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Mycorrhizae of outplanted conifer seedlings on eastern Vancouver Island Roth, Aaron Lyle 1990

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MYCORRHIZAE OF OUTPLANTED CONIFER SEEDLINGS ON EASTERN VANCOUVER ISLAND By AARON LYLE ROTH B.Sc, The Uni v e r s i t y of B r i t i s h Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n . THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF SOIL SCIENCE We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 199 0 ©Aaron Lyle Roth, 1990 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 DE-6 (2/88) ABSTRACT Mycorrhizal c o l o n i z a t i o n and types of mycorrhizae that formed on container-grown Douglas-fir, western hemlock, and western red cedar seedlings were determined i n a nursery near Nanaimo, B.C. and under a range of f i e l d conditions on eastern Vancouver Island. Methods included a root c l e a r i n g , bleaching, and s t a i n i n g procedure that allowed for accurate estimates of percentage c o l o n i z a t i o n and some advantages i n mycorrhiza c h a r a c t e r i z a t i o n . The percentage of Douglas-fir and western hemlock short roots colonized by ectomycorrhizal fungi i n the nursery was highly variable but over 99 percent of the mycorrhizae were formed by Thelephora terrestris. A f t e r one f i e l d season mycorrhizal c o l o n i z a t i o n l e v e l s were between 7 2 and 9 3 percent on the new roots formed. The most d i f f i c u l t to regenerate s i t e had the lowest percentage c o l o n i z a t i o n and number of ectomycorrhizal types. T. terrestris mycorrhizae s t i l l had the highest r e l a t i v e abundance followed by Rhizopogon vinicolor (on Douglas-fir only), Cenococcum geophilum, Mycelium radicus atrovirens, Tujber-like, Sndogone-like, and 38 minor types of ectomycorrhizae. Some types of ectomycorrhizae were only present or common on s p e c i f i c s i t e s . This included a type that formed spore-like structures on the mantle c y s t i d i a and a type that produced red-brown hyphal exudates. Douglas-fir seedlings a r t i f i c i a l l y inoculated with R. vinicolor i n an Oregon nursery were t a l l e r than control seedlings when outplanted but no height or weight diff e r e n c e was found a f t e r one f i e l d season. The 17 types of mycorrhizae that formed on the c o n t r o l seedlings were dominated i n r e l a t i v e abundance by a type that was morphologically i d e n t i c a l to that formed on the seedlings that were a r t i f i c i a l l y inoculated with R. vinicolor. Western red cedar did not form mycorrhizae i n the nursery but formed low l e v e l s of vesicular-arbuscular mycorrhizae i n the f i e l d that included both f i n e and coarse . endophytes. TABLE OF CONTENTS Abstract i i Table of Contents i v L i s t of Tables v L i s t of Figures v i i Acknowledgements x i i 1 Introduction 1.1 Mycorrhizal symbiosis 1 1.2 Objectives of the study 5 2 L i t e r a t u r e Review 2.1 The importance of mycorrhizae i n f o r e s t r y 6 2.2 Mycorrhizal c o l o n i z a t i o n p o t e n t i a l 15 2.3 S i l v i c u l t u r e and the management of mycorrhizae 3 8 2.4 C l a s s i f i c a t i o n of ectomycorrhizae 4 6 3 Materials and Methods 3.1 Species and seedlots 61 3.2 Study s i t e s and s o i l sampling 63 3.3 Sampling of seedlings 74 3.4 Root preparation 76 3.5 Determination of percentage c o l o n i z a t i o n 77 3.6 Types of mycorrhizae 79 3.7 Data analysis 82 4 Results 4.1 Seedling status before and a f t e r outplanting 83 4.2 Variable c o r r e l a t i o n s 97 4.3 Ectomycorrhizal types i n the nursery 102 4.4 Ectomycorrhizal types formed a f t e r outplanting 103 4.5 Key to the types of ectomycorrhizae encountered.... 