0.70 0.48 0.61 1.39 0.88 0.94 0.73 0.97 0.89 0.89 1.01 1.00 1.26 0.81 0.98 1.16 3.2 (1.3) 4.1 (1.8) 3.6 (1.5) 4.1 (1.2) 3.5 (1.5) 4.0 (1.6) 4.5 (1.8) Mean 0.53 0.89 1.00 3.8 NOTE: Pn data were log-transformed prior to analysis. Means in original units are shown. N = 5 and 10, respectively, for the 5 and 10 week data. 10-week Pn data averaged across s o i l temperature. N — 30 for WUE; means averaged across time and temperature. 148 Inoculation treatments influenced Pn at 10 weeks (Table 5.22) but not at 12 weeks. At 10 weeks Pn was highest in the L. bicolor and T. terrestris treatments and least i n the control and H. crustuliniforme treatments. In contrast, s o i l temperature effects on Pn increased with time. In the 6°C s o i l , values for Pn at 5, 10 and 12 weeks were 0.53, 0.85 and 0.57 /imol C02/m2s; equivalent values for the 12°C s o i l were 0.89, 1.19, and 1.85 /imol C02/m2s. At 5 weeks, the rate of Pn correlated positively with mature shoot N (N - 14, r - 0.70, P = <0.01) and Ca (N - 14, r - 0.80, P < 0.001) concentrations; but not with shoot P or Fe concentrations. Pn data were not correlated (P > 0.40) with the nutritional status of shoot tissues at the 10 and 12 week measurements. Pn correlated positively with new foliage biomass (NFB) at 5 weeks (N =70, r = 0.53, P < 0.001); but not with NFB at 10 and 12 weeks. Caliper growth (0 to 12 weeks) correlated positively with the rate of Pn at 5 weeks (N - 14, r - 0.76, P = 0.002) and weakly with the rate of Pn at 12 weeks (N - 14, r - 0.46, P - 0.10). In order to make inferences about the role of carbohydrate accumulation or drain on the rate of net photosynthesis (Herold 1980), 2 specific leaf dry weight (mg needle mass/cm all-sided surface area) or SLDW, was calculated from the dry weight and surface area of the needles used to measure Pn. This index which estimates the status of leaf carbohydrate (Ehret and J o l l i f f e 1985) was strongly affected by s o i l temperature (ANOVA P < 0.001), time (P <0.001), an interaction between time and temperature (P <0.001) and to a lesser degree by inoculation treatment (P - 0.02) and an interaction between inoculation and temperature treatments (P = 0.01). Mean values of SLDW by temperature and time treatments are shown in Table 5.23. Table 5.23. 149 Effect of s o i l temperature and time on specific leaf dry o weight (mg/cm ) Temperature Week 5 10 12 6°C 5.4 (0.7) 4.8 (0.7) 5.4 (0.8) 12°C 4.7 (1.0) 4.2 (0.7) 3.9 (0.7) NOTE: N — 56, standard deviations in parentheses. 18. Instantaneous values of nitrogen- (INUE') and phosphorus-(IPUE') use  efficiency INUE and IPUE showed no effect of inoculation treatment (P > 0.75). Soil temperature had a significant effect on both NUE and PUE at 5 and 12 weeks (P < 0.001). PUE and NUE values followed the same trend; therefore only NUE values are presented. At 5 weeks, NUE doubled i n the warmer s o i l , increasing from 28 to 52 /xmol C02/mol N; comparable values at 12 weeks were 30 and 86 nmol C02/mol N. Values of NUE were similar to those estimated by Field et a l . (1983) for 5 species of California evergreens, i.e., 28 to 68 /imol C02/mol N. 150 19. WUE (instantaneous water-use efficiency) WUE showed significant effects of s o i l temperature (P < 0.001), inoculation treatment (P - 0.006), time (P < 0.001), and an interaction between s o i l temperature and time (P < 0.001). Mean WUE (averaged across time and temperature) for the various inoculation treatments ranged from 3.2 to 4.5 /mol C02/mmol H2O (Table 5.22). The mean value for non-inoculated seedlings (3.2) was lower (P - 0.01) than the mean for inoculated seedlings (4.0, averaged across 6 inocula). Seedlings inoculated with H. crustuliniforme or E-strain had lower (P - 0.01) mean values of WUE (3.5 and 3.6, respectively than did seedlings inoculated with T. terrestris (4.5), forest s o i l (4.1), L. bicolor (4.1), and A. bvssoides (4.0). The values of WUE were similar to those reported by Fie l d et a l . (1983) for 5 California evergreen shrubs but higher than those reported by MacDonald and Lieffers (1990) for current foliage of black spruce trees. In the latte r study, WUE ranged between 1.5 and 3.0 /imol C02/mmol H2O. Averaged across time and temperature, mean values of WUE for the inoculation treatments correlated positively (P <0.03) with mean values of mature shoot XP and %N. Mean WUE values correlated weakly with new foliage production (r - 0.31, N - 70, P - 0,01) and caliper growth (r - 0.36, N = 35, P - 0.03) in the 6°C s o i l ; but were not correlated (P > 0.50) with these growth parameters in the 12°C s o i l . 20. Plant Moisture Stress At 5 weeks, plant moisture stress estimated from xylem pressure potential (XPP) varied with inocula (P < 0.001) and s o i l temperature (P < 0.001). These effects were modified by a significant interaction between 151 inocula and temperature (P - 0.01). Averaged across a l l inocula, plant moisture stress was lower i n the 12°C s o i l . Ranking of inocula, however, differed for the two s o i l temperatures (Table 5.24). XPP showed no effect of inoculation at either s o i l temperature at 10 and 12 weeks; and XPP data did not correlate with growth or nutrient data. Table 5.24. Effect of inoculation treatment on mean xylem pressure potential (-MPa) 5 weeks after transplanting Soil temperature 6°C 12°C Control 0.63 0.33 Forest s o i l 0.68 0.53 E-strain 0.53 0.30 L. bicolor 0.63 0.41 H. crustuliniforme 0.61 0.57 A. bvssoides 0.53 0.46 T. terrestris 0.58 0.39 Mean 0.52 0.36 NOTE: N = 5. Data transformed prior to analysis; means i n original units (MPa) are shown. 152 Discussion I emphasize that the results of this study apply to specific edaphic conditions (i.e., acidic, cool moist soi l s with a good potential for indigenous ECM infection) and specific plant material (i.e., cold-stored white spruce seedlings with below optimum shoot concentrations of nitrogen). In addition, comparisons of s o i l temperature and inoculation treatments were made i n a controlled environment to minimize fluctuations in environmental conditions other than s o i l temperature. 1. Interactions between Soil Temperature and Inoculation With the exception of root growth, growth and physiology of white spruce seedlings were not appreciably altered by interactions between s o i l temperature and inoculation treatments. Averaged across a l l inocula, root growth (dry weight, short root production) was greater, as expected, i n the 12°C s o i l . The optimum s o i l temperature for root growth of many conifer species l i e s between 18 and 24°C (Heninger and White 1974, Nambiar et a l . 1979, Ritchie and Dunlap 1980, Andersen et a l . 1986). However, seedlings inoculated with E-strain or H. crustuliniforme showed no detectable increase i n root growth in the 12°C s o i l . In a study of white spruce seedling response to s o i l temperature, Dobbs and McMinn (1977) found root function declined at a threshold temperature between 10 and 15°C. E-strain and H. crustuliniforme inoculation appeared to raise this threshold temperature compared with other inocula. Alternatively, these inocula may lower the root growth capacity of white spruce seedlings regardless of s o i l temperature. The nutrient data suggest that important interactions occurred between s o i l temperature and inoculation effects on total nutrient uptake 153 and the rate of uptake per unit root per unit time, particularly i n the 5 to 12 week period. Two inocula, T. terrestris and L. bicolor. improved root efficiency or specific absorption of N at both s o i l temperatures compared with non-inoculated seedlings; but their effect on specific absorption was much greater i n the 12°C s o i l . In the 6°C s o i l , the ranking of inoculation treatments i n descending order of root efficiency was: T. terrestris > L. bicolor > A. bvssoides and non-inoculated > forest floor > E-strain > H. crustuliniforme. For the 12°C s o i l the equivalent ranking was T. terrestris > L. bicolor > forest floor and E-strain > A. bvssoides > non-inoculated > H. crustuliniforme. Unfortunately, the lack of replicate nutrient data for the different inoculation and temperature treatment combinations precluded testing the s t a t i s t i c a l significance of these interactions. 2. Effects of Soil Temperature on Seedling Growth and Physiology The reductions i n shoot growth and the rate of net photosynthesis observed in the cooler s o i l (6°C) are in agreement with the numerous studies (Nielsen 1971, Lavender and Overton 1972, Rook and Hobbs 1975, Lopushinsky and Kaufmann 1984, Delucia 1986) that have demonstrated a positive correlation between s o i l temperature, shoot growth and photosynthesis. Reduced shoot growth i n cool so i l s has been attributed to various factors including (1) decreased a v a i l a b i l i t y , uptake and translocation of nutrients (Power et a l . 1963, Bowen 1970, Cooper 1973), (2) increased plant moisture stress resulting from increases in water viscosity and root resistance to water flow (Kramer 1940, Sutton 1969, Running and Reid 1980, Grossnickle and Blake 1985), and (3) reduced synthesis and transport of plant growth regulators such as cytokinin and 154 gibberellin from root to shoot tissues (Ritchie and Dunlap 1980, Atkin et a l . 1973). In the f i r s t 5 weeks after transplanting, differences in shoot growth at the two s o i l temperatures was not related to nutrient a v a i l a b i l i t y and uptake. A significant reduction (about 40%) i n the rate of net photosynthesis and new foliage production occurred i n the 6°C s o i l even though there was no detectable effect of s o i l temperature on N and P uptake or content. Several observations suggest that water stress contributed to poor shoot growth during this period. Mean values of xylem pressure potential, water-use efficiency and calcium absorption, which i s related to the mass flow of water (Marschner 1986), were lower in the 6°C s o i l . The level of plant moisture stress (0.7 MPa) measured in the 6°C s o i l i s considered sufficient to inhibit turgor-mediated processes such as root or leaf area growth in many plant species (Hsiao 1973). During the 5-12 week period, seedling moisture status showed no appreciable effect of s o i l temperature. In contrast, f o l i a r nutrient concentrations, total nutrient uptake, and root efficiency (specific absorption of nutrients) were strongly influenced by s o i l temperature. Newly-flushed foliage of seedlings grown in the 6°C s o i l was chlorotic, a visual symptom of nutrient deficiency commonly exhibited by plant species grown at sub-optimum s o i l temperatures (Cooper 1973). Foliar analysis indicated very severe and mild deficiencies, respectively of N and Fe. The specific absorption of nitrogen (uptake per unit root per time) increased by 72% i n the 12°C s o i l . In addition, root biomass was approximately 20% greater i n the 12°C s o i l . These results indicate that the a v a i l a b i l i t y , absorption or translocation of nutrients limited shoot growth in the 6°C s o i l either 155 directly or indirectly, e.g., through alterations in the balance of plant growth substances. The nutritional status of plants, particularly that of nitrogen, alters the quantity and composition of plant growth regulators synthesized i n root tissues (Marschner 1986 and references therein). Because of the par a l l e l decline in stomatal conductance and net photosynthesis with decreasing s o i l temperature, many researchers (e.g., Anderson and McNaughton 1973, Kaufmann 1977) assume that plant water stress, through i t s effect on CO2 a v a i l a b i l i t y , i s the major factor limiting net photosynthesis in cool s o i l . However, three observations suggest that net photosynthesis in the 6°C s o i l was not limited by water stress. F i r s t , the level of plant moisture stress measured in the 6°C s o i l (xylem pressure potential of -0.7 MPa) was not sufficient to reduce net photosynthesis significantly; the rate of net photosynthesis i s rela t i v e l y constant over a wide range of xylem pressure potentials above -1.5 MPa for many plants, including spruce seedlings (Hsiao 1973, Turner and Jarvis 1975, Beadle et a l . 1981). Second, mean xylem pressure potential differed by less than 0.2 MPa between 6 and 12°C s o i l . Third, internal concentrations of CO2 were not lower in the 6°C s o i l as would be expected i f stomatal conductance limited CO2 a v a i l a b i l i t y . Internal CO2 concentrations were actually higher (P < 0.001) in the 6°C s o i l ; averaging 283 and 253 Mg/g) respectively for the 6 and 12°C s o i l s . In most studies, s o i l or root-zone temperatures are reduced rapidly and plants are exposed to different temperatures for very b r i e f periods, generally less than 24 hours. This exposure time is too short to study the acclimation response of plants to cool s o i l s . The mechanisms underlying short-term and long-term plant response to s o i l temperatures may d i f f e r considerably (Delucia 1986, Setter and Greenway 1988). After exposing 156 Englemann spruce seedlings to seven days of cold s o i l temperatures, Delucia (1986) concluded that net photosynthesis was limited mainly by non-stomatal factors, such as carbohydrate accumulation in needle tissues when root sink a c t i v i t y was reduced by cool temperatures. The rate of net photosynthesis may be regulated by the demand for carbohydrates created in non-photosynthetic tissue (Herold 1980). Alternatively, i n cool so i l s the export of plant growth regulators, such as cytokinins, from root to shoot tissues may be reduced. These substances are thought to influence net photosynthetic rates by altering enzyme synthesis, membrane permeabilities and translocation patterns (Wareing et a l . 1968). A nutritional explanation for the temperature-induced changes in photosynthesis during the 5-12 week period is supported by the positive correlation between net photosynthetic rate and mature shoot N or P. However, i t is also possible that sink ac t i v i t y influenced the rate of net photosynthesis (Herold 1980) as proposed by Delucia (1986). The specific dry weight (leaf dry weight/leaf area) of mature needle tissues was higher in the cooler s o i l ; perhaps reflecting an increased accumulation of needle carbohydrates (Ehret and J o l l i f f e 1985) or a lowering of root sink ac t i v i t y (Harley and Smith 1983 and references therein). Nutrient-use efficiency estimated as net photosynthesis per unit shoot N and P (PNUE) was 2- to 3-fold greater i n the 12°C s o i l . Because PNUE was derived from the ratio of net carbon assimilation to shoot nutrient content, increases in PNUE may refle c t either (1) a decrease i n shoot N or P, or (2) an increase in net photosynthetic efficiency at a particular level of shoot N or P. The temperature-induced increase i n PNUE observed i n this study was not accompanied by a decrease i n shoot N or P. Concentrations of shoot N and P in the 12°C s o i l were similar (at 5 weeks) 157 or higher (at 12 weeks; 0.5% N at 6°C versus 0.8% at 12°C) than i n the 6°C s o i l . Moreover, PNUE increased as shoot N and P concentrations increased. The poor nutritional status of seedlings in 6°C s o i l probably contributed to the low PNUE of these seedlings. Needles with low nitrogen concentrations may have low nutrient-use efficiencies because a high proportion of their nitrogen is bound to non-photosynthetic compounds involved i n c e l l regulation and respiration (Chapin et a l . 1987) Decreasing s o i l temperature from 12 to 6°C reduced root growth more than shoot growth; consequently shoot/root ratios were higher in the 6°C s o i l . A similar response was reported by Barney (1951) for pine germinants grown at s o i l temperatures ranging from 5 to 20°C. However, the opposite trend, i.e., shoot/root ratios increasing with s o i l temperature, i s more commonly reported for many plant species (Davis and Lingle 1961, Davidson 1969, Lavender and Overton 1972). The variable results of s o i l temperature studies probably r e f l e c t the use of different growing environments, s o i l temperatures and plant material. In a study of Douglas-fir seedlings in their f i r s t growing season, Lavender and Overton (1972) observed that root growth of Douglas-f i r seedlings was relatively unaffected by s o i l temperatures between 10 and 20°C. However, a sharp decline in root growth could alter shoot/root allometry i n soils below 10°C. Root function and growth of many conifer species declines sharply at s o i l temperatures below 10°C (Kramer 1940, Babalola et a l . 1968, Kaufmann 1975, Running and Reid 1980, Delucia 1986). The low light intensities (approx. 10% f u l l sunlight) common to s o i l temperature studies conducted in growth chambers may also influence shoot/root ratios. Under these conditions, root growth is limited by photosynthate a v a i l a b i l i t y and may be less responsive to changes in s o i l 158 temperature. Light intensity was higher, approximately 25% of f u l l sunlight, i n this study. The interaction between root and shoot growth is often viewed as a competitive source-sink relationship with root growth limited by carbohydrates from the shoot and shoot growth limited by nutrients from the root (Loomis 1953). According to this hypothesis, when nutrient a v a i l a b i l i t y and the efficiency of nutrient absorption are limited by i n f e r t i l e , cold or droughty s o i l s , shoot growth is reduced more than root growth and shoot/root ratios decrease. The pattern of plant growth is adjusted to maximize nutrient uptake. The results of this study do not support this hypothesis since shoot/root ratios increased even though seedling nutrient uptake and root efficiency (nutrient absorption per unit root dry weight) decreased in the cooler s o i l . In most s o i l temperature studies, experimental plants are annuals or perennials i n their f i r s t growing season. Shoot growth of perennial cold-stored seedlings is less l i k e l y to be depend on current root a c t i v i t y or growing environment. Shoot tissue formed i n previous years can supply considerable amounts of nutrients (Fife and Nambiar 1984) and carbohydrates (Gordon and Larson 1968, Webb 1977) to new shoot growth of conifer seedlings. During the f i r s t .5 weeks, the N and P content of mature shoot tissue decreased by about 20%. Some of these nutrients were probably translocated to new foliage as described for pine seedlings by Fife and Nambiar (1984). The coincidence of higher shoot/root ratios and lower f o l i a r nutrient concentrations i s not in agreement with a growth analysis of s o i l temperature effects reported by Margolis and Brand (1990). In their study, both shoot/root ratios and f o l i a r nutrient concentrations of outplanted eastern white pine seedlings were inversely related to s o i l temperature. They attributed this effect to an increase i n root efficiency (nutrient uptake per unit root) with decreasing s o i l temperature. However, numerous studies have shown that root efficiency i s maximum at s o i l temperatures i n the 20 and 30°C range and that i t decreases at s o i l temperatures below this range (Nielsen and Humphries 1966, Bowen 1970, Cooper 1973). Alternatively, their results may reflec t (1) the confounding of s o i l temperature treatments with s o i l f e r t i l i t y , or (2) the inadequacy of the particular index they used to estimate root efficiency. Warm microsites were created by removing organic s o i l horizons and surface debris; a practice which also reduces s o i l f e r t i l i t y . Margolis and Brand (1990) used the ratio of f o l i a r (not total) N to root weight (N/RW) as an indicator of the a b i l i t y of a given amount of root to supply nitrogen. N/RW calculated from data i n the present study, showed no effect of s o i l temperature (P > 0.30). Moreover, this index was closely related (r > 0.80, P < 0.001) to the nutritional status of seedling at the beginning of the measurement period. The distribution of dry matter between shoots and roots varies with environmental conditions. These changes may be interpreted as adaptive, with the plant maintaining an optimum balance between the nutrient or water absorbing capacity of the root and the photosynthetic capacity of the shoot in a particular environment (Davidson 1969, Troughton 1980, Chapin et a l 1987). However, the decrease in shoot/root ratio observed i n the 6°C s o i l would seem to have limited or no adaptive value. The potential for seedling moisture or nutrient de f i c i t s would be increased by the high proportion of shoot to root growth in an environment which limits root efficiency. It is more common for the shoot/root ratios of spruce seedlings to decrease after their f i r s t growing season (Hermann 1977). 160 Alternatively, the inverse relationship observed during the 12-week study may not be an adaptive response. It may simply be the result of s o i l temperatures that are sub-optimum for root growth; a i r temperatures that are optimum for shoot growth; and the a b i l i t y , at least i n the short-term, for new shoot growth of determinate species to be sustained by nutrients translocated from mature shoot tissues. 3. Seedling response to "any" mycorrhizae at the time of transplanting Averaged across a l l inocula, the presence of mycorrhizae at the time of transplanting, did not improve spruce seedling growth. This result does not support the premise that the presence of "any" fungus is better than "no" fungus for the particular study conditions, i.e., cool moist acidic forest s o i l with good potential for root colonization by indigenous ECM fungi. Mycorrhizal infection, however, did alter root morphology by reducing the number of short roots per seedling and per unit root weight. On average, mycorrhizal infection increased the water-use efficiency of mature white spruce needles by approximately 25%. Inoculated seedlings tended to have higher rates of net photosynthesis but lower rates of transpiration. Water-use efficiency was not associated with improved growth in the particular study conditions. However, the improved water-use efficiency of mycorrhizal seedlings suggests that "any" fungus might be better than "none" in cool, droughty s o i l s . Water-use efficiency was related positively with shoot N and P indicating improved nutrition was in part responsible for the effect of mycorrhizal infection on water-use efficiency. Averaged across temperature and time, inoculated seedlings had higher mean values of P and N i n mature shoot tissues (respectively, 0.18 and 0.60%) compared to non-inoculated seedlings (respectively, 0.14 and 0.51%). Nutritionally-induced changes in the rate of net photosynthesis, the hydraulic conductivity of the root system, the se n s i t i v i t y of stomata to abscisic acid (ABA), or the balance between ABA and cytokinins in needle tissue (Marschner 1986) would also affect water-use efficiency. Guehl et a l . (1990) also found that inoculation with a variety of ECM fungi increased the water-use efficiency of Italian stone pine seedlings grown in moist s o i l s . Although they attributed this effect to the improved nutritional status of inoculated seedlings, the nutritional status of non-inoculated and inoculated seedlings was not compared. However, a positive relationship between the water-use efficiency of conifer seedlings and f o l i a r N and P have been reported by Sheriff et a l . (1986). Due to insufficient replication, i t was not possible to test s t a t i s t i c a l l y the effect of inoculation on nutrient uptake by white spruce seedlings. The raw data suggest that inoculation increased nutrient uptake and efficiency of uptake (rate of uptake per unit root per unit time) in the 12°C but not the 6°C s o i l , even though the inoculant fungi grew and infected new roots in the 6°C s o i l . This conclusion is supported by 32 experiments conducted by Mejstrik (1970). He found that differences i n P uptake between excised non-mycorrhizal and ectomycorrhizal root segments increased with solution temperature in the range of 5 to 30°C. Inoculation, on average, had the greatest influence on nitrogen uptake, which was the most limiting of the analyzed nutrients. Nitrogen uptake by inoculated seedlings (averaged across a l l inocula) was 65% greater than that of non-inoculated seedlings in the 12°C s o i l . There was no appreciable difference i n P or Ca uptake between non-inoculated or inoculated seedlings at either s o i l temperature. Fe data were incomplete and could not be used to compare uptake by inoculated and non-inoculated seedlings. However, inoculation (averaged across 4 inocula) increased the Fe content of new foliage by about 55% at both s o i l temperatures, suggesting comparison of Fe uptake by mycorrhizal and non-mycorrhizal seedlings warrants further study. The results are in agreement with studies conducted with VA mycorrhizae (Smith and Roncadori 1986) and excised ectomycorrhizae (Harley and Wilson 1959, Edmonds et a l . 1976) showing that the benefits of the mycorrhizal symbiosis i n terms of nutrient uptake decrease in cool s o i l s . Smith and Roncadori (1986) reported that VA mycorrhizae increased the phosphorus and copper uptake of cotton plants at s o i l temperatures of 24°C and 36°C; but mycorrhizae had no effect on nutrient uptake at sub-optimum s o i l temperatures of 18°C. Harley and Smith (1983) speculated that the optimum temperature for nutrient uptake by mycorrhizae is lower than the optimum for fungal growth. The distribution of N and Ca between shoot and root tissues was not affected by inoculation; but the proportion of P in shoot tissues was increased, on average, by inoculation. Studies of VA (Powell 1975, Smith and Daft 1978) and ECM infections (Black 1986) have demonstrated that mycorrhizal symbioses alter the distribution of nutrients within the host plant, especially when plant nutrients are deficient. In contrast to this study, Black (1986) found that mycorrhizal infection increased the proportion of N and P In root tissues of Douglas-fir seedlings. His nutrient analyses coincided with sporocarp production by the inoculant fungus (L. laccata) suggesting to him that nutrients stored i n fungal tissues were preferentially used by the mycorrhizal fungi during i t s reproductive phase. Unfortunately, i n this study, no attempt was made to 163 monitor spore production by the inoculant fungi. 4. Seedling response to specific fungi The presence of specific mycorrhizae (e.g., forest s o i l , L. bicolor or T. terrestris) at the time of transplanting did improve spruce seedling growth and/or nutrition; supporting the second premise of a r t i f i c i a l inoculation programs, i.e., some ECM fungi are more beneficial to host plants than others in cool s o i l s . These benefits occurred even though most non-inoculated seedlings were infected with indigenous ECM fungi by the f i f t h week after transplanting. Of the inocula tested, forest s o i l was one of the most effective in enhancing spruce seedling growth. Seedlings inoculated with forest s o i l grew better than non-inoculated seedlings even though (1) non-inoculated seedlings were rapidly infected with forest fungi 5 weeks after they were transplanted into forest s o i l and (2) the size and nutritional status of non-inoculated seedlings and those inoculated with forest s o i l were similar at the time of transplanting. Soil inoculum has been used successfully around the world (Mikola 1970); and in some cases, i t has proved more effective than pure culture inoculum (Ri f f l e and Tinus 1982). The greater diversity of mycorrhizae (more than 3 morphological types) infecting the root systems of seedlings inoculated with forest floor may have contributed to the efficacy of this inoculum (Perry et a l . 1987). Naturally-regenerated seedlings typically have more than one fungal partner (Trappe 1977). Sinclair (1974) reported that Douglas-fir seedlings grew better i n bare-root nursery s o i l when infected with more than one type of ECM fungus. It i s also possible that microorganisms associated with the mycorrhizosphere contributed to the growth promoting effect of the forest s o i l inoculum. Soil collected from an arbutus stand was shown to increase 164 N 2 fixation i n the rhizosphere of planted Douglas-fir seedlings (Amaranthus et a l . 1990) . . ... L. bicolor was an effective pure culture inoculum, increasing a l l aspects of seedling growth. Under nursery conditions, Laccaria spp. have been shown to depress seedling growth (Molina 1982, Shaw et a l . 1982, Danielson et a l . 1984c). In outplanting studies, response to inoculation with Laccaria sp. has been variable. Shaw et a l . (1987b) and Loopstra et a l . (1988) reported no growth response to L. laccata: others (e.g., Wilson et a l . 1987, Danielson and Visser 1989) reported positive responses with L. proxima infection. Also Richter and Bruhn (1989) found that L. bicolor infection increased the survival of container-grown red and jack pine seedlings planted i n sandy, xeric s o i l . Laccaria spp. may be especially tolerant of acidic s o i l s . Gagnon et a l . (1987, as cited by Kropp and Langlois 1990) successfully inoculated conifer seedlings with L. bicolor in peat-based growing media, with pH values as low as 3.5. L. laccata inoculation has been shown to increase the growth of Sitka spruce seedlings on acidic organic and mineral s o i l s (Thomas and Jackson 1983, Mason et_ a l . 1983). T. terrestris was effective especially i n promoting nutrient uptake and caliper growth. In North America, this fungus is considered a "weed fungus" which contaminates inoculation experiments and naturally infects conifer seedlings in both bare-root and container nurseries (Mikola 1989, Castellano and Molina 1989, Danielson and Visser 1990). T. terrestris had proven less effective than Pisolithus tinctorius on planting sites with a s o i l moisture d e f i c i t (Marx and Cordell 1987); thus T. terrestris is assumed to be adapted to the f e r t i l e , moist conditions of nurseries and not to the more stressful environments of planting sites (Marx et a l . 1984). 165 However, seedling stress and poor growth can also result from an excess, rather than a lack of s o i l moisture. T. terrestris is better adapted to acidic poorly-drained forest so i l s than are other ECM fungi including P.  tinctorius (Cruz 1974, Thomas and Jackson 1983, Wilson et a l . 1987). Coutts and N i c o l l (1990b) noted that mycelial strands of T. terres t r i s are extremely tolerant of flooding, surviving 149 days of waterlogged s o i l s . Inoculation with E-strain reduced new foliage production relative to non-inoculated seedlings and most other ECM fungi. A similar response was reported by Danielson et a l . (1984a) when jack pine seedlings were inoculated with E-strain and grown over a wide range of f e r t i l i z e r regimes in a container nursery environment. The thin mantle and coarse Hartig net development characteristic of E-strain mycorrhizae and the coincidence of E-strain infection with poor seedling growth, suggested to Levisohn (1954) and Bjorkman (1949, cited in Mikola 1965) that E-strain infections were parasitic on conifer seedlings. In contrast, Mikola (1965) found there was no correlation between seedling growth and E-strain infection. Published results of conifer response to E-strain inoculation suggest that the efficacy of this fungus may vary with s o i l acidity. On a reclamation site i n northern Alberta, shoot growth of Jack pine seedlings inoculated with E-strain was more than double that of non-inoculated seedlings (Danielson and Visser 1989) two years after planting. The seedlings were planted in peat-amended o i l sands with pH values ranging from 6.3-7.5. In contrast, Holden et a l . (1983) observed that E-strain was less effective i n promoting the growth of Sitka spruce seedlings than were other ECM fungi, including T. terrestris and Laccaria spp. Their experiment was conducted in a greenhouse with an acidic humus-type forest s o i l (pH 4.8). Similarly, i n greenhouse and f i e l d experiments conducted by 166 Thomas and Jackson (1983), E-strain was found to be less effective in an acidic forest s o i l compared to T. terrestris and Laccaria laccata. Mikola (1965) noted that bare-root nurseries located on acidic s o i l s (pH 4.0-6.2) have less E-strain infection compared with those located on more neutral s o i l s (pH 5.1-6.6). Danielson and Pruden (1989) reported that E-strain was the most common fungal symbiont of young and mature urban spruce growing in moderately alkaline so i l s (pH 7.3-8.1). H. crustuliniforme significantly depressed the growth of white spruce seedlings i n this study. Previous studies have shown the efficacy of H. crustuliniforme i n promoting host growth is poor relative to other fungi (Chu-Chou 1985, Tyminska et a l . 1986, Danielson and Visser 1989). Its efficacy may be greater i n soils that are dryer or less acidic than those used in this study. Stenstrom et a l . (1990) noted that nursery seedlings inoculated with H. crustuliniforme appeared to tolerate drought better than non-inoculated seedlings. In culture, H. crustuliniforme mycelium tolerates pH values as high as 9 (Dennis 1985); and i n s o i l s , H. crustuliniforme mycorrhizae tolerate dry, moderately alkaline so i l s (Danielson and Pruden 1989). Soil type may also strongly influence the efficacy of H. crustuliniforme. Mason et a l . (1983) observed that birch trees were naturally infected with H. crustuliniforme i n mineral so i l s but not i n organic s o i l s . Inoculation with A. bvssoides had negligible effects on the growth of seedlings under the conditions of this study. I am not aware of any other studies of A. bvssoides efficacy. Inoculation with A. bvssoides i n previous studies has not resulted in successful root infection (Danielson et a l . 1984c, Danielson and Visser 1989). Like E-strain and H. crustuliniforme. Amphinema mycorrhizae tolerate dry, alkaline s o i l 167 conditions (Danielson and Pruden 1989) and i t may be a more effective symbiont i n these conditions. 5. Possible reasons for differences in Mycorrhizal Efficacy The lack of a positive host response to mycorrhizal inoculation can result from low levels of root infection by the inoculant fungi (Ruehle et a l . 1981, Lee and Koo 1983, McAfee and Fortin 1986, Wilson et a l . 1987, Last et a l . 1990). Ruehle et a l . (1981) and Last et a l . (1990) found that the response of conifer seedlings to inoculation was negligible unless more than 50% of short roots were infected by the inoculant fungus. However, in this study, variable spruce seedling response to inoculation could not be attributed to the degree of root colonization by the inoculant fungi at the time of transplanting. With the exception of the A. bvssoides treatment, inoculant fungi colonized more than 95% of the short roots of inoculated seedlings during the nursery production phase. Furthermore, the growth of seedlings inoculated with bvssoides was not related (P > 0.25) to percent infection at the time of transplanting (estimated from percent of original root plug colonized by A. bvssoides). Nor was seedling growth related to the source of inocula. The least (H. crustuliniforme) and most effective (L. bicolor) inocula tested in this study were collected from northern f i e l d sites. Of the two nursery isolates, one (A. bvssoides) had a negligible effect on seedling growth; the other (T. terrestris) improved seedling growth. 168 Variable host response to specific fungi was related to inocula-induced differences in four parameters: (1) the nutritional status of seedlings at the time of planting, (2) rate of net photosynthesis, (3) nutrient uptake after planting and (4) nutrient-use efficiency. These factors are discussed below in four sections. I n i t i a l nutritional status of seedlings New foliage biomass, 65% of which occurred i n the f i r s t 5 weeks after transplanting, was strongly influenced by seedling nutrition at the time of transplanting. The amount of new foliage biomass in spruce seedlings i s to a large extent predetermined and, therefore, can be expected to be more responsive than root or caliper growth to inocula-induced changes in seedling nutrition during the nursery production phase. However, other factors may also have been involved since T. terrestris seedlings with an i n i t i a l nutrient status similar (i.e., low) to H. crustuliniforme seedlings produced much more new foliage. New foliage production was also related to the proportion of total seedling N and P contained in shoot tissues. However, E-strain seedlings produced much less new foliage than seedlings with similar total N and P contents (e.g., Aj. bvssoides and control seedlings) at the time of transplanting. The smaller proportion of total seedling N and P contained in the shoot tissues of E-strain seedlings l i k e l y contributed to their infer i o r production of new foliage. Regardless of s o i l temperature, inoculant fungi (e.g., forest floor, T. terrestris and L. bicolor) which increased total seedling dry weight also increased the proportion of dry matter allocated to shoot relative to root tissues. Ectomycorrhizal infection has been previously shown to 169 increase the rate of shoot growth relative to root growth (Hatch 1937, Cline and Reid 1982, Black 1986), an effect which is usually attributed to an improvement in the nutritional status of the host plant. Mycorrhizal infection i s considered to increase the efficiency (nutrient uptake/unit root) and therefore, to reduce the amount of root biomass required for nutrient absorption (Cline and Reid 1982, Black 1986). In this study, shoot/root allometry was not correlated with N and P uptake or with root efficiency, but was influenced by the i n i t i a l nutrient content of shoot tissues and to a lesser degree by total seedling nutrient content. Rate of net photosynthesis Inoculation with forest floor, L. bicolor and T. terrestris increased seedling net photosynthetic rates at 5 and 10 weeks. This increase was accompanied by greater new foliage production and percent caliper growth. Enhanced rates of net photosynthesis i n response to mycorrhizal infection have been observed in previous studies (Ekwebelam and Reid 1983, Harley and Smith 1983, Reid et a l . 