117 5 Discussion 5.1 Mycorrhiza formation before outplanting 123 5.2 Mycorrhiza formation a f t e r outplanting 124 5.3 A r t i f i c i a l l y inoculated Douglas-fir seedlings 131 5.4 Ectomycorrhizal inoculum p o t e n t i a l .....134 5.5 Other ectomycorrhizal types formed on new roots....139 5.6 Further research into mycorrhiza management 148 5.7 Characterization of ectomycorrhizae 149 5.8 Conclusions 154 Li t e r a t u r e c i t e d 157 Appendix 1: Percent cover and mycorrhizal status of non-crop species and n a t u r a l l y regenerated conifers 176 Appendix 2: Means of estimating dry root weights from fresh root weights 183 Appendix 3: Analysis of variance summary tables 186 Appendix 4: Data c o l l e c t e d 192 Appendix 5: Characterization of ectomycorrhizal types 225 Appendix 6: Glossary of te c h n i c a l terms 295 i v LIST OF TABLES Table I. Seedling production information f o r selected seedlots from MacBean Nursery 62 Table I I . Selected seedlots and study s i t e information 63-65 Table I I I . Form used to record c h a r a c t e r i s t i c s of each type of ectomycorrhiza encountered 81 Table IV. Fd9509: mycorrhizal c o l o n i z a t i o n and seedling growth i n potting mix previously fumigated with methyl bromide 83 Table V. Comparison of Fd9766 and Fd4503 from nursery and S i t e 3 85 Table VI. Fd9766: mycorrhizal c o l o n i z a t i o n and seedling growth 90 Table VII. Fd4503: mycorrhizal c o l o n i z a t i o n and seedling growth 91 Table VIII. Hw7321: mycorrhizal c o l o n i z a t i o n and seedling growth 92 Table IX. Cw4511: mycorrhizal c o l o n i z a t i o n and seedling growth 93 Table X. Fd3290C (control) versus Fd3290I (inoculated): mycorrhizal c o l o n i z a t i o n and seedling growth 96 Table XI. S i g n i f i c a n c e of variable c o r r e l a t i o n s under nursery conditions 99 Table XII. Significance of v a r i a b l e c o r r e l a t i o n s under f i e l d conditions 100-101 Table XIII. Percentage of seedlings sampled from each p l o t with each type of ectomycorrhiza on plug and new root 115-117 v i LIST OF FIGURES FIG. 1. S i t e 1C 68 FIG. 2. S i t e II 68 FIG. 3. S i t e 2F 69 FIG. 4. S i t e 2H 69 FIG. 5. S i t e 2R 70 FIG. 6. S i t e 4 70 FIG. 7. S i t e 5 71 FIG. 8. S i t e 6 71 FIG. 9. S i t e 7H 72 FIG. 10. S i t e 7C 72 FIG. 11. Nonmycorrhizal and ectomycorrhizal Douglas-fir short roots a f t e r s t a i n i n g procedure 78 FIG. 12. Nonmycorrhizal hemlock root t i p 78 FIG. 13. Fine and coarse endophyte forming v e s i c u l a r -arbuscular mycorrhizae with western red cedar 78 FIG. 14. Mycorrhizal c o l o n i z a t i o n of Fd9766 87 FIG. 15. Mycorrhizal c o l o n i z a t i o n of Fd4503 87 FIG. 16. Seedling weight of Fd9766 88 FIG. 17. Seedling weight of Fd4503 88 FIG. 18. Mycorrhizal co l o n i z a t i o n of Douglas-fir seedlings a r t i f i c i a l l y inoculated with Rhizopogon vinicolor versus co n t r o l seedlings 95 FIG. 19. Seedling weight of Douglas-fir seedlings a r t i f i c i a l l y inoculated with Rhizopogon vinicolor versus co n t r o l seedlings 95 v i i FIG. 20. Colonization pattern of Type 1 (Thelephora terrestris) mycorrhizae on nursery seedlings 104 FIG. 21. Occurrence of Type 1 (Thelephora terrestris) mycorrhizae on f i e l d c o l l e c t e d seedlings 106 FIG. 22. Relative abundances of the major types and combined r e l a t i v e abundance of the minor types of ecto-mycorrhizae encountered on f i e l d c o l l e c t e d seedlings 107 FIG. 23. Occurrence of Type 2 (Rhizopogon vinicolor) mycorrhizae on f i e l d c o l l e c t e d seedlings 109 FIG. 24. Relative abundances of ectomycorrhizal types on plug roots and new roots of Douglas-fir from S i t e 1 I l l FIG. 25. Relative abundances of the minor types of ecto-mycorrhizae encountered on f i e l d c o l l e c t e d seedlings 113 FIG. 26. Type 1 (Thelephora terrestris) mycorrhizae: habit i n nursery 227 FIG. 27. Type 1 mycorrhizae: part of sporocarp 227 FIG. 28. Type 1 mycorrhizae: habit i n f i e l d 228 FIG. 29. Type 1 mycorrhizae: mature t i p 228 FIG. 30. Type 1 mycorrhizae: senescent t i p 228 FIG. 31. Type 1 mycorrhizae: rhizomorph a x i l 229 FIG. 32. Type 1 mycorrhizae: stained c y s t i d i a 229 FIG. 33. Type 1 mycorrhizae: unstained c y s t i d i a 230 FIG. 34. Type 1 mycorrhizae: mycelial strand 230 FIG. 35. Type 1 mycorrhizae: mid mantle pattern 231 FIG. 36. Type 1 mycorrhizae: inner mantle pattern 231 FIG. 37. Type 1 mycorrhizae: cross-section 232 FIG. 38. Type 1 mycorrhizae: Hartig net 232 v i i i FIG. 39. Type 1 mycorrhizae: Habit of Type 1 contrasted with Type 2 mycorrhizae before s t a i n i n g 236 FIG. 40. Type 1 mycorrhizae: Habit of Type 1 contrasted with Type 2 mycorrhizae a f t e r s t a i n i n g 236 FIG. 41. Type 2 (Rhizopogon vinicolor) mycorrhizae: habit 237 FIG