1983). Numerous studies have shown that net photosynthetic rate increases with increasing levels of f o l i a r N and P (Marschner 1986) and that mycorrhizal infection increases nutrient uptake (Harley and Smith 1983). Improved nutrition probably explains the differences among inoculated seedlings i n net photosynthesis at the 5-week measurement. At 5 weeks, the rate of net photosynthesis correlated positively with the level of N and P in mature shoot tissue; and i n i t i a l and 5-week shoot N and P were influenced by inoculation treatments. However, net photosynthetic rate at 10 weeks showed no relationship to the nutrient status of mature shoot tissues at 0, 5, or 12 weeks. Possibly, a nutrient other than N, P, Ca or Fe limited the rate of net 170 photosynthesis. Alternatively, the various inocula may have altered source-sink relationships or the balance of plant growth regulators affecting carbon assimilation. Reid et a l . (1983) speculated that the carbon cost of mycorrhizal infections might stimulate the rate of net photosynthesis in the host plant. If this is the case, one might expect 10-week net photosynthetic rate to be inversely related to specific leaf dry weight (i . e . , needle dry weight/surface leaf area). Specific leaf dry weight has been shown to increase when leaf carbohydrates accumulate (Ehret and J o l l i f f e 1985). However, there was no (P > 0.70) relationship between these two parameters. Nutrient uptake after transplanting Seedlings inoculated with different fungi varied widely in their a b i l i t y to absorb nutrients, particularly N at low s o i l temperatures. Root and caliper growth, 50% or more of which occurred in the 5-12 week period, correlated positively with nutrient uptake but not with the i n i t i a l nutritional status of seedlings. Inoculation with T. t e r r e s t r i s . L. bicolor and forest s o i l significantly improved seedling caliper growth and nutrient uptake. New foliage production, the majority of which occurred in the f i r s t 6 weeks after transplanting, was not correlated with nutrient uptake. T. terrestris and H. crustuliniforme seedlings which were nutritionally inferior to other inoculated and control seedlings at the time of transplanting absorbed, respectively, the most and least N for 12 weeks after transplanting. Averaged across the two s o i l temperatures, there was a 3-fold difference in N uptake between seedlings infected with T. terre s t r i s and those infected with H. crustuliniforme. Large 171 differences (as great as 10-fold) in nutrient uptake by various kinds of excised mycorrhizae in solution culture have been recorded (Mejstrik 1970, Langlois and Fortin 1978). Harley and Smith (1983) suggest that nutrient uptake by different kinds of mycorrhizae is strongly influenced by the amount of external hyphae (i.e., absorbing surface area) extending into the rooting medium. The relatively low nutrient uptake by E-strain and H. crustuliniforme mycorrhizae does not support this hypothesis. These two types of mycorrhizae had, respectively, sparse and very abundant development of external hyphae. Chu-Chou (1985) also observed that H. crustuliniforme was less effective i n enhancing N and P uptake of radiata pine compared with other ECM fungi. Although H^ . crustuliniforme may be less effective than other fungi i n promoting nutrient uptake, seedlings inoculated with H.  crustuliniforme have been found to absorb more ammonium- and nitrate-nitrogen i n solution culture than non-mycorrhizal conifer seedlings (Rygiewicz et a l . 1984a,b). It is important to note that most control seedlings in this study were infected with mycorrhizal fungi 5 weeks after transplanting; therefore they were not non-mycorrhizal. Washing of root samples prior to oven-drying for chemical analysis removed external hyphae attached to mycorrhizae, therefore total nutrient uptake for the mycorrhizal symbiosis was underestimated particularly for the H. crustuliniforme infection with i t s abundant external mycelium. The low content of nutrients in H. crustuliniforme seedlings may have resulted, in part, from the retention of nutrients i n external hyphae. Low temperatures are thought to depress the a b i l i t y of vesicular-arbuscular mycorrhizae to absorb phosphorus and also to transport phosphorus to the host plant (Harley and Smith 1983 and references therein). 172 Nutrient-use efficiency Nutrient-use efficiency, or the relative gain i n dry weight per unit of seedling N or P, accounted for the greatest proportion of v a r i a b i l i t y in relative growth rates among inoculation treatments. This variation was not due to inoculation effects on nutrition during the nursery production phase, nor to effects on seedling water status (e.g, water-use efficiency, xylem pressure potential, calcium uptake) after transplanting. Nor were there differences among inocula in net photosynthetic rate per unit f o l i a r N or P, as reported by Bethlenfalvay et a l . (1987) for mycorrhizal soybean plants. The variation may have resulted from nutrients which were not analyzed or from non-nutritional factors, e.g., alterations in carbon metabolism or i n the balance of plant growth regulators. The amount of carbon lost through root respiration or the development of fungal tissues (external mycelium , f r u i t bodies) may have varied among inocula. ECM infection has been shown to increase root respiration and the translocation of photosynthate to root systems (Tranquillini 1964, Reid et a l . 1983, Ingestad et a l . 1986). Root respiration losses can be significant with as much as 90% of the assimilated carbon lost through root respiration, particularly when plants are nitrogen-deficient (Hermann 1977). Significant quantities of host photosynthate can also be incorporated into fungal structures. Inhibition of conifer seedlings i n nursery environments has been attributed to use of host photosynthate for external hyphae (Langlois 1983, cited in Gagnon et a l . 1987) and sporocarp production (Shaw et a l . 1982). Various plant growth regulators, e.g., cytokinins and auxins, are produced by ECM fungi in culture (Ek et a l . 1983, Ho 1987). 173 Although there i s no direct evidence for transfer of these substances to the host plant, they may affect the host plant or rhizosphere population. Alternatively, ECM fungi may stimulate or inhibit host plant production of certain plant growth regulators. The low nutrient-use efficiency of seedlings inoculated with H. crustuliniforme supports a non-nutritional explanation for the differences among inocula. In previous studies the poor growth response of seedlings to infection by H. crustuliniforme has been attributed to the carbon cost of i t s extensive mycelium (Tyminska et a l . 1986), i t s relative i n a b i l i t y to synthesize plant growth regulators (Tyminska et a l . 1986), and i t s high respiration rate (Marshall and Perry 1987). Reid et a l . (1983) and Ingestad et a l . (1986) proposed that the higher carbon cost of mycorrhizal systems was compensated for by increased rates of net photosynthesis since the growth of mycorrhizal seedlings was equal to or greater than that of non-mycorrhizal seedlings. In this regard, i f H. crustuliniforme infection represented a high carbon cost to spruce seedlings, there was no compensatory increase in the rate of net photosynthesis. Differences i n the timing of bud break whether hormone- or nutrient-induced may also have contributed to inocula-induced differences in nutrient-use efficiency. The percentage of seedlings (data from Chapter IV for a l l inocula except A. bvssoides') flushed at 21 days correlated positively with mean nitrogen-use efficiency (r - 0.88, N = 6, P =0.02). Seedlings with high values of nutrient-use efficiency (control, forest floor) also flushed earlier than other seedlings. Differences i n the speed of bud break may be due to inocula-mediated alterations i n the a v a i l a b i l i t y of carbohydrates and nutrients for bud expansion or to shifts in the balance of plant growth regulators. Fungal infections alter the 174 translocation patterns of host plants (Smith et a l . 1969). Ingestad et a l . (1986) estimated that 30-40% more carbon was translocated to ectomycorrhizal root systems than to non-mycorrhizal systems when nitrogen was deficient (shoot N ranged from 0.7 to 0.9%) as i n this study. During the 5-12 week period, the nitrogen-use efficiency of L. bicolor seedlings increased f i v e - f o l d in the warmer s o i l (12°C), while the nitrogen-use efficiency of E-strain seedlings showed no response to s o i l temperature. The reason for the low nutrient-use efficiency of this fungus are not clear. Nitrogen uptake during the 5-12 week period was similar to that of inoculation treatments (e.g., control, A. bvssoides) with higher values of nutrient-use efficiency. The low nitrogen-use efficiency of E-strain seedlings was reflected in the elevated levels of nitrogen i n new foliage (1.7%). Phosphorus uptake was relatively low i n comparison to N uptake and i t is possible that an imbalance of N and P decreased nitrogen-use efficiency. Alternatively, at 12°C, the E-strain fungus may have been stimulated to produce spores, increasing carbon translocation to fungal tissues. 175 Summary Seedling growth and net photosynthetic rates were lower at 6°C than at 12°C. Root growth was reduced proportionally more than shoot growth with the result that seedlings grown at 6°C had higher shoot/root ratios. Reductions in net photosynthetic rate were attributed to plant moisture stress in the short-term and to nutrient stress i n the long-term. White spruce seedlings showed no response in the particular study conditions ( i . e . , cool, wet, acidic forest soils) to the presence of "any" mycorrhizae at the time of transplanting. In contrast, they responded positively to inoculation by specific inocula (e.g., T^ . t e r r e s t r i s . L.  bicolor and forest floor); and negatively to others (e.g., H.  crustuliniforme and E-strain). Positive responses to inoculation included increased growth, nutrient uptake, nutrient-use efficiency and allocation of biomass to shoot tissues. Since plant growth rate is highly correlated with the proportion of carbon invested i n leaf area (Potter and Jones 1977), the increased partitioning of dry matter to shoot tissues suggests that the inoculation effects observed in this study would persist into another growing season. Bowen (1973) and Black (1986) emphasize that nutrient relations of plants involve not only the uptake, but also the distribution and u t i l i z a t i o n of nutrients. The results support further investigation of mycorrhizal effects on nutrient distribution and nutrient-use efficiency. 176 CHAPTER VI IMPLICATIONS FOR APPLIED FORESTRY AND RESEARCH The dissertation began by underscoring the need to improve our knowledge of the efficacy of ectomycorrhizal fungi in different s o i l environments. Past research has shown that ectomycorrhizal inoculation has variable success i n promoting conifer seedling f i e l d performance (Bledsoe et a l . 1982, Shaw et a l . 1987b, Trofymow and van den Driessche 1991). The overall objective of the dissertation was to examine the effects of cool s o i l temperature on mycorrhizal efficacy. Trappe (1977) stressed the importance of s o i l temperature to the efficacy of mycorrhizal fungi and the need for more knowledge of the adaptation of specific ectomycorrhizal fungi to s o i l temperatures at the planting si t e . Cool temperatures have been shown to eliminate the benefits normally derived from infection by vesicular-arbuscular mycorrhizal fungi (Moawad 1978, Smith and Roncadori 1986) . Similar research has not been conducted for the ectomycorrhizal symbiosis. White spruce seedlings were chosen as host plants. It is well known that the growth of planted white spruce in the sub-boreal forest region is often poor for several years after planting (Mullin 1963, Vyse 1981, Burdett et a l . 1984, Butt 1986), and that cool s o i l temperatures contribute to the poor growth of spruce seedlings (Butt 1986, Binder et a l . 1987). A r t i f i c i a l inoculation with specific ectomycorrhizal fungi has the potential to promote growth in white spruce planted i n cool s o i l s during this period. Research conducted by McAfee and Fortin (1989) suggests that a r t i f i c i a l inoculation with selected fungi w i l l benefit spruce seedlings more than pine seedlings. They planted 6-week-old non-mycorrhizal black spruce and jack pine seedlings together on ten diverse reforestation sites in Quebec. Two months after planting, percent colonization of spruce roots by indigenous fungi was much weaker (< 25%) compared with that of pine roots (> 50%). In addition, the growth of spruce seedlings was positively correlated with the degree of root colonization. Previous chapters addressed the relationship of s o i l temperature to four aspects of mycorrhizal efficacy: the a b i l i t y to colonize roots of nursery seedlings (Chapter II); survival and colonization of new roots i n forest s o i l (Chapter III); the a b i l i t y to accelerate the acclimation of seedlings to cool soi l s during the transplant stress period (Chapter IV); and the a b i l i t y to increase growth and nutrition of seedlings during a simulated f i e l d growing season (Chapter V). The purpose of this chapter is to discuss the implications of the main findings of the dissertation for applied forestry and research. Implications for Applied Forestry The main findings of the dissertation research support the perspective that s o i l temperature has an important influence on a l l aspects of ectomycorrhiza efficacy. Although the results should be interpreted with caution because the experiments were conducted in controlled environment, they have some relevance to the production of inoculated seedlings i n container nurseries and to the reforestation of cool forest sites. 178 1. Establishment of inoculant fungi in container nurseries Growing mix temperature strongly influenced the extent of infection established on containerized seedlings. Overall, percent root infection was best for most fungal isolates, regardless of origin, at 16°C, with 6°C more detrimental to infection than 26°C. Primary infection (i.e., from "free" mycelial inocula) was inhibited less than secondary infection (i.e., from established mycorrhizae) in 6°C s o i l , indicating that nursery establishment of inoculant fungi would be inhibited by cool root-zone temperatures i n the f i r s t three months after sowing. To determine the potential limitation of temperature to primary root colonization, i t w i l l be necessary to quantify infection success in more detail between 5 and 20°C root-zone temperatures. It is important to know the lower limits of the optimal temperature range for specific fungi. In culture, fungal growth decreases rapidly once this threshold temperature i s reached (Dennis 1985, Samson and Fortin 1986). The origin ( f i e l d or nursery) of an isolate was not a predictor of response to temperature. Therefore, individual isolates must be screened for their response to low or high temperature i n peat:vermiculite growing medium. This conclusion must be considered with caution because of (1) the limited number of isolates for each species of fungus, and (2) the different ages and cultural history of the various isolates used in the study. Nevertheless, the results are consistent with the results of in vi t r o studies of temperature response variation in ectomycorrhizal fungi (Dennis 1985, Samson and Fortin 1986, Cline et a l . 1987), i.e., the temperature response of isolates i s not related to their geographic origin. Knowledge of the temperature tolerance of specific fungi w i l l be useful in manipulating nursery infection. Rapid colonization by inoculant 179 fungi is important to prevent contamination by fungi indigenous (e.g., T. terrestris) to container nurseries. F e r t i l i z e r regimes have been shown to affect the composition of mycobionts on roots of container seedlings (Hunt 1989). Growing mix temperature could also be an important factor influencing the relative competitiveness of inoculant and indigenous nursery fungi. The two isolates of T. terrestris had a higher rate of mycorrhiza formation at 26°C than did most other inocula, indicating that this fungus would be more competitive at high growing mix temperatures. In the f i r s t three months after sowing, growing mix temperatures in southern B.C. container nurseries can exceed 30°C (Husted and Barnes 1987). 2. Importance of container stock with favorable root egress patterns In general, inoculant fungi colonized a high proportion of the new roots formed outside the original container root mass during the three months after transplanting. This was attributed to several factors including (1) the successful establishment of inoculant fungi in the nursery and (2) the pattern of l a t e r a l root egress from the container plugs. New roots emerged from the entire root plug surface f a c i l i t a t i n g the spread of the inoculant fungi from old to new roots. Nursery practices which encourage this pattern of root growth should be an integral component of a r t i f i c i a l inoculation programs, and probably container seedling production i n general. Lateral root egress from the entire root plug would also speed the colonization of non-mycorrhizal container-grown seedlings by native s o i l ECM fungi. 180 3. Effect of inocula-induced changes in nutrition or size during the  nursery production phase Under the conditions of this study (i.e., slight nitrogen deficiency, cool s o i l s ) , inoculation with specific mycorrhizae had more effect on seedlings physiology during the transplant stress period than did the s o i l temperature treatments (6 versus 12°C). Inoculation treatments influenced seedling acclimation i n the 6°C s o i l as determined by net photosynthetic rate and resistance to water flow. These effects were related to differences i n root size, nutrient content and distribution of nutrients between root and shoot tissues at the time of transplanting. Root biomass and shoot N and P were inversely related, respectively, to resistance to water flow and net photosynthetic rate. The study results emphasize the importance of root size and morphology at the time of transplanting. In a review of seedling characteristics which correlate with f i e l d performance, Lavender (1988) noted that the growth of conifer seedlings after outplanting was often more closely related to root mass or volume at the time of planting than to root growth capacity, especially when new root growth was limited by droughty or cool s o i l s . Mycorrhizal inoculation has variable effects on the growth of container-grown seedlings, often resulting i n smaller seedlings compared to non-inoculated seedlings (Castellano and Molina 1989). Cultural regimes may have to be modified to produce naturally or a r t i f i c i a l l y inoculated mycorrhizal seedlings which meet the c u l l standards for nutrition and size. In particular, fungi which produce abundant sporocarps in the nursery may have detrimental effects on seedling nutrition, requiring compensatory f e r t i l i z a t i o n . 181 Differences among inoculation treatments (including the control, non-inoculated) i n resistance to water flow might have been greater i f storage conditions had been more severe or i f the seedlings had been deficient in phosphorus. It is important to recognize (1) that the seedlings were cold-stored i n rel a t i v e l y mild conditions compared to standard practices i n B.C., (2) that shoot nitrogen concentrations were low and (3) root and shoot phosphorus concentrations were adequate. Beneficial effects of VA infection on root hydraulic conductance have been attributed to improvements in phosphorus nutrition by the symbiotic association (Nelsen and Safir 1982). The magnitude of inoculation effects on net photosynthetic rate would l i k e l y decrease for seedlings with higher levels of shoot nitrogen (> 1.8%). 4. Cold-storage of white spruce seedlings Binder et a l . (1987) noted that "hot planting" (i.e., with no cold storage) of white spruce seedlings in late summer is very successful, in part because this practice avoids f a l l - l i f t i n g and lengthy storage. Regardless of mycorrhizal infection, the results provide evidence that long periods of cold storage at below-freezing temperatures reduce the a b i l i t y of white spruce seedlings to acclimate to cool s o i l s . Acclimation of a l l test seedlings was faster and levels of seedling resistance to water flow lower when compared to other studies (Grossnickle and Blake 1985, Grossnickle 1987, 1988) i n which spruce seedlings were cold-stored for long periods at below-freezing temperatures. Comparisons of the water relations of pine and spruce species made by these and other researchers may be seriously confounded by differences in storage treatments between the two species. 182 B i n d e r e t a l . (1987) a l s o sugges ted t h a t the h i g h s h o o t / r o o t r a t i o s o f w h i t e spruce n u r s e r y s t o c k are no t a p p r o p r i a t e f o r c o o l f o r e s t s i t e s . Fur thermore , they h y p o t h e s i z e d t h a t the shoot growth check observed i n spruce p l a n t a t i o n s may be a p e r i o d d u r i n g which s e e d l i n g s a l l o c a t e more carbon to r o o t growth r a t h e r than shoot growth . The r e l a t i v e l y h i g h shoot to r o o t growth i n 6°C s o i l v e r sus 12°C s o i l suggests t h a t w h i t e spruce s e e d l i n g s may have a l i m i t e d a b i l i t y to ad ju s t t h e i r growth p a t t e r n s to maximize n u t r i e n t and water up take . Carbon a l l o c a t i o n i n spruce s e e d l i n g s needs f u r t h e r i n v e s t i g a t i o n . 5. Compe t i t i venes s o f i n o c u l a n t f u n g i a t low s o i l temperatures I n the absence o f ind igenous inocu lum, the spread o f i n o c u l a n t f u n g i to new r o o t s formed o u t s i d e the c o n t a i n e r r o o t was independent o f the two t e s t s o i l temperatures (6 , 1 2 ° C ) ; whereas, i n the presence o f ind igenous inocu lum, the percentage o f new r o o t s c o l o n i z e d by i n o c u l a n t f u n g i was l e s s i n 12°C s o i l than i n 6°C s o i l . I n the warmer s o i l , a c c e l e r a t e d r o o t e l o n g a t i o n i n c r e a s e d the l i k e l i h o o d t ha t s h o r t r o o t s a t the t i p s o f e l o n g a t i n g l a t e r a l s were c o l o n i z e d by n a t i v e s o i l f u n g i . The r e s u l t s suggest t h a t the c o m p e t i t i v e n e s s (based on pe rcen t r o o t i n f e c t i o n ) o f i n o c u l a n t f u n g i may be i n v e r s e l y p r o p o r t i o n a l to s o i l temperature a t r o u t i n e r e f o r e s t a t i o n s i t e s . The g e n e r a l i t y o f t h i s r e s u l t s h o u l d be t e s t e d w i t h r e p l i c a t i o n over a w i d e r range o f s o i l temperatures and i n a v a r i e t y o f s o i l environments i n c l u d i n g those i n wh ich ind igenous funga l inocu lum i s a t t a ched to l i v i n g p l a n t r o o t s . The ind igenous inoculum i n t h i s s tudy was " f r e e " ( i . e . , no t a t t a c h e d to l i v i n g p l a n t r o o t s ) as would l i k e l y be the case on b a c k l o g r e f o r e s t a t i o n s i t e s . Root c o l o n i z a t i o n by " f r ee" inoculum i s p r o b a b l y more s e n s i t i v e to s o i l temperature than 183 established inoculum; the latte r inoculum has a supply of carbon and does not have to compete with rhizosphere organisms for carbohydrates during the i n i t i a l stages of root colonization. It i s often assumed that inoculant fungi w i l l be replaced by indigenous fungi after planting. The results of this study suggest that the replacement process w i l l be slow i n cool s o i l s , and mycorrhizae established i n the nursery w i l l continue to dominate the root systems at least for the f i r s t growing season. This finding has two implications for a r t i f i c i a l inoculation programs. F i r s t , a r t i f i c i a l inoculation with specific fungi has greater potential to influence seedling performance, either positively or negatively, on cool northern or high altitude reforestation sites compared with warmer southern sites. There is greater likelihood that selected fungi w i l l dominate the root system after planting, especially on backlog reforestation sites which may have lower and less predictable amount of indigenous inoculum than recently logged sites or more southern reforestation sites (Danielson 1985). Second, selection of fungi e f f i c i e n t in promoting conifer growth w i l l be more important for outplanting on cool sites than in warmer ones. Mycorrhizae established in the nursery may continue to dominate the root system even though they may not benefit, or even negatively affect, seedlings planted in cool s o i l s . A high p r i o r i t y should be given to examining the efficacy i n cool s o i l s of fungi which naturally infect container-grown spruce seedlings (e.g., Thelephora t e r r e s t r i s . E-strain and Amphinema bvssoides) in cool, wet s o i l s . Although the isolates of E-strain and H. crustuliniforme used in this study met the f i r s t two c r i t e r i a of effective fungi (aggressive i n nursery, persistence in field ) they did not promote the growth of spruce seedlings, at least in the f i r s t three months 184 after outplanting, in cool s o i l s . 6. Superiority of specific inocula Averaged across a l l inocula, the presence of mycorrhizae at the time of transplanting, did not significantly improve the growth of spruce seedlings transplanted into cool s o i l s . This result does not support the premise that the presence of "any" fungus is better than "no" fungus for the particular study conditions, i.e., non-saturating l i g h t intensity, cool moist acidic forest s o i l with good potential for root colonization by indigenous ectomycorrhizal fungi. On average, mycorrhizal infection increased seedling nutrition and thus the water-use efficiency of mature white spruce needles. This suggests that "any" fungus might be better than "none" i n cool, dry soil s or on sites with a high evapotranspiration potential. The controlled environment seedlings were subject to less moisture stress compared to outplanted spruce seedlings in northern B.C. Mid-day xylem pressure potentials of approximately -1.6 MPa have been recorded for outplanted white spruce seedlings in late spring and early summer (Binder et a l . 1987), whereas mid-day xylem pressure potentials in this study were above -1.0 MPa. Study results do support the second premise of a r t i f i c i a l inoculation programs, i.e., some species of ectomycorrhizal fungi on certain sites are more beneficial than other fungi. The findings suggest mycorrhizal infections established in the nursery w i l l influence, both negatively and positively, the f i e l d performance of spruce seedlings planted i n cool soils (6-12°C). It i s important to recognize that establishment of forest s o i l fungi in the nursery benefited seedling growth after transplanting into cool so i l s even though non-inoculated seedlings were rapidly infected by forest s o i l fungi after transplanting. Inoculation treatments (e.g., 185 forest floor, T. terrestris and L. bicolor') which most benefited total seedling biomass also increased the proportion of assimilated carbon invested i n new photosynthetic capacity, suggesting that the growth benefits would continue into a second growing season. Forest s o i l inoculum should be considered as an alternative to pure culture inoculum, i f funding and technology is not available to investigate the efficacy of specific native ECM fungi. Seedlings inoculated with forest s o i l may benefit from the greater diversity of ectomycorrhizal fungi and rhizosphere organisms contained in s o i l inoculum compared to pure culture inoculum (Perry et a l . 1987). However, there are several disadvantages to forest s o i l inoculum: (a) the need to handle large volumes of forest s o i l , (b) the risk of introducing soil-borne pathogens into container nurseries (c) the lack of control over the specific fungi forming mycorrhizae and (d) the a b i l i t y of forest s o i l inoculum from cool sites to aggressively colonize roots i n a warm container environment. The operational problems associated with handling forest s o i l may be i t s major limitation as an inoculum source. I am not aware of any published reference documenting the introduction of pathogens with forest s o i l inoculum. In fact, forest s o i l may inhibit pathogens in nurseries, especially when the growing medium is relatively s t e r i l e ^ . Smith (1967) observed that Fusarium infection of pine seedlings declined after the seedlings were transplanted into forest s o i l . If a diverse population of ectomycorrhizal fungi is advantageous, the operational problems of applying forest s o i l need to be weighed against the d i f f i c u l t y of achieving mixed-species root infection from pure culture or spore inocula. ^ Personal communication with J. Sutherland, Forestry Canada, Victoria, B.C. 186 Forest s o i l inoculum co l o n i z e d roots w e l l i n 16 and 26°C peat : v e r m i c u l i t e mixes. However, i t i s important to note that i t was a p p l i e d to 8-week-old germinants. In an o p e r a t i o n a l nursery, f o r e s t s o i l would l i k e l y be added to the growing mixture p r i o r to sowing, and the inoculum would have to remain v i a b l e u n t i l germinants developed short r o o t s . Further study i s needed to determine the degree of root i n f e c t i o n that could be expected i f f o r e s t s o i l was a p p l i e d under these c o n d i t i o n s . Of the fungal i s o l a t e s studied. L. b i c o l o r and T. t e r r e s t r i s appear to be most promising f o r f u r t h e r t e s t i n g i n c o o l , a c i d i c moist f o r e s t s o i l s . Although not too much should be i n f e r r e d from the use of one i s o l a t e of each species, nevertheless these fungi were easy to e s t a b l i s h i n c u l t u r e and i n the nursery, s t r o n g l y c o l o n i z e d new roots i n c o o l s o i l s and promoted s e e d l i n g growth i n c o o l s o i l s . L. b i c o l o r i s a p a r t i c u l a r l y promising ectomycorrhizal fungus f o r a r t i f i c i a l i n o c u l a t i o n and i t s b a s i c physiology and genetics are being researched at the U n i v e r s i t e Laval (Kropp and F o r t i n 1988). This information w i l l l ead to the breeding of superior s t r a i n s of L. b i c o l o r . In North America, T. t e r r e s t r i s i s often considered a "weed fungus" which contaminates i n o c u l a t i o n experiments and n a t u r a l l y i n f e c t s c o n i f e r seedlings i n both bare-root and container n u r s e r i e s . T. t e r r e s t r i s had proven l e s s e f f e c t i v e than other fungi, e s p e c i a l l y P i s o l i t h u s t i n c t o r i u s . on p l a n t i n g s i t e s with a s o i l moisture d e f i c i t (Marx and C o r d e l l 1987). However, T. t e r r e s t r i s may be b e t t e r adapted to a c i d i c poorly-drained f o r e s t s o i l s than are other ectomycorrhizal fungi i n c l u d i n g I\_ t i n c t o r i u s (Cruz 1974, Thomas and Jackson 1983, Wilson et a l . 1987). In t h i s study, T. t e r r e s t r i s i n f e c t i o n s i g n i f i c a n t l y increased n i t r o g e n uptake even i n the 6°C s o i l . T. t e r r e s t r i s occurs over a wide range of tree ages and has good 187 potential to persist on outplanted seedlings (Thomas e_t a l . 1983, Wilson et a l . 1987). Thomas et a l . (1983) observed T. terrestris mycorrhizae on nursery, 4- and 50-year-old outplanted Sitka spruce. 6. Selecting e f f i c i e n t mycorrhizal fungi The efficacy of a particular ectomycorrhizal fungus w i l l depend on three factors: (1) i t s aggressiveness i n conifer nurseries (degree of root colonization), (2) i t s survival and colonization of new roots after planting and (3) i t s inherent a b i l i t y to benefit the host plant (Trappe 1977) . The main findings of this study emphasize the importance of gathering s i t e - s p e c i f i c information on mycorrhiza efficacy. The well-established principle that forestry practices must be based on site-specific information must be extended to the mycorrhizal symbiosis. The effect of slow-release f e r t i l i z e r on persistence of the E-strain isolate also points to the need to select specific fungi that interact favorably with s i l v i c u l t u r a l regimes. In both the 6 and 12°C s o i l s , inoculant fungi continued to be the dominant fungi colonizing new roots for the three months after transplanting. Persistence on new roots was positively correlated with the percentage of the container root plug colonized by the inoculant fungi before transplanting, emphasizing the importance of Trappe's f i r s t c r i t e r i o n of efficacy, i.e., fungi selected for a r t i f i c i a l inoculation programs must be able to colonize roots aggressively in conifer nurseries. The a b i l i t y of established mycorrhizae to infect new roots formed after nursery-grown seedlings are planted into cool soils should be used as a screening variable when selecting potential fungal isolates for a r t i f i c i a l inoculation of high altitude or latitude reforestation sites. 188 Reforestation of European sub-alpine forests has been conducted using "low temperature" strains of ectomycorrhizal fungi (Moser 1958). Studies of the temperature relations of naturally occurring ectomycorrhiza of Betula spp. growing on mine reclamation sites (Ingleby et a l . 1985), suggest that spring and f a l l temperatures are more important to root colonization than are summer temperatures. These data should be collected i n forest so i l s with indigenous microflora since the temperature response of fungi varies with the biological and chemical properties of the growing medium (Theodorou and Bowen 1971, Marx 1981). Differences between mycorrhizal fungi cannot be predicted from their growth in culture (Ingleby et a l . 1985). It is important to distinguish between the effects of s o i l temperature on primary and secondary infection processes. Primary infection of roots from "free" inoculum was inhibited more by cool s o i l temperatures than was secondary infection of the root system from mycorrhizal fungi already established on the root system. For example, neither T. terrestris isolate formed mycorrhizae in the 6°C peat:vermiculite mix, suggesting that this fungus may not colonize roots well in cool forest s o i l . However, established infections of T. terrestris successfully colonized new root growth of cold-stored seedlings transplanted into 6°C s o i l . The main findings of the study showed that the f i r s t two c r i t e r i a are appropriate only when the third c r i t e r i o n i s sati s f i e d . In this study, established H. crustuliniforme and E-strain fungi survived and colonized new roots in 6 and 12°C so i l s but did not promote the growth or nutrition of white spruce seedlings at these temperatures, supporting the conclusion of Marx et a l . (1970) that ectomycorrhizal fungi do not benefit host plant 189 growth over the entire range of s o i l temperatures at which they form mycorrhizae. Marx and coworkers hypothesized that the physiological response of the host plant to particular s o i l temperatures determined whether a mycorrhizal symbiosis would be beneficial. White spruce seedlings did benefit from infection by other fungi (e.g., L. bicolor) in cool s o i l s , indicating that interactions between the host plant and specific fungi are equally, or more important, than host plant physiology alone, i n determining the benefits derived from the symbiosis in cool s o i l s . If E-strain and H. crustuliniforme had been less aggressive, i n the nursery and after transplanting, allowing greater colonization by the indigenous fungal population, their depressive effect on seedling growth may have been less pronounced. Aggressive strains of fungi established i n the nursery may impose an excessive carbon drain on outplanted seedlings (Stenstrom et a l . 1990), particularly when carbon assimilation i s restricted by low nutrient a v a i l a b i l i t y , droughty or cool s o i l s . In addition, they can inhibit the colonization of new roots by a diversity of indigenous fungi adapted to the planting environment (Stenstrom et a l . 1990). Implications for Forestry Research These w i l l be disussed under three broad areas. The f i r s t focuses on the need to broaden the prevailing concept of the mycorhizal symbiosis to include a wider range of host response. The second discusses the kinds of information reported i n mycorrhizal studies, the methods used to assess mycorrhizae and the implications of the dissertation results to other areas of physiological research. The third area of concern is the current 190 emphasis on physiological and biochemical investigations of mycorrhizal efficacy. 1. Need for more detailed investigation of the f u l l range of host  response to mycorrhizal infection The mycorrhizal symbiosis is commonly viewed as a mutualistic relationship between a fungus and host plant i n which both partners benefit. It is commonly accepted that the mycorrhizal symbiosis is a good one in nature. However, the detrimental effects of specific fungi observed in this study are not without precedent. In their review of the mycorrhizal literature, Harley and Smith (1983) found published and unpublished reports documenting cases where the host plant did not benefit and even those where the growth of the host plant was depressed. Harley and Smith (1983) and Wilcox (1983) emphasized the need to examine the fungus-host plant relationship in a broader context using the original definition of a symbiotic relationship. This definition includes (1) relationships i n which one partner benefits but the other neither benefits nor loses (commensalism), and (2) those in which one partner benefits at the expense of the other (parasitism). Harley (1969) hypothesized that the degree of benefits to the host plant depended on the balance between two factors (1) the benefit of improved nutrition and (2) the cost of carbon use by the fungal partner. Environmental conditions appear to influence the benefits accruing to each partner i n the symbiosis. Growth depressions are most l i k e l y to occur in low ligh t conditions and in very low or high nutrient regimes (Hatch 1937, Trappe 1977, Harley and Smith 1983 and references therein) where the benefits of improved nutrition may note compensate for the carbon cost of 191 the symbiosis. In low light environments (e.g., conifer plantation with brush competition), the rate of net photosynthesis may be too low to compensate for the carbon demand of mycobionts. This also may be the case when net photosynthesis and nutrient a v a i l a b i l i t y are reduced by cool s o i l temperatures. On average, mycorrhizal infection did not improve nutrient uptake in the 6°C s o i l but did in the 12°C s o i l . Researchers have tended to ignore reports of negative or no host plant response to mycorrhizal infection. Harley and Smith (1983, p. 186) noted: "Results of this kind which show no effect of mycorrhizal infection or even a decrease in growth rate may be found scattered through the literature but are not often stressed." More rigorous investigation of the effects of environment on carbon and nutrient exchanges between symbionts, and on the degree of host plant response to infection, are essential i n order to estimate the potential benefits of mycorrhizal infection and to apply mycorrhizal technology to particular reforestation problems. Identification of environments or conditions which are l i k e l y to reduce host plant response to infection provides focus for a r t i f i c i a l inoculation programs. Selection of specific fungi for these conditions is potentially rewarding. The results show that although some fungi were detrimental i n the experimental conditions (cool, acidic, moist s o i l s ) ; others were very beneficial, increasing spruce seedling growth by 20 to 30%. 2. Experimental technique When I began comparing my results to other studies, I quickly realized that published results of f i e l d and controlled environment studies 192 (including my own) would be more valuable to other workers i f they included descriptions of the i n i t i a l nutritional status of seedlings, the relative growth rates of the various inoculation treatments, and the environmental conditions of the experiment. It is misleading to generalize about the benefits of a specific mycorrhizal fungus to a particular host plant without describing the nutritional status of the seedlings and the environmental conditions (e.g., acidity, temperature, f e r t i l i t y , moisture status of s o i l ) of the study. I n i t i a l size differences are usually reported. However, the results of this study do not support the contention of Mexal (1980) that the effects of various inoculation treatments on the survival and growth of outplanted seedlings are primarily due to differences i n the i n i t i a l size of seedlings. Equally important to growth in this study were i n i t i a l nutrient contents and distribution of nutrients, and relative growth rates after transplanting. Reliable assessments of mycorrhiza formation are an integral part of mycorrhizal studies. At least for small spruce seedlings, visual or low power magnification assessments may not accurately c l a s s i f y infected and uninfected short roots. At low power magnification, short roots of spruce seedlings with a Hartig net but lacking a well-developed mantle may be c l a s s i f i e d as non-mycorrhizal. Formation of the Hartig net, which is the diagnostic c r i t e r i a for mycorrhiza formation, preceded mantle formation. The early stages of infection, have been shown to influence host plant physiology (Nylund and Unestam 1982) and their detection is important to understanding the mycorrhizal symbiosis. Gross characteristics (e.g., lack of root hairs, distinctive colours, branching and swelling) commonly used to identify infected roots visually or at low power magnification can be found on non-mycorrhizal root systems (Nylund and Unestam 1982) and are not 193 reliable indicators of mycorrhiza formation. Finally, the results of this study have implications for any study of seedling physiology. Inoculation treatments accounted for a significant proportion of the v a r i a b i l i t y i n physiogical parameters (e.g., net photosynthetic rate, xylem pressure potential) especially in the f i r s t five weeks of the experiment. It is important for physiologists to be aware of this source of v a r i a b i l i t y and i t s influence on physiological parameters when they select "uniform" populations of seedlings for experiments and when they interpret physiogical responses. 3. Direction for future research The inconsistent results of a r t i f i c i a l inoculation experiments reflects many factors including differences i n (1) the physiology of ectomycorrhizal fungi (Harley and Smith 1983), (2) the adaptation of fungal symbionts to the environment of the planting site (Trappe 1977, Parke 1985), and (3) the i n a b i l i t y of inoculant fungi to persist i n the f i e l d in the presence of indigenous inoculum (Trappe 1977) or fungal grazers (Fitter 1985). Research on mycorrhizal efficacy has focused on the physiology of mycorrhizae in culture or in symbiosis (usually excised mycorrhizae) with the aim of discovering the characteristics which cause one fungal species or strain to be more effective than others in promoting the growth or nutrient uptake of host plants. One long-term goal of a r t i f i c i a l inoculation research is to select and breed fungi which have superior a b i l i t i e s to promote host plant growth. Knowledge of the biochemical and physiological attributes which endow fungi with superior efficacy is considered essential to achieve this goal (Harley 1985). 194 So far, physiological research has not revealed the underpinnings of efficiency. In a review of past mycorrhizal research, Harley (1985, p. 28) concludes: "But regardless of more than 50 years of research on their cultural behavior and growth physiology we do not know, at a l l , what properties an e f f i c i e n t mycorrhizal fungus should possess. Although we know that they vary i n effectiveness both between species and between variants of a single species, i t is clear that we have not yet asked the right questions." Harley (1985) recommends asking more questions in the areas of biochemistry and intermediate physiology of mycorrhizal fungi. I wonder, however, i f these questions are the most "profitable" ones to meet the stated applied objectives of selecting superior strains of fungi. F i r s t , physiological and biochemical characteristics (e.g., high rates of hormone, acid phosphatase synthesis) may not be useful c r i t e r i a for selecting fungi. The rates of physiological processes, such at photosynthetic rate, are often poorly correlated with plant productivity; plant productivity is more dependent on the amount of photosynthetic capacity (leaf area) and the length of time i t is functional (Hunt 1982). Second, breeding for specific physiological characteristic may not be a desirable goal unless the host plant lives in a physical and biological environment which is uniform i n time and space. Jones (1983) cautioned that breeding for specific attributes, such as drought resistance, is risky for plants grown in fluctuating environments. Breeding for lower hydraulic conductivity, for example, w i l l improve water conservation in droughty years but w i l l not be beneficial to plant growth in years when water is adequate. Third, differences i n efficacy between ectomycorrhizal fungi are just as l i k e l y to result from difference in adaptation to different environments 195 as from differences i n fungal physiology. We know that there is considerable ecological variation within and between fungal species and we have the opportunity to select fungi which are adapted to a broad range or particular s o i l environments. Although i t is recognized that i t is essential to select fungi that are adapted to particular environments (Trappe 1977, Harley and Smith 1983, Perry et a l . 1987), there has been l i t t l e research directed to studying the mycorrhizal symbiosis in different s o i l environments or the efficacy of specific ectomycorrhizal fungi i n different s o i l environments. As a result: "we are s t i l l i n the "Dark Ages" with regard to applying these features of ecological specialization to agriculture, forestry or i n restoring native vegetation" (Parke 1985, p. 107). Given the relatively small amount of research directed to the ecology of mycorrhizal symbiosis, i t can be argued that questions concerning ecology may have more potential to improve our understanding of efficacy and the selection of superior fungi than those concerned with the biochemistry or physiology of mycorrhizal fungi. Fie l d studies of the seasonal periodicity of mycorrhiza development, root and shoot growth of conifer seedlings would improve our understanding (1) of the processes affecting persistence of inoculant fungi after planting and (2) the interactions between fungal and host plant development and mycorrhizal symbioses. 1 9 6 REFERENCES Abbott, L.K., and Robson, A.D. 1984. Assessing the potential for widescale VA mycorrhizal inoculation. In Molina, R. (ed.) Proceedings of the Sixth North American Conference on Mycorrhizae, June 25-29, 1984, Bend. OSU Forest Research Laboratory, Corvallis, Oregon. Alexander, I.J. 1981. Picea sitchensis and Lactarius rufus mycorrhizal association and i t s effects on seedling growth and development. Trans. Br.. Mycol. Soc. 76:417-423. Alexander, I.J., and Fairley, R.I. 1983. Effects of N f e r t i l i s a t i o n on populations of fine roots and mycorrhizas i n spruce humus. Plant Soil 71:49-53. Alexander, I.J., and Fairley, R.I. 1986. Growth and nitrogen uptake rates of ectomycorrhizal spruce seedlings. In Gianinazzi-Pearson, V., and Gianinazzi, S. (eds.) Mycorrhizae:physiology and genetics. Proc. 1st ESM, Dijon, 1-5 July. INRA, Paris, pp.377-382. Allen, M.F., Smith, W.K., Moore Jr., T.S., and Christensen, M. 1981. Comparative water relations and photosynthesis of mycorrhizal and non-mycorrhizal Bouteloua gr a c i l i s H.B.K. Lag ex Steud. New Phytol. 88:683-693. Amaranthus, M.P., and Perry, D.A. 1987. Effect of s o i l transfer on ectomycorrhiza formation and the survival and growth of conifer seedlings on old, nonreforested clearcuts. Can. J. For. Res. 17:944-950. Amaranthus, M.P., L i , CY., and Perry, D.A. 1990. Influence of vegetation type and madrone s o i l inoculum on associative nitrogen fixation in Douglas-fir rhizospheres. Can. J. For. Res. 20:368-371. Andersen, CP., Markhart III, A.H. , Dixon, R.K. , and Sucoff, E.I. 1988. Root hydraulic conductivity of vesicular-arbuscular mycorrhizal green ash seedlings. New Phytol. 109:465-471. Andersen, CP., Sucoff, E.I., and Dixon, R.K. 1986. Effects of root zone temperature on root i n i t i a t i o n and elongation in red pine seedlings. Can.J.For.Res. 16:696-700. Anderson, J.E., and McNaughton, S.J. 1973. Effects of low s o i l temperature on transpiration, photosynthesis, leaf relative water content, and growth among elevationally diverse plant populations. Ecology 54:1220-1233. Atkin, R.K., Barton, G.E., and Robinson, D.K. 1973. Effect of root growing temperature on growth substances i n xylem exudate in Zea mays. J. Exp. Bot. 24:475-487. 197 Babalola, 0., Boersma, L., and Youngberg, C.T. 1968. Photosynthesis and transpiration of Monterey pine seedlings as a function of s o i l water suction and s o i l temperature. Plant Physiol. 43:515-521. Ballard, T.M. 1985. Nutritional demands of planted spruce. In Proceedings, Interior spruce seedling performance: state of the art. Northern Silviculture Committee Meeting, February 1985, Prince George, B.C. Ballard, T.M., and Carter, R.E. 1986. Evaluating forest stand nutrient status. B.C. Min. For., Land Manage. Rep. No. 20. Victoria, B.C. Bancroft, T.A. 1964. Analysis and inference for incompletely specified models involving the use of preliminary test(s) of significance. Biometrics 20:427-442. Barber, S.A. 1984. Soi l Nutrient Bioavailability. John Wiley and Sons Inc., New York. Barney, CW. 1951. Effects of s o i l temperature and light intensity on root growth of l o b l o l l y pine seedlings. Plant Physiol. 26:146-163. Beadle, C.L. , Neilson, R.E., Jarvis, P.C, and Talbot, H. 1981. Photosynthesis as related to xylem water potential and carbon dioxide concentration in Sitka spruce. Physiol. Plant. 52:391-400. Beaton, J.D., Moss, A., MacRae, I., Konkin, J.W., McGhee, W.P.T., and Kosick, N. 1965. Observations on foliage nutrient content of several coniferous tree species i n Br i t i s h Columbia. For. Chron. 41:222-236. Benecke, U., and Gobi, F. 1974. The influence of different mycorrhizae on growth, nutrition and gas-exchange of Pinus mugo seedlings. Plant Soil 40:21-32. Benzian, B., and Smith, H.A. 1973. Nutrient concentrations of healthy seedlings and transplants of Picea sitchensis and other conifers grown in English forest nurseries. Forestry 46:55-69. Benzian, B., Brown, R.M., and Freeman, S.C.R. 1974. Effect of late-season top-dressings of N (and K) applied to conifer transplants in the nursery on their survival and growth on Br i t i s h forest sites. Forestry 47:153-184. Bethlenfalvay, G.J., Brown, M.S., and Newton, W.E. 1987. Photosynthetic water- and nutrient-use efficiency in a mycorrhizal legume. In Sylvia, D.M., Hung, L.L., and Graham, J.J. (eds.). Mycorrhizae in the next decade-practical applications and research p r i o r i t i e s . 7th NACOM, 3-8 May, 1987. Gainesville, Florida. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. pp. 231-233. Binder, W.D., Spittlehouse, D.L., and Draper, D.A. 1987. Post Planting studies of the physiology of white spruce 1984-1985. Progress Report No. 5, E.P. 966, Research Branch, Ministry of Forests, B.C. 198 Bjorkman, E. 1949. The ecological significance of the ectotrophic mycorrhizal association i n forest trees. Sv. Bot. Tidskr. 43:223-262. (I was unable to see this a r t i c l e and r e l i e d on Mikola 1965 for information on Bjorkman) Bjorkman, E. 1962. The influence of ectotrophic mycorrhiza on the development of forest tree plants after planting. Proceedings of the 13th International Union Forest Research Organization, 1961. Part 2. Vol. 1. Sect. 24-1. (I was unable to obtain this reference and have re l i e d on Kropp and Langlois 1990 for information on Bjorkman) Black, CH. 1986. The effects of mycorrhizal colonization and phosphorus f e r t i l i z a t i o n on Douglas-fir seedling growth, morphology, and photosynthesis. Ph.D. Thesis. S o i l Science Dept., Oregon State University, Corvallis, Oregon. Blake, T.J. 1983. Transplanting shock in white spruce; effect of cold storage and root pruning on water relations and stomatal conditioning. Physiol. Plant. 57:210-216. Bledsoe, C.S., Tennyson, K., and Lopushinsky, W. 1982. Survival and growth of outplanted Douglas-fir seedlings inoculated with mycorrhizal fungi. Can. J. For. Res. 12:720-723. Borges, R.C, and Chaney, W.R. 1989. Root temperature affects mycorrhizal efficacy in Fraxinus pennsylvanica Marsh. New Phytol. 112:411-417. Bowen, CD. 1970. Effects of s o i l temperature on root growth and on phosphate uptake along Pinus radiata roots. Aust. J. Soil Res. 8:31-42. Bowen, CD. 1973. Mineral nutrition of ectomycorrhizae. In Marks, G.C, and Kozlowski, T.T. (eds.) Physiological Ecology. Academic Press, New York. pp. 151-205. Boyle, CW. , Robertson, W.J., and Salonius, P.O. 1987. Use of mycelial slurries of mycorrhizal fungi as inoculum for commercial tree seedling nurseries. Can. J. For. Res. 17:1480-1486. Brand, D.G. 1987. Estimating the surface area of spruce and pine foliage from displaced volume and length. Can. J. For. Res. 17:1305-1308. Brand, D.C, and Janas, P.S. 1988. Growth and acclimation of planted white pine and white spruce seedlings i n response to environmental conditions. Can. J. For. Res. 18:320-329. Bray, R.H., and Kurtz, L.T. 1945. Determination of total, organic, and available forms of phosphorus in s o i l s . Soil Sci. 59:39-45. Brix, H. 1979. Effects of plant water stress on photosynthesis and survival of four conifers. Can. J. For. Res. 9:160-165. 199 Brix, H. 1981. Effects of nitrogen f e r t i l i z e r source and application rates on f o l i a r nitrogen concentration, photosynthesis, and growth of Douglas-fir. Can. J. For. Res. 11:775-780. Brown, A.C., and Sinclair, W.A. 1981. Colonization and infection of primary roots of Douglas-fir seedlings by the ectomycorrhizal fungus Laccaria  laccata. For. Sci. 27:111-124. Burdett, A.N. 1983. Quality control i n the production of forest planting stock. For. Chron. 59:132-138. Burdett, A.N., Herring, L.J., and Thompson, C.F. 1984. Early growth of planted spruce. Can. J. For. Res. 14:644-651. Butt, G. 1986. Backlog forest land rehabilitation i n the SBS and BWBS zones in the northern interior of Br i t i s h Columbia. FRDA Report No.023. Camm, E.L., and Harper, G.J, [1991]. Temporal variations in cold sen s i t i v i t y of root growth in cold-stored white spruce seedlings. University of Br i t i s h Columbia. Submitted for publishing. Carlson, W.C. 1986. Root system considerations in the quality of l o b l o l l y pine seedlings. South. J. Appl. For. 10:87-92. Castellano, M.A., and Molina, R. 1989. Mycorrhizae. In Landis, T.D., Tinus, R.W., McDonald, S.E., and Barnett, J.P. (eds.) The Container Tree Nursery Manual, Volume 5. Agric. Handbk. 674. Washington, DC: U.S. Dept. of A g r i c , Forest Service, pp. 101-167. Castellano, M.A., and Trappe, J.M. 1985. Ectomycorrhizal formation and plantation performance of Douglas-fir nursery stock inoculated with Rhizopogon spores. Can. J. For. Res. 15:613-617. Chalupa, V., and Fraser, D.A. 1968. Effect of s o i l and air temperature on soluble sugars and growth of white spruce (Picea glauca) seedlings. Can. J. Bot. 46:65-69. Chapin, F.S., III, and Kedrowski, R.A. 1983. Seasonal changes in nitrogen and phosphorus fractions and autumn retranslocation in evergreen and deciduous taiga trees. Ecology 62:376-391. Chapin, F.S.,III, Bloom, A.J., Field, C.B., and Waring, R.H. 1987. Plant responses to multiple environmental factors. Bioscience 37:49-57. Chilvers, G.A. 1968. Some distinctive types of eucalypt mycorrhiza. Aust. J. Bot. 16:49-70. Chu-Chou, M. 1979. Mycorrhizal fungi of Pinus radiata in New Zealand. Soil Biol. Biochem. 11:557-562. Chu-Chou, M. 1985. Effect of different mycorrhizal fungi on Pinus radiata seedling growth. In Molina, R. (ed.) Proceedings 6th NAC0M, June 25-29, 1984, Bend, Oregon. Oregon State University, Forest Research Laboratory, Corvallis, OR. p.208. 200 Chu-Chou, M., and Grace, L.J. 1981. Mycorrhizal fungi of Pseudotsuga menziesii i n the North Island of New Zealand. Soil Biol, and Biochem. 13:247-249. Cline, M.L., and Reid, CP.P. 1982. Seed source and mycorrhizal fungus effects on growth of containerized Pinus contorta and Pinus ponderosa seedlings. For. Sci. 28:237-250. Cline, M.L., France, R.C and Reid, CP.P. 1987. Intraspecific and interspecific growth variation of ectomycorrhizal fungi at different temperatures. Can. J. Bot. 65:869-875. Coleman, M., Bledsoe, CS., and Smit-Spinks, B. 1987. Ectomycorrhizae decrease Douglas-fir root hydraulic conductivity. In Sylvia, D.M., Hung, L.L., and Graham, J.H. (eds.) Proceedings 7th NACOM, May 3-8, 1987, Gainesville, FL. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. p.243. Coleman, M.D., Bledsoe, C.S., and Smit, B.A. 1990. Root hydraulic conductivity and xylem sap levels of zeatin riboside and abscisic acid i n ectomycorrhizal Douglas f i r seedlings. New Phytol. 115:275-284. Colombo, S.J., and Asselstine, M.F. 1989. Root hydraulic conductivity and root growth capacity of black spruce (Picea mariana) seedlings. Tree Physiol. 5:73-81. Cooper, A.J. 1973. Root temperature and plant growth. Res. Rev. N. 4, Commonwealth Bureau of Horticulture and Plantation Crops, East Mailing Kent, England. Coutts, M.P., and N i c o l l , B.C. 1990a. Growth and survival of shoots, roots, and mycorrhizal mycelium in clonal Sitka spruce during the f i r s t growing season after planting. Can. J. For. Res.20:861-868. Coutts, M.P., and N i c o l l , B.C. 1990b. Waterlogging tolerance of roots of Sitka spruce clones and of strands from Thelephora terrestris mycorrhizas. Can. J. For. Res. 20:1894-1899. Crowley, D.E., Maronek, D.M., and Hendrix, J.W. 1986. Effect of slow release f e r t i l i z e r s on formation of mycorrhizae and growth of container grown pine seedlings. J. Environ. Hort. 4:97-101. Cruz, R.E. de l a . 1974. The effects of mycorrhizal treatments and nitrogen-phosphorous f e r t i l i z a t i o n on f i e l d growth of slash pine seedlings. Ph.D. Thesis, University of Florida [Abstract]. Dissertation Abstracts International, B (1975) 35(8) 4052. Dahlberg, A. 1990. Effect of s o i l humus cover on the establishment and development of mycorrhiza on containerised Pinus sylvestris L. and Pinus contorta ssp. l a t i f o l i a Englem. after outplanting. Scand. J. For. Res. 5:103-112. 201 Danielson, R.M. 1982. Taxonomic a f f i n i t i e s and c r i t e r i a for identification of the common ectendomycorrhizal symbiont of pines. Can. J. Bot. 60:7-18. Danielson, R.M. 1984a. Ectomycorrhiza formation by the operculate discomycete Sphaerosporella brunnea (Pezizales). Mycologia 76:454-461. Danielson, R.M. 1984b. Ectomycorrhizal associations in jack pine in northeastern Alberta. Can. J. Bot. 62:932-939. Danielson, R.M. 1985. The ectomycorrhizal status of white spruce seedlings transplanted from a bareroot nursery to a clearcut with three site preparation treatments. Rep. Project EP 966, Victoria, B.C: B.C. Ministry of Forests. 10 pp. Danielson, R.M., and Pruden, M. 1989. The ectomycorrhizal status of urban spruce. Mycologia 81:335-341. Danielson, R.M., and Visser, S. 1984. Mycorrhizal status of container-grown conifers in the Pine Ridge Provincial Nursery. Annual report submitted to Research Management Division of Alberta Environment, Edmonton. Danielson, R.M., and Visser, S. 1988. Ectomycorrhizae of jack pine and green alder: assessment of the need for inoculation, development of inoculation techniques and outplanting t r i a l s on o i l sand tai l i n g s . Report RRTAC 88-5, Alberta Land Conservation and Reclamation Council, Edmonton. Danielson, R.M., and Visser, S. 1989. Host response to inoculation and behavior of introduced and indigenous ectomycorrhizal fungi of jack pine grown on oil-sands tai l i n g s . Can. J. For-. Res . 19 :1412-1421. Danielson, R.M., and Visser, S. 1990. The mycorrhizal and nodulation status of container-grown trees and shrubs reared in commercial nurseries. Can. J. For. Res. 20:609-614. Danielson, R.M., G r i f f i t h s , C.L., and Parkinson, D. 1984a. Effects of f e r t i l i z a t i o n on the growth and mycorrhizal development of container-grown jack pine seedlings. For. Sci. 30:828-835. Danielson, R.M., Visser, S., and Parkinson, D. 1984b. The effectiveness of mycelial slurries of mycorrhizal fungi for the inoculation of container-grown jack pine seedlings. Can. J. For. Res. 14:140-142. Danielson, R.M., Visser, S., and Parkinson, D. 1984c. Production of ectomycorrhizae on container-grown jack pine seedlings. Can. J. For. Res. 14:33-36. Danielson, R.M. , Zak, J.C, and Parkinson, D. 1984d. Mycorrhizal inoculum in a peat deposit formed under a white spruce stand i n Alberta. Can. J. Bot. 63:2557-2560. 202 Daughtridge, A.T., Pallardy, S.G., Garrett, H.G. and Sander, I.L. 1986. Growth analysis of mycorrhizal and nonmycorrhizal black oak ("Quereus  velutina Lam.) seedlings. New Phytol. 103:473-479. Davidson, R.L. 1969. Effect of root/leaf temperature differentials on root/shoot ratios in some pasture grasses and clover. Ann. Bot. 33:561-569. Davis, R.M., and Lingle, J.C. 1961. Basis of shoot response to root temperature i n tomato. Plant Physiol. 36:153-162. Day, R.J., and MacGillivray, G.R. 1975. Root regeneration of f a l l - l i f t e d white spruce nursery stock in relation to s o i l moisture content. For. Chron. 75:196-199. Delucia, E.H. 1986. Effect of low root temperature on net photosynthesis, stomatal conductance and carbohydrate concentration i n Engelmann spruce (Picea engelmannii Parry ex Engelm.) seedlings. Tree Physiol. 2:143-154. Dennis, J. 1985. Effect of pH and temperature on in vitro growth of ectomycorrhizal fungi. Canadian Forestry Service, Pacific Forestry Centre Info. Rep. BC-X-273. Dobbs, R.C. 1976. Effect of i n i t i a l mass of white spruce and lodgepole pine planting stock on f i e l d performance in the Br i t i s h Columbia interior. Can. For. Sci. Centre, Victoria, B.C. BC-X-149. Dobbs, R.C, and McMinn, R.C. 1977. Effects of scalping on s o i l temperature and growth of white spruce seedlings. 6th B.C. Soil Science Workshop, B.C. Min. A g r i c , Victoria, pp. 66-73. Dosskey, M.C, Linderman, R.C, and Boersma, L. 1990. Carbon-sink stimulation of photosynthesis in Douglas f i r seedlings by some ectomycorrhizas. New Phytol. 115:269-274. Eckblad, F.-E. 1968. The genera of the operculate discomycetes- a re-evaluation of their taxonomy, phylogeny and nomenclature. Norw. J. Bot. 15:1-184. Edgington, E.S. 1987. Randomization tests. Marcel Dekker Inc., New York. Edmonds, A.S., Wilson, J.M., and Harley, J.L.. 1976. Factors affecting potassium uptake by beech mycorrhizas. New Phytol. 76:307-315. Eg l i , S., and Kalin, I. 1985. Dynamics of mycorrhizae after inoculation in non-disinfected nursery s o i l s . In R. Molina (ed.) Proceedings of the 6th North American Conference on Mycorrhizae, June 25-29, 1984, Bend, Oregon, U.S.A. Forest Research Laboratory, Oregon State University, Corvallis, p. 226. Ehret, D.L., and J o l l i f f e , P.A. 1985. Leaf injury to bean plants grown in carbon dioxide enriched atmospheres. Can. J. Bot. 63:2015-2020. 203 Eisenhart, C. 1947. The assumptions underlying the analysis of variance. Biometrics 3:1-21. Ek, M., Ljungquist, P.O., and Stenstrom, E. 1983. Indole-3-acetic acid production by mycorrhizal fungi determined by gas chromatography-mass spectrometry. New Phytol. 94:401-407. Ekwebelam, S.A., and Reid, CP.P. 1983. Effect of light, nitrogen f e r t i l i z a t i o n , and mycorrhizal fungi on growth and photosynthesis of lodgepole pine seedlings. Can. J. For. Res. 13:1099-1106. Elfving, D.C, Kaufmann, M.R. , and Hall, A.E. 1972. Interpreting leaf water potential measurements with a model of the soil-plant-atmosphere continuum. Physiol. Plant. 27:161-168. Eriksson, J., and Ryvarden, L. 1973. The Corticiaceae of north Europe. Vol. 2. Aleurodiscus-Confertobasidium. Fungiflora, Oslo. (I was unable to see this a r t i c l e and re l i e d on Danielson et a l . 1984d for information on this a r t i c l e . ) Farr, W.A., Smith, H.A., and Benzian, B. 1977. Nutrient concentrations in naturally regenerated seedlings of Picea sitchensis in Southeast Alaska. Forestry 50:103-112. Field, C , Merino, J., and Mooney, H.A. 1983. Compromises between water-use efficiency and nitrogen-use efficiency in five species of California evergreens. Oecologia 60:384-389. Fife, D.N., and Nambiar, E.K.S. 1984. Movements of nutrients i n radiata pine i n relations to the growth of shoots. Ann. Bot. 54:303-314. Fiscus, E.L. 1981. Effects of abscisic acid on the hydraulic conductance and the total ion transport through Phaseolus root systems. Plant Physiol. 68:169-174. Fitt e r , A.H. 1985. Functioning of vesicular-arbuscular mycorrhizas under f i e l d conditions. New Phytol. 99:257-265. Fleming, L.V., Deacon, J.W., Last, F.T., and Donaldson, S.J. 1984. Influence of propagating s o i l on the mycorrhizal succession of birch seedlings transplanted to a f i e l d s i t e . Trans. Br.. Mycol. Soc. 82:707-711. Furlan, V., and Fortin, J.A. 1973. Formation of endomycorrhizae by Endogone  calospora on Allium cepa under three different temperature regimes. Nat. Can. (Que.) 100:467-477. Gagnon, J., Langlois, C C , and Fortin, J.A. 1987. Growth of containerized jack pine seed inoculated with different ectomycorrhizal fungi under a controlled f e r t i l i z a t i o n schedule. Can. J. For. Res. 17:840-845. Garbaye, J. 1983. Premiers resultats de recherches sur l a competitivite des champignons ectomycorhiziens. Plant Soil 71:303-308. 204 Garbaye, J. 1986. Effects of mycorrhizal status on bud-break of beech and oak seedlings. In Gianinazzi-Pearson, V., and Gianinazzi, S. (eds.) Mycorrhizae:physiology and genetics. Proc. 1st ESM, Dijon, 1-5 July. INRA, Paris, pp.493-496). Garbaye, J., and Wilhelm, M.E. 1985. Facteurs limitants et aspects dynamiques de l a mycorhization controlee de Fagus s i l v a t i c a Lin. par Hebeloma crustuliniforme (Bull, ex Saint-Amans) Quel, sur tourbe f e r t i l i s e e . Ann. Sci. For. 42:53-68. Gardner, W.R, and Ehlig, C.F. 1962. Some observations on the movement of water to plant roots. Agron. J. 54:453-456. Godbout, C., and Fortin, J.A. 1985. Synthesized ectomycorrhizae of aspen: fungal genus level of structural characterization. Can. J. Bot. 63:252-262. Goldstein, G.H., Brubaker, L.B., and Hinckley, T.M. 1985. Water relations of white spruce (Picea glauca (Moench) Voss) at tree line i n north central Alaska. Can. J. For. Res. 15:1080-1087. ' Gordon, J.C, and Larson, P.R. 1968. Seasonal course of photosynthesis, respiration, and distribution of C i n young Pinus resinosa trees as related to wood formation. Plant Physiol. 43:1617-1624. Graham, J.H., and Syvertsen, J.P. 1984. Influence of vesicular-arbuscular mycorrhiza on the hydraulic conductivity of roots of two citrus rootstocks. New Phytol. 97:277-284. Grossnickle, S.C 1987. Influence of flooding and s o i l temperature on the water relations and morphological development of cold-stored black spruce and white spruce seedlings. Can. J. For. Res. 17:821-828. Grossnickle, S.C. 1988. Planting stress i n newly planted jack pine and white spruce. 1 Factors influencing water uptake. Tree Physiol. 4:71-83. Grossnickle, S.C, and Blake, T.B. 1985. Acclimation of cold-stored jack pine and white spruce seedlings: effect of s o i l temperature on water relation patterns. Can. J. For. Res. 15:544-550. Grossnickle, S.C, and Reid, CP.P. 1983. Ectomycorrhiza formation and root development patterns of conifer seedlings on a high-elevation mine sit e . Can. J. For. Res. 13:1145-1158. Grossnickle, S.C, and Russell, J.H. 1990. Water movement in yellow-cedar seedlings and rooted cuttings: comparison of whole plant and root system pressurization methods. Tree Physiol. 6:57-68. Guehl, J.M., Mousain, D., Falconnet, C , and Gruez, J. 1990. Growth, carbon dioxide assimilation capacity and water-use efficiency of Pinus pinea L seedlings inoculated with different ectomycorhizal fungi. Ann. Sci. For. 47:91-100. 205 Hackett, C. 1969. A study of the root system of barley: II. Relationships between root dimensions and nutrient uptake. New Phytol. 68:1023-1030. Hacskaylo, E., Palmer, J.G., and Vozzo, J.A. 1965. Effect of temperature on growth and respiration of ectotrophic mycorrhizal fungi. Mycologia 57:748-756. Harley, J.L. 1969. Mycorrhiza and nutrient uptake in forest trees. In Physiology of tree crops. Academic Press, London, pp. 163-179. Harley, J.L. 1985. Mycorrhizal studies: past and future. In Gianinazzi-Pearson, V., and Gianinazzi, S. (ed.) Proceedings of the 1st European Symposium on Mycorrhizae, 1-5 July 1985, Dijon, INRA, pp. 25-33. Harley, J.L., and Smith, S.E. 1983. Mycorrhizal symbiosis. Academic Press, New York. Harley, J.L., and Wilson, J.M. 1959. The absorption of potassium by beech mycorrhizas. New Phytol. 58:281-298. Hatch, A.B. 1937. The physical basis of mycotrophy in Pinus. Black Rock Forest Bulletin No. 6. Cornwall-on-the-Hudson, New York. Haug, I., and Oberwinkler, F. 1987. Some distinctive types of spruce mycorrhizae. Trees 1:172-188. Hayman, S. 1974. Plant growth responses to vesicular-arbuscular mycorrhiza. IV. Effect of lig h t and temperature. New Phytol. 73:71-80. Heninger, R.L., and White, D.P. 1974. Tree seedling growth at different s o i l temperatures. For. Sci. 20:363-367. Hermann, R.K. 1977. Growth and production of tree roots: a review. In Marshall, J.K. (ed.) The belowground ecosystem- a synthesis of plant-associated processes. Range Science Series No. 26, Colorado State University, Fort Collins. Herold, A. 1980. Regulation of photosynthesis by sink a c t i v i t y -the missing link. New Phytol. 86:131-144. Hicks, CR. 1973. Fundamental concepts in the design of experiments. Holt, Rinehart, and Winston, New York. Ho, I. 1987. Comparison of eight Pisolithus tinctorius isolates for growth rate, enzyme activity, and phytohormone production. Can. J. For. Res. 17:31-35. Hogberg, P. 1989. Growth and nitrogen inflow rates in mycorrhizal and non-mycorrhizal seedlings of Pinus svlvestris. For. Ecol. Manage. 28:7-17. 206 Holden, J.M., Thomas, G.W., and Jackson, R.M. 1983. Effect of mycorrhizal inocula on the growth of Sitka spruce seedlings i n different s o i l s . Plant S o i l 71:313-317. Hsiao, T.C. 1973. Plant responses to water stress. Annu. Rev. Plant Physiol. 24:519-570. Hung, L-L. L., and Molina, R. 1986. Temperature and time i n storage influence the efficacy of selected isolates of fungi i n commercially produced ectomycorrhizal inoculum. For. Sci. 32:534-545. Hung, L.-L., and Trappe, J.M. 1983. Growth variation between and within species of ectomycorrhizal fungi i n response to pH in vi t r o . Mycologia 75:234-241. Hunt, G. 1989. Effect of controlled-release f e r t i l i z e r s on growth and mycorrhizae i n container-grown Engelmann spruce. West. J. Appl. For. 4:129-131. Hunt, R. 1973. A method of estimating root efficiency. J. Appl. Ecol. 10:157-164. Hunt, R. 1982. Plant growth curves. Edward Arnold, London. Husted, L., and Barnes, S. 1987. Dieback of container-grown Douglas-fir seedlings. A Contribution Agreement Report for the Canada-British Columbia Forest Resource Development Agreement. Huxley, J.S. 1932. Problems of relative growth. Methuen and Co., London. Ingestad, T. 1959. Studies on the nutrition of forest trees seedlings. II. Mineral nutrition of spruce. Physiol. Plant. 12:568-593. Ingestad, T., Arveby, A.S., and Kahr, M. 1986. The influence of ectomycorrhiza on nitrogen nutrition and growth of Pinus svlvestris seedlings. Physiol. Plant. 68:575-582. Ingleby, K., Last, F.T., and Mason, P.A. 1985. Vertical distribution and temperature relations of sheathing mycorrhizas of Betula spp. growing on coal spoil. For. Ecol. and Manage. 12:279-285. Jarvis, P.G. 1976. The interpretation of the variations in leaf water potential and stomatal conductance found i n canopies in the f i e l d . Philos. Trans. R. Soc. London Ser. B. 273:593-610. Jones, H.G. 1983. Plants and microclimate. Cambridge University Press, Cambridge. Kaufmann, M.R. 1975. Leaf water stress in Engelmann spruce. Plant Physiol. 56:841-844. Kaufmann, M.R. 1977. Soil temperature and drying cycle effects on water relations of Pinus radiata. Can. J. Bot. 55:2413-2418. 207 Koide, R. 1985. The effect of VA mycorrhizal infection and phosphorus status on sunflower hydraulic and stomatal properties. J. Exp. Bot. 36:1087-1098. Kramer, P.J. 1940. Root resistance as a cause of decreased water absorption by plants at low s o i l temperatures. Plant Physiol. 15:63-79. Kramer, P.J., and Bullock, H.C. 1966. Seasonal variations in the proportions of suberized and unsuberized roots of trees i n relation to the absorption of water. Am. J. Bot. 53:200-204. Kramer, P.J., and Kozlowski, T.T. 1979. Physiology of woody plants. Academic Press, New York. Kropp, B.R., and Fortin, J.A. 1988. The incompatibility system and relative ectomycorrhizal performance of monokaryons and reconstituted dikaryons of Laccaria bicolor. Can. J. Bot. 66:289-294. Kropp, B.R., and Langlois, C.-G. 1990. Ectomycorrhizae in reforestation. Can. J. For. Res. 20:438-451. Kropp, B.R., Castellano, M.A., and Trappe, J.M. 1985. Performance of outplanted western hemlock (Tsuga heterophvlla (Raf.) Sarg.) seedlings inoculated with Cenococcum geophilum. Tree Plant. Notes 36:13-16. Kuhns, M.R., Garrett, H.E., Teskey, R.O. and Hinckley, T.M. 1985. Root growth of black walnut trees related to s o i l temperature, s o i l water potential and leaf water potential. For. Sci. 31:617-629. Lamb, R.J. 1979. Factors responsible for the distribution of mycorrhizas of Pinus in eastern Australia. Aust. For. Res. 9:25-34. Langlois, CG. 1983. Recherches fondamentales et appliquees sur les ectomycorhizes des coniferes. Ph.D. thesis, Universite Laval, Ste-Foy. [Reference unavailable, r e l i e d on information from Gagnon et a l . 1987] 32 Langlois, C C , and Fortin, J.A. 1978. Absorption of phosphorus ( P) by excised ectomycorrhizae i n balam f i r (Abies balsamea (L.) Mill) from low concentrations of H2PO4. Nat. Can. (Que.) 105:417-424. Last, F.T., Mason, P.A., and Wilson, J. 1984. Controlled inoculation of Sitka spruce with sheathing (ecto-) mycorrhizal fungi-a commercial experience in 1982. Scot. For. 38:75-77. Last, F.T., Wilson, J., and Mason, P.A. 1990. Numbers of mycorrhizas and growth of Picea sitchensis-what i s the relationship. Proceedings 2nd European Symposium on Mycorrhizae, 14-20 Aug. 1988, Prague. Agric. Ecosyst. and Environ. 28:293-298. 208 Lavender, D.P. 1988. Characterization and manipulation of the physiological quality of planting stock. In Proceedings, 10th North American Forest Biology Workshop, 1988, University of Br i t i s h Columbia. Lavender, D.P., and Overton, W.S. 1972. Thermoperiods and s o i l temperatures as they affect growth and dormancy of Douglas-fir seedlings of different geographic origin. Research Paper 13, Forest Research Laboratory, School of Forestry, Oregon State University. Lavender, D.P., Sweet, G.B., Zaerr, J.B., and Hermann, R.K. 1973. Spring shoot growth i n Douglas-fir may be i n i t i a t e d by gibberellins exportd from the roots. Science (Washington, D.C.) 182:838-839. Lawrence, W.T., and Oechel, W.C. 1983a. Effects of s o i l temperature on the carbon exchange of taiga seedlings. I. Root respiration. Can. J. For. Res. 13:840-849. Lawrence, W.T., and Oechel, W.C. 1983b. Effects of s o i l temperature on the carbon exchange of taiga seedlings. II. Photosynthesis, respiration and conductance. Can. J. For. Res. 13:850-859. Lee, K.J. and Koo, CD. 1983. Inoculation of pines in a nursery with Pisolithus tinctorius and Thelephora terrestris in Korea. Plant Soil 71:325-329. Levisohn, I. 1954. Aberrant root infections of pine and spruce seedlings. New Phytol. 53:284-290. Levy, Y., and Krikun, J. 1980. Effect of vesicular-arbuscular mycorrhiza in Citrus iambhiri water relations. New Phytol. 85:25-31. Levy, Y., Syvertsen, J.P., and Nemec, S. 1983. Effect of drought stress and vesicular-arbuscular mycorrhiza on citrus tranpiration and hydraulic conductivity of roots. New Phytol. 93:61-66. Leyton, L. 1948. Mineral nutrient relationships of forest trees. Forestry Abstracts 9:399-408. Lippu, J., and Puttonen, P. 1989. Effects of s o i l temperature on gas exchange and morphological structure of shoot and root i n 1 yr old Scots pine (Pinus sylvestris L.) seedlings. Ann. Sci. For. 46(s): 459s-463s. Loomis, W.E. 1953. Growth correlation. In Loomis, W.E. (ed.) Growth and Differentiation of Plants. Iowa State College Press, Ames. pp. 197-217. Loopstra, E.M. , Shall III, CG. , Sidle, R.C. 1988. Ectomycorrhizal inoculation f a i l s to improve performance of Sitka spruce seedlings on clearcuts in southeastern Alaska. West. J. Appl. For. 3:110-112. Lopushinsky, W., and Kaufmann, M.R. 1984. Effects of cold s o i l on water relations and spring growth of Douglas-fir seedlings. For. Sci. 30:628-634. 2 0 9 Macdonald, S.E., and Lieffers, V.J. 1990. Photosynthesis, water relations, and f o l i a r nitrogen of Picea mariana and Larix l a r i c i n a from drained and undrained peatlands. Can. J. For. Res. 20:995-1000. Marais, L.J., and Kotze, J.M. 1978. Effect of s o i l temperature on mycorrhizal development and growth of seedlings of Pinus patula Schlecht. et Cham. S. Afr. For. J. 106:34-36. Margolis, H.A., and Brand, D.G. 1990. An ecophysiological basis for understanding plantation establishment. Can. J. For. Res. 20:375-390. Markhart III, A.H. 1984. Amelioration of chilling-induced water stress by abscisic acid-induced changes i n root hydraulic conductance. Plant Physiol. 74:81-83. Marks, G.C, and Foster, R.C. 1967. Succession of mycorrhizal associations on individual roots of radiata pine. Aust. For. Res. 31:193-201. Marschner, H. 1986. Mineral nutrition of higher plants. Academic Press, London. Marshall, J., and Perry, D.A. 1987. Basal and maintenance respiration of mycorrhizal and nonmycorrhizal root systems of conifers. Can. J. For. Res. 17:872-878. Marx, D.H. 1969. Antagonism of mycorrhizal fungi to root pathogenic fungi and s o i l bacteria. Phytopathology 59:153-163. Marx, D.H. 1977. The role of mycorrhizae in forest production. Tappi Conf. Pap., Annu. Mtg., Feb. 1977, Atlanta, Ga. pp. 151-161. Marx, D.H. 1979. Synthesis of Pisolithus tinctorius ectomycorrhizae on White Oak seedlings in fumigated nursery s o i l . For. Service Res. Note, USDA, SE 280. Marx, D.H. 1981. V a r i a b i l i t y i n ectomycorrhizal development and growth among isolates of Pisolithus tinctorius as affected by source, age, and reisolation. Can. J. For. Res. 11:168-174. Marx, D.H., and Bryan, W.C 1971. Influence of ectomycorrhizae on survival and growth of aseptic seedlings of Loblolly pine at high temperature. For. Sci. 17:37-41. Marx, D.H. , and Cordell, C E . 1987. Ecology and management of ectomycorrhizal fungi in regenerating forests in the eastern United States. In Sylvia, D.M., Hung, L.L., and Graham, J.J. (eds.). Mycorrhizae in the next decade-practical applications and research p r i o r i t i e s . 7th NACOM, 3-8 May, 1987. Gainesville, Florida. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. pp.69-71. 2 1 0 Marx, D.H., and Kenney, D.S. 1982. Production of ectomycorrhizae fungus inoculum. In Schenck, N.C. (ed.) Methods and Principles of Mycorrhizal Research. American Phytopathological Society:St. Paul, Minnesota, pp. 131-146. Marx, D.H., Cordell, C.E., Kenney, D.S., Mexal, J.G., Artman, J.D., R i f f l e , J.W., and Molina, R.J. 1984. Commercial vegetative inoculum of Pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on bare-root tree seedlings. For. Sci. Monogr. 25. Marx, D.H., Craig, W.C, and Davey, CB. 1970. Influence of temperature on aseptic synthesis of ectomycorrhizae by Thelephora terrestris and Pisolithus tinctorius in l o b l o l l y pine. For. Sci. 16:424-431. Marx, D.H., Hatch, A.B., and Mendicino, J.F. 1977. High s o i l f e r t i l i t y decreases sucrose content and susceptibility of l o b l o l l y pine to ectomycorrhizal infection by Pisolithus tinctorius. Can. J. Bot. 55:1569-1574. Marx, D.H., Hedin, A., and Toe, S.F.P. 1985. IV. Fiel d performance of Pinus  caribaea var hondurensis seedlings with specific ectomycorrhizae and f e r t i l i z e r after three years on a savanna in Liberia. For. Ecol. Manage. 13:1-25. Mason, P.A., Wilson, J., Last, F.T., and Walker, C. 1983. The concept of succession in relation to the spread of sheathing mycorrhizal fungi on inoculated tree seedlings growing in unsterile s o i l s . Plant Soil 71:247-256. McAfee, B.J., and Fortin, J.A. 1986. Competitive interactions of ectomycorrhizal mycobionts under f i e l d conditions. Can. J. Bot. 64:848-852. McAfee, B.J., and Fortin, J.A. 1988. Comparative effects of the s o i l microflora on ectomycorrhizal inoculation of conifer seedlings. New Phytol. 108:443-449. McAfee, B.J., and Fortin, J.A. 1989. Ectomycorrhizal colonization on black spruce and jack pine seedlings outplanted in reforestation sites. Plant S o i l 116:9-17. Mejstrik, V. 1970. The uptake of ^P by different kinds of ectotrophic mycorrhiza of Pinus. New Phytol. 69:295-298. Mexal, J.C 1980. Root Growth Studies, Part "F". Aspects of mycorrhizal inoculation in relation to reforestation. N.Z. J. For. Sci.10:208-217. Mikola, P. 1965. Studies on the ectendotrophic mycorrhiza of pine. Acta For. Fenn. 79.2:1-56. Mikola, P. 1970. Mycorrhlzal inoculation in afforestation. Int. Rev. For. Res. 3:123-196. 211 Mikola, P. 1989. The role of ectomycorrhiza in forest nurseries. Agric. Ecosyst. Environ. 28:343-350. Moawad, M. 1978. Ecophysiology of vesicular-arbuscular mycorrhiza in the tropics. In Harley, J.L., and Russell, R. (eds.) The soil-root interface. Academic Press, New York. Molina, R. 1982. Use of the ectomycorrhizal fungus Laccaria laccata in forestry. I. Consistency between isolates in effective colonization of containerized conifer seedlings. Can. J. For. Res. 12:469-473. Molina, R., and Trappe, J.M. 1982. Applied aspects of ectomycorrhizae. In Rao, S. (ed.) Advance in agricultural microbiology. N.W. Oxford and IBH Publishing Co.-.New Delhi. Morrison, I.K. 1974. Mineral nutrition of conifers with special reference to nutrient status interpretation: a review of the literature. Canadian Forestry Forest Service Pub. No. 1343, Ottawa. Moser, M. 1958. Der einfluss l i e f e r temperaturen auf des Wachstum und Lebenstadigheit hoherer Pilze mit spezieller Beruchsichtigung von Mykorrizapilzen. Sydowia 12:386-399. (I was unable to see this a r t i c l e and r e l i e d on Harley and Smith 1983 for information on Moser) Mullin, R.E. 1963. Planting check in spruce. For. Chron. 39:252-259. Nambiar, E.K.S., Bowen, G.D., and Sands, R. 1979. Root regeneration and plant water status of Pinus radiata D.Don seedlings transplanted to different s o i l temperatures. J. Exp. Bot. 30:1119-1131. Nelsen, C E . and Safir, CR. 1982. Increased drought tolerance of mycorrhizal onion plants caused by improved phosphorus nutrition. Planta 154:407-413. Neter, J., and Wasserman, W. 1974. Applied linear s t a t i s t i c a l models. Richard D. Irwin Inc., Homewood, I l l i n o i s . Newman, E.I. 1966. A method of estimating the total length of root in a sample. J. Appl. Ecol. 3:139-145. Nielsen, K.F. 1971. Roots and roots temperatures. In Carson, E.W. (ed.) The plant root and i t s environment. University Press of Virginia. Charlottesville, pp. 29-61. Nielsen, K.F., and Humphries, E.C 1966. Effects of root temperature on plant growth. Soils and F e r t i l i z e r s 29:1-7. Nightingale, CT. 1935. Effects of temperature on growth, anatomy, and metabolism of apple and peach roots. Bot. Gaz. 96:581-637. Nylund, J.-E., and Unestam, T. 1982. Structure and physiology of ectomycorrhizae I. The process of mycorrhizae formation i n Norway spruce in v i t r o . New Phytol. 91:63-79. 212 Orlander, G., and Due, K. 1986. Water relations of seedlings of Scots pine grown in peat as a function of s o i l water potential and s o i l temperature. Stud. For. Suec. No. 175. 13 pp. Orlov, A.Y. 1957. Observations on absorbing roots of spruce (Picea excelsa Link) i n natural conditions. Bot. Zh. SSSR. 42:1172-1181 (Translated from Russian, Israel Program Sci. Transl., Jerusalem). (I was unable to read this reference and rel i e d on Slankis 1974 for information on Orlov) Pacovsky, R.S., Paul, E.A., and Bethlenfalvay, G.J. 1986. Comparisons between phosphorus-fertilized and mycorrhizal plants. Crop Sci. 26:151-156. Parke, J.L. 1983. Factors affecting the inoculum potential of VA and ectomycorrhizal fungi i n forest soils of southwest Oregon and northern California. Ph.D. Thesis, Oregon State University, Corvallis, OR. Parke, J.L. 1985. Effects of environment on mycorrhizae: leaving the Dark Ages. In Molina, R. (ed.) Proceedings 6th NACOM. June 25-29, 1984, Bend. OSU Forest Research Laboratory, Corvallis, Oregon, pp. 107-109. Parke, J.L., Linderman, R.G. , and Black, CH. 1983a. The role of ectomycorrhizas in drought tolerance of Douglas-fir seedlings. New Phytol. 95:83-95. Parke, J.L., Linderman, R.C, and Trappe, J.M. 1983b. Effect of root zone temperature on ectomycorrhiza and vesicular-arbuscular mycorrhiza formation i n disturbed and undisturbed forest so i l s of southwest Oregon. Can. J. For. Res. 13:657-665. Parkinson, J.A., and Allen, S.E. 1975. A wet oxidation procedure for the determination of nitrogen and mineral nutrients in biological material. Commun. Soil Sci. Plant Anal. 6:1-11. Passioura, J.B. 1982. Water in the soil-plant-atmosphere continuum. In Pirson, A., and Zimmerman, M.H. (eds.) Encyclopedia of plant physiology New Series 12B. Springer, Berlin Heidelberg, New York, pp. 5-33. Passioura, J.B. 1988. Water transport i n and to roots. Annu. Rev. Plant Physiol. Plant Mol. B i o l . 39:245-265. Peech, M. 1965. Methods of Soi l Analysis. Part 2 - Chemical and Microbiological Properties. Am. Soc. Agron. Madison, Wisconsin, pp. 922-923. Perry, D.A., Molina, R., and Amaranthus, M.P. 1987. Mycorrhizae, mycorrhizospheres, and reforestation: current knowledge and research needs. Can. J. For. Res. 17:929-940. 213 Philipson, J.J. 1988. Root growth in Sitka spruce and Douglas-fir transplants: dependence on the shoot and stored carbohydrates. Tree Physiol. 4:101-108. Potter, J.R., and Jones, J.W. 1977. Leaf area partitioning as an important factor in growth. Plant Physiol. 59:10-14. Powell, C.L. 1975. Potassium uptake by endotrophic mycorrhizas. In Sanders, F.E., Mosse, B., and Tinker, P.B. (eds.) Endomycorrhizas. Academic Press, London, pp. 461-468. Power, J.F., Grunes, D.L., W i l l i s , W.O., and Reichman, G.A. 1963. Soil temperature and phosphorus effects on barley growth. Agron. J. 5:389-392. Radin, J.W., and Boyer, J.S. 1982. Control of leaf expansion by nitrogen nutrition in sunflower plants. Role of hydraulic conductivity and turgor. Plant Physiol. 64:771-775. Radin, J.W., and Eidenbock, M.P. 1984. Hydraulic conductance as a factor limiting leaf expansion of phosphorous-deficient cotton plants. Plant Physiol. 75:372-377. Ratnayake, M., Leonard, R.T., and Menge, J.A. 1978. Root exudation i n relation to supply of phosphorus and i t s possible relevance to mycorrhizal formation. New Phytol. 81:543-552. Reid, CP.P., Kidd, F.A. , and Ekwebelam, S.A. 1983. Nitrogen nutrition, photosynthesis and carbon allocation in ectomycorrhizal pine. Plant So i l 71:415-432. Richter, D.L., and Bruhn, J.N. 1989. Fiel d survival of containerized red and jack pine seedlings inoculated with mycelial slurries of ectomycorrhizal fungi. New For. 3:247-258. R i f f l e , J.W., and Tinus, R.W. 1982. Ectomycorrhizal characteristics, growth, and survival of a r t i f i c i a l l y inoculated ponderosa and Scots pine in a greenhouse and plantation. For. Sci. 28:646-660. Ritchie, CA. , and Dunlap, J.R. 1980. Root growth potential: i t s development and expression i n forest tree seedlings. N.Z. J. For. Sci. 10:218-248. Ritchie, G.A., and Hinckley, T.M. 1975. The pressure chamber technique as an instrument for ecological research. Adv. Ecol. Res. 9:165-254. Robertson, N.F. 1954. Studies on the mycorrhiza of Pinus sylvestris. I. The pattern of development of mycorrhizal roots and i t s significance for experimental studies. New Phytol. 53:253-283. Rook, D.A., and Hobbs, J.F.F. 1975. Soil Temperatures and growth of rooted cuttings of radiata pine. N.Z. J. For. Sci. 5: 296-305. Rovira, A.D. 1969. Plant root exudates. Bot. Rev. 35:35-57. 214 Ruehle, J.L. 1982. Fiel d performance of container-grown l o b l o l l y pine seedlings with specific ectomycorrhizae on a reforestation site in South Carolina. South. J. Appl. For. 6:30-33. Ruehle, J.L. 1983. The relationship between lateral-root development and spread of Pisolithus tinctorius ectomycorrhizae after planting of container-grown l o b l o l l y pine. For. Sci. 29:519-526. Ruehle, J.L. 1985. Lateral-root development and spread of Pisolithus tinctorius ectomycorrhizae on bare-root and container-grown l o b l o l l y pine seedlings after planting. For. Sci. 31:220-225. Ruehle, J.L., Marx, D.H., Barnett, J.P., and Pawuk, W.H. 1981. Survival and growth of container-grown and bare-root shortleaf pine seedlings with Pisolithus and Thelephora ectomycorrhizae. South. J. of Appl. For. 5:20-23. Running, S.W. 1976. Environmental control of leaf water conductance i n conifers. Can. J. For. Res. 6:104-112. Running, S.W., and Reid, CP.P. 1980. Soil temperature influences on root resistance of Pinus contorta seedlings. Plant Physiol. 65:635-640. Rygiewicz, P.T., Bledsoe, C.S., and Zasoski. R.J. 1984a. Effects of ectomycorrhizae and solution pH on [ N]ammonium uptake by coniferous seedlings. Can. J. For. Res. 14:885-892. Rygiewicz, P.T., Bledsoe, C.S., and Zasoski. R.J. 1984b. Effects of ectomycorrhizae and solution pH on [ N]nitrate uptake by coniferous seedlings. Can. J. For. Res. 14:893-899. Safir, G.R., Boyer, J.S., and Gerdemann, J.W. 1972. Nutrient status and mycorrhizal enhancement of water transport in soybean. Plant Physiol. 49:700-703. Salt, G.A. 1979. The increasing interest in "minor pathogens". In Schippers, B., and Gams, W. (eds.) Soil-borne Pathogens. Academic Press, New York. Samson, J., and Fortin, J.A. 1986. Ectomycorrhizal fungi of Larix l a r i c i n a and the interspecific and intraspecific variation in response to temperature. Can. J. Bot. 64:3020-3028. Sands, R., and Theodorou, C. 1978. Water uptake by mycorrhizal roots of radiata pine seedlings. Aust. J. Plant Physiol. 5:301-309. Sands, R. , Fiscus, E.L. , and Reid, CP.P. 1982. Hydraulic properties of pine and bean roots with varying degrees of suberization, vascular differentiation and mycorrhizal infection. Aust. J. Plant Physiol. 9:559-569. Schenck, N.C, and Schroder, V.N. 1974. Temperature response of Endo gone mycorrhiza on soybean roots. Mycologia 66:600-605. 215 Schenck, N.C, and Smith, CS. 1982. Responses of six species of vesicular-arbuscular mycorrhizae fungi and their effects on soybean at four s o i l temperatures. New Phytol. 92:193-201. Schramm, J.R. 1966. Plant colonization studies on black wastes from anthracite mining in Pennsylvania. Trans. Am. Philos. Soc. 56:1-194. Setter, T.L. and Greenway, H. 1988. Growth reductions of rice at low root temperature: decreases in nutrient uptake and development of chlorosis. J. Exp. Bot. 39:811-829. Shaw, C C , III, Jackson, R.M. , and Thomas, G.W. 1987a.. F e r t i l i z e r levels and fungal strain influence the development of ectomycorrhizae on Sitka spruce seedlings. New For. 1:215-224. Shaw, C C , III, Molina, R. , and Walden, J. 1982. Development of ectomycorrhizae following inoculation of containerized Sitka and white spruce seedlings. Can. J. For. Res. 12:191-195. Shaw, C.C.III, Sidle, R.C, and Harris, A.S. 1987b. Evaluation of planting sites common to a southeast Alaska clearcut. III. Effects of microsite type and ectomycorrhizal inoculation on growth and survival of Sitka spruce seedlings. Can. J. For. Res. 17:334-339. Sheriff, D.W., Nambiar, E.K.S., and Fife, D.N. 1986. Relationships between nutrient status, carbon assimilation and water use efficiency in Pinus radiata (D.Don) needles. Tree Physiol. 2:73-88. Siegel, S. 1956. Nonparametric s t a t i s t i c s for the behavorial sciences. McGraw-Hill Book Company, Inc., New York. Sinclair, W.A. 1974. Development of ectomycorrhizae in a Douglas-fir nursery: I. Seasonal characteristics. For. Sci. 20: 51-56. Slankis, V. 1974. Soil factors influencing formation of mycorrhizae. Annu. Rev. Phytopathol. 12:437-457. Small, E. 1972. Photosynthetic rates in relation to nitrogen recycling as an adaptation to nutrient deficiency i n peat bog plants. Can. J. Bot. 50:2227-2233. Smit-Spinks, B. 1983. Investigation of the cold hardiness of Pinus  svlvestris L. Ph.D. Thesis, University of Minnesota. Smith, D.C, Muscatine, L. , and Lewis, D.H. 1969. Carbohydrate movement from autotrophs to heterotrophs in parasitic and mutualistic symbioses. Biol. Rev. 44:17-90. Smith, CS., and Roncadori, R.W. 1986. Responses of three vesicular-arbuscular mycorrhizal fungi at four s o i l temperatures and their effects on cotton growth. New Phytol. 104:89-95. 216 Smith, R.S., Jr. 1967. Decline of Fusarium oxvsporum i n the roots of Pinus  lambertiana seedlings transplanted into forest s o i l s . Phytopathology 57:1265. Smith, S.E. 1985. The concept of effectiveness in symbiotic relationships. In Molina, R. (ed.) Proceedings 6th NACOM, June 25-29, 1984, Bend. OSU Forest Research Laboratory, Corvallis, Oregon, pp. 1146-1149. Smith, S.E., and Daft, M.J. 1978. The effect of mycorrhizas on the phosphate content, nitrogen fixation and growth of Medicago sativa. Aust. J. Plant Physiol. 4:403-413. Smith, S.S.E. 1980. Mycorrhizas of autotrophic higher plants. Bi o l . Rev. 55:475-510. Smolander, H., and Oker-Blom, P. 1989. The effect of nitrogen content on the photosynthesis of Scots pine needles and shoots. Ann. Sci. For. 46(s):473s-475s. Steel, R.G.D., and Torrie, J.H. 1980. Principles and procedures of st a t i s t i c s . McGraw-Hill Book Co., New York. Stenstrom, E., and Unestam, T. 1987. Ecological variation in pine mycorrhiza. In Sylvia, D.M. , Hung, L.L., and Graham, J.H. (eds.) Mycorrhiza in the next decade, practical applications and research p r i o r i t i e s . 7th NACOM, May 3-8, 1987. Gainseville, Florida. Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL. p. 104. Stenstrom, E., Ek, M., and Unestam, T. 1990. Variation in f i e l d response of Pinus svlvestris to nursery inoculation with four different ectomycorrhizal fungi. Can. J. For. Res. 20:1796-1803. Summerbell, R.C. 1987. The inhibitory effect of Trichoderma species and other s o i l microfungi on formation of mycorrhiza by Laccaria bicolor in v i t r o . New Phytol. 105:437-448. Sutherland, D.C, and Day, R.J. 1988. Container volume affects survival and growth of white spruce, black spruce, and jack pine seedlings: a literature review. North. J. of Appl. For. 5:185-189. Sutton, R.F. 1969. Form and development of conifer root systems. Commonwealth Forestry Bureau, Technical Comm. No. 7, Oxford, England. Sutton, R.F. 1980. Root system morphogenesis. N.Z. J. For. Res. 10:264-292. Swan, H.S.D. 1962. The mineral nutrition of the Grand'Mere plantations. Woodlands Research Index No. 131. Pulp and Paper Res. Inst, of Canada. Swan, H.S.D. 1971. Relationship between nutrient supply, growth and nutrient concentrations in the foliage of white and red spruce. Pulp Pap. Res. Inst. Can., Woodlands Rep. 40. 217 Technicon Industrial Systems 1977. Autoanalyzer II Methodology: Individual/simultaneous determination of nitrogen and phosphorous i n BD acid digests. Industrial Method No. 329-74W/B for phosphorus and Industrial Method No. 334-74W/B+ for nitrogen. Technicon Industrial Systems, Tarrytown,NY. Tennant, D. 1975. A test of a modified line intersect method of estimating root length. J. Ecol. 63:995-1001. Teskey, R.O., Hinckley,T.M., and Grier, CC. 1984. Temperature-induced change i n the water relations of Abies amabilis (Dougl.) Forbes. Plant Physiol. 74:77-80. Theodorou, C , and Bowen, CD. 1971. Influence of temperature on the mycorrhizal associations of Pinus radiata D.Don. Aust. J. Bot. 19:13-20. Thomas, G.W., and Jackson, R.M. 1979. Sheathing mycorrhizas of nursery grown Picea sitchensis. Trans. Br.. Mycol. Soc. 73:117-125. Thomas, CW. , and Jackson, R.M. 1983. Growth responses of Sitka spruce seedlings to mycorrhizal inoculation. New Phytol. 95:223-229. Thomas, G.W., Rogers, D., and Jackson, R.M. 1983. Changes in the mycorrhizal status of Sitka spruce following outplanting. Plant Soil 71:319-323. Tranquillini, W. 1964. Photosynthesis and dry matter production of trees at high altitudes. In Zimmerman, M.H. (ed.) The Formation of Wood in Forest Trees. Maria Moors Cabot Foundation for Botanical Research, New York. pp. 606-618., Trappe, J.M. 1967. Principles of classifying ectotrophic mycorrhizae for identification of fungal symbionts. Proc. 14th Congress Report of the International Union of Forest Research Organisations, Munich, Part V, Section 24, pp.46-59. Trappe, J.M. 1977. Selection of fungi for ectomycorrhizal inoculation in nurseries. Annu. Rev. Phytopathol. 15:203-222. Troeng, E., and Linder, S. 1982. Gas exchange in a 20-year-old Scots pine. I. Net photosynthesis of current and one-year-old shoots within and between seasons. Physiol. Plant. 54:7-14. Trofymow, J.A., and van den Driessche, R. (1991). Mycorrhizas. In van den Driessche, R. (ed.) Handbook of Conifer Seedling Nutrition. CRC Press, Boca Raton FL, Chapter 8. Troughton, A. 1980. Environmental effects upon root-shoot relationships. In Sen, D.N. (ed.) Environment and Root Behaviour. Gesbios International, Jodhpur, India, pp. 25-41. 218 Turner, N.C, and P.C Jarvis. 1975. Photosynthesis in Sitka spruce [Picea sitchensis (Bong.) Carr.]. IV. Response to s o i l temperature. J. Appl. Ecol. 12:561-576. Tyminska, A., Le Tacon, F., and Chadoeuf, J. 1986. Effect of three ectomycorrhizal fungi on growth and phosphorus uptake of Pinus  s i l v e s t r i s at Increasing phosphorus levels. Can. J. Bot. 64:2753-2757. Tyron, P.R., and Chapin, F.S., III. 1983. Temperature control over root growth and root biomass in taiga forest trees. Can. J. For. Res. 13:827-833. Van Cleve, K. , Oechel, W.C, and Horn, J.L. 1990. Response of black spruce (Picea mariana) ecosystems to s o i l temperature modification in interior Alaska. Can. J. For. Res. 20:1530-1535. Visser, S. 1986. Ectomycorrhizal inoculation of containerized lodgepole pine and white spruce in the Pine Ridge nursery at Smoky Lake, Alberta. Kananaskis Centre for Environmental Research, University of Calgary. Contract research report prepared for Forest Research Branch, Alberta Energy and Natural Resources. Vogt, K.A. , R.L. Edmonds, C C Grier, and S.R. Piper. 1980. Seasonal changes in mycorrhlzal and fibrous-textured root biomass in 23- and 180-year-old Pacific s i l v e r f i r stands in western Washington. Can. J. For. Res. 10:523-529. Vyse, A. 1981. Growth of young spruce plantations in interior B r i t i s h Columbia. For. Chron. 57:174-180. Wareing, P.F., Khalifa, M.M., andTreharne, K.J. 1968. Rate-limiting processes i n photosynthesis at saturating light intensities. Nature (London) 220:453-457. Waring, S.A., and Bremner, J.M. 1964. Ammonium production in s o i l under waterlogged conditions as an index of nitrogen a v a i l a b i l i t y . Nature 201 (London):951-952. Webb, W.L. 1977. Seasonal allocation of photoassimilated carbon in Douglas f i r seedlings. Plant Physiol. 60:320-322. Welbank, P.J. 1962. The effects of competition with Agropyron repens and of nitrogen- and water supply on the nitrogen content of Impatiens  parviflora. Ann. Bot. 26:361-373. Wilcox, H.E. 1983. Fungal parasitism of woody plant roots from mycorrhizal relationships to plant disease. Annu. Rev. Phytopath. 21:221-242. Wilcox, H.E., and Ganmore-Neumann, R. 1975. Effects of temperature on root morphology and ectendomycorrhizal development i n Pinus resinosa Ait. Can. J. For. Res. 5:171-175. 219 Wilkinson, L. 1988. SYSTAT: The System for S t a t i s t i c s . Evanston, IL:SYSTAT, Inc. Wilson, J., Mason, P.A., Last, F.T., Inglebey, K., and Munro, R.C. 1987. Ectomycorrhiza formation and growth of Sitka spruce seedlings on fi r s t - r o t a t i o n forest sites in northern Britain. Can. J. For. Res. 17:957-963. Zak, B. 1973. Classification of ectomycorrhizae. In Marks, G.C, and Kozlowski, T.T. (eds.) Ectomycorrhizae: their ecology and physiology. Academic Press, New York. pp. 43-78. APPENDIX A INDEX OF COMMON AND SCIENTIFIC NAMES arbutus ash green ash birch s i l v e r birch Douglas-fir f i r amabilis f i r hemlock western hemlock pine eastern white pine Itali a n stone pine jack pine l o b l o l l y pine lodgepole pine radiata pine red pine Scots pine slash pine Arbutus menziesii Pursh. Fraxinus pennsvlvanica Marsh. Betula pendula Roth Pseudotsuga menziesii (Mirb.) Franco Abies amabilis Dougl. ex Forbes Tsuga heterophvlla (Raf.) Sarg. Pinus strobus L. P. pinea L. P. banksiana Lamb. P. taeda L. P. contorta Dougl. ex. Loud. P. radiata D. Don P. resinosa A i t . P. svlvestris L. P. e l l i o t i i Engelm. spruce black spruce Engelmann spruce Sitka spruce white spruce Picea mariana (Mill.) B.S.P. P. engelmannii Parry P. sitchensis (Bong.) Carr. P. glauca (Moench) Voss soybean Glycine max L. Merr. 221 APPENDIX B KEY CHARACTERISTICS OF MYCORRHIZA FORMED BY INOCULANT AND INDIGENOUS FUNGI 1. Inoculant Fungi E-strain or Complexipes moniliformis Walker (Danielson 1982, Danielson and Pruden 1989) Mycorrhizae: simple, medium to dark brown, glabrous with sparse hyphae. Mantle: thin, discontinuous and textura intrica t a . Extramatrical (EM) hyphae: 4-5 /tm diam., simple septate with Woronin bodies v i s i b l e i n young hyphae. Hyphae s t i f f , pale and smooth when young to tawny coloured with b l i s t e r - l i k e ornaments on mature hyphae. Amphinema bvssoides (Danielson and Pruden 1989) Mycorrhizae: simple, covered with abundant to dense whitish to yellow mycelium. EM hyphae: 2-3 (-4) /tm in diam. with keyhole clamps, finely ornamented, color varying from pale cream to yellow. Mycelial strands common, whitish to yellow. Other: hyphae, mycelial strands and ectomycorrhizae turning bright yellow in 3% KOH. Hebeloma crustuliniforme 5249 Mycorrhiza: simple, woolly, covered with abundant to dense mycelium, white. Mantle: loose, textura intricata. EM hyphae: abundant, binding roots and s o i l together, 3-4 /tm diam., most septa clamped, hyaline, smooth to verrucose with fine, deciduous ornaments; no colour change in 3% KOH. 222 Laccaria bicolor Mycorrhizae: Mantle: EM hyphae: smooth to subfloccose, plump, pale violet-brown to deep brown. outer layer-textura intricata, densely interwoven, frequently forked, septate hyphae 3-5 urn diam. smooth, hyaline, clamped, 2.5-4 /im diam, frequent branches, frequent elbow-like protrusions. inner layer-textura epidermoidea, hyphae 4-10 /tm i n diam. Mycelial strands: uncommon, not organized. Thelephora terres t r i s Mycorrhizae: EM hyphae: Mantle: Cystidia: simple, ligh t to grey-brown, cystidia frequent. 3-4 um diam. outer layer-loose textura intricata, hyphae 2-7 /im diam., septate with few clamp connections. inner layer-textura epidermoidea, hyphae 5-7 /tm in diam. , no clamps. 2-3 um diam., up to 100 um, septate with a clamp connection at the mantle surface, some retraction septa as described by Schramm (1966). Mycelial strands: frequent, undifferentiated; cystidia present. 2. ECM Indigenous to Forest Soil Mixture Cenococcum geophilum (Chilvers 1968, Danielson and Pruden 1989) Mycorrhizae: black, simple, club-shaped with stiff,dark hyphae radiating from the mantle. Mantle: stellate pattern of c e l l s in plan view. EM hyphae: dark brown (500x), 4-6 um diam., smooth, simple-septate with Woronin bodies. 223 Tuber-like (Danielson and Pruden 1989) Mycorrhizae: plump, pal l id, becoming darker reddish-brown with age; surface smooth or bearing hyaline cystidia. Mantle: Cystidia: textura "jigsaw" to textura epidermoidea on young tips with cells up to 30 x 4 /im. thin-walled, simple-septate at base, 4 /im at base, up to 110 /im long and pointed at the tip. Tomentella-like with c y s t i d i a (Danielson and Pruden 1989) Mycorrhizae: Mantle: Cystidia simple, dark brown; many hyphoid cystidia radiating from tip. textura angularis to textura "jigsaw" encrusted with discontinuous pigment; cells up to 4 x 25 /im. light gold, up to 60 /im x 2-3 /im. Tomentella-like without cystidia (Danielson et a l . 1984d, Danielson and Pruden 1989) Mycorrhizae: Mantle: EM hyphae: simple, dark brown. textura angularis to textura "j igsaw" encrusted with discontinuous pigment; cells up to 4 x 25 /im. 3-5 /xm, simple septate or clamped with thick yellow-brown walls; hyphae emerge from inflated cells (15-20 /im diam.) at the mantle surface; s t i f f and rarely branched. Mycelial strands: rare, undifferentiated. Hebeloma-like (Danielson and Pruden 1989) Mycorrhiza: simple, covered with abundant to dense mycelium, white to tan. Mantle: EM hyphae: textura intricata. 3-4 /im diam., most septa clamped, hyaline; no colour change in 3% KOH. 224 Mycelium radicis atrovirens (Visser 1986, Thomas and Jackson 1979) Mycorrhizae: Mantle: EM hyphae: Unknown type 1 Mycorrhizae: Mantle: dark brown to black, simple, often covered with a loose net of dark hyphae. irregular, discontinuous textura epidermoidea, hyphae associate with mantle are typically sinuous, smooth, and 3-4 urn diam. olive-brown, 2-2.5 /im, verrucose, simple septa. simple, lig h t yellow brown. discontinuous, textura intricata, strands of hyphae (formed by pa r a l l e l hyphae) form a loose interwoven net, hyphae 1.5 um diam. EM hyphae: 1-1.5 fim diam, hyaline, septate, no clamps. Sphaerosporella-like (Danielson 1984a) Mycorrhizae: simple, brown. Mantle: discontinuous textura epidermoidea, hyphae smooth, 4-15 um. EM hyphae: coarse, cinnamon-brown, smooth or verrucose, 4-12 /im diam., indented at septa. APPENDIX C NUTRIENT CONCENTRATIONS AT 0, 5, AND 12 WEEKS Week 6°C s o i l 12°C s o i l N P Ca Fe N P Ca Fe Uninocu la ted seedl ings mature shoot t i s s u e 0 1.1 0.26 0.49 74 1. ,2 0. ,24 0, ,57 70 5 0.5 0.10 0.32 40 0. .5 0. ,12 0, ,33 40 12 0.5 0.12 0.32 50 0. ,6 0. ,20 0. ,48 41 root t i s s u e 0 1.6 0.28 0.43 1. .2 0. ,23 0. .37 5 1.3 0.18 0.32 1. .1 0. ,18 0. ,33 12 1.4 0.20 0.34 1. .2 0. ,18 0, ,35 new f o l i a g e 5 0.7 0.19 0.12 22 0. .7 0. ,19 0. ,20 23 12 0.7 0.17 0.27 25 0. .9 0. ,22 0, ,42 32 Seedl ings i n o c u l a t e d wi th f o r e s t s o i l -mature shoot t i s s u e 0 1.4 0.35 0.49 69 1. .4 0. .36 0, ,56 65 5 0.5 0.14 0.28 24 0. .6 0. .17 0. ,40 30 12 0.5 0.15 0.27 32 0. .8 0, ,21 0, .40 44 root t i s s u e 0 1.6 0.35 0.42 1. .6 0. ,35 0. .42 5 1.3 0.18 0.32 1. .4 0, ,19 0, .31 12 1.3 0.21 0.32 1. .1 0. ,18 0, .28 new f o l i a g e t i s s u e 5 0.8 0.20 0.07 23 0, .8 0. ,17 0 .16 21 12 0.6 0.15 0.20 25 1. .0 0, ,21 0 .44 40 Seedl ings i n o c u l a t e d wi th E - s t r a i n mature shoot t i s s u e 0 0.9 0. 28 0.46 46 0. 9 0. 25 0. ,42 55 5 0.5 0. 13 0.29 40 0. 5 0. 12 0. 29 35 12 0.4 0. 10 0.30 33 0. 8 0. 23 0. ,49 52 root t i s s u e 0 1.9 0. 48 0.42 1. 8 0. 36 0. ,46 5 1.5 0. .24 0.34 1. 4 0. 27 0. ,33 12 1.4 0. ,21 0.32 1. 7 0. 23 0. ,35 new f o l i a g e t i s s u e 5 0.8 0. ,20 0.17 0. 7 0. 19 0. ,17 12 0.5 0. ,13 0.23 1. 7 0. ,22 0, ,57 Seedl ings i n o c u l a t e d wi th L . b i c o l o r mature shoot t i s s u e 0 1.2 0. ,39 0.48 39 1. ,1 0. ,34 0. ,51 58 5 0.5 0. ,17 0.33 59 0. ,7 0. ,22 0, .40 34 12 0.6 0. ,19 0.30 30 0. ,8 0. ,28 0. .45 38 root t i s s u e 0 1.4 0. ,33 0.41 1. ,2 0. ,25 0. .37 5 1.9 0. .29 1. ,7 0, ,30 12 1.7 0. .23 0.28 1, ,5 0. .22 0, .32 new f o l i a g e t i s s u e 5 0.8 0, .22 0.09 20 0, ,8 0. .20 0, .17 19 12 0.7 0. .20 0.23 19 0. ,9 0, .26 0 .47 30 Seedl ings i n o c u l a t e d wi th H. c r u s t u l i n i f o r m e mature shoot t i s s u e 0 0.8 0 .20 0.41 62 0, .9 0 .27 0 .48 68 5 0.5 0, .14 0.28 35 0. .5 0, .16 0 .37 35 12 0.4 0 .13 0.35 30 0, .7 0 .20 0 .47 90 root t i s s u e 0 1.3 0 .25 0.38 1, .4 0, .25 0 .43 _ _ 5 1.4 0 .20 0.33 1, .3 0 .20 0 .34 12 1.3 0 .17 0.35 1, .3 0 .16 0 .36 227 Seedl ings i n o c u l a t e d wi th H. c r u s t u l i n i f o r m e (cont . ) new f o l i a g e t i s s u e 5 0.8 0.24 0.16 - - 0.7 0.17 0.16 12 0.5 0.14 0.26 - - 0.9 0.20 0.46 Seedl ings i n o c u l a t e d wi th A. bvssoides mature shoot t i s s u e 0 1. ,1 0. 27 0.40 60 1. ,1 0. 25 0. ,43 64 5 0. 6 0. 13 0.32 38 0. ,6 0. ,16 0. .36 41 12 0. 5 0. 17 0.33 39 0. 8 0. 22 0, ,50 45 roo t t i s s u e 0 1. .4 0. ,30 0.41 1. ,5 0. 30 0. .39 5 1. .5 0. ,19 0.33 1. ,4 0. ,19 0, .34 12 1. ,5 0. ,19 0.35 1. ,3 0. ,17 0. .31 . - -new f o l i a g e t i s s u e 5 0. ,8 0. ,21 0.09 24 0. ,8 0. ,19 0, .16 23 12 0. ,8 0. ,19 0.22 21 1. ,0 0. ,23 0, .41 33 Seedl ings i n o c u l a t e d wi th T . t e r r e s t r i s mature shoot t i s s u e 0 0. ,8 0. ,21 0.42 77 0. ,8 0, ,24 0. .42 71 5 0. ,5 0. ,14 0.26 30 0, ,6 0, .21 0, .34 39 12 0. .6 0. ,18 0.28 51 1. .0 0. ,29 0, .47 56 root t i s s u e 0 1. .3 0. .25 0.41 1. ,1 0. .23 0 .39 5 1. .5 0, .23 0.30 1, .4 0. .24 0 .34 12 1, .5 0, .20 0.27 1, .8 0, .29 0 .34 new f o l i a g e t i s s u e 5 0. .8 0. .23 0.08 25 0, .8 0. .18 0 .17 24 12 0, .8 0. .18 0.24 42 1, .2 0, .25 0 .43 38 NOTE: S u f f i c i e n t t i s s u e was not a v a i l a b l e to analyze f o r a c t i v e Fe i n a l l root and new f o l i a g e samples. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0302170/manifest

Comment

Related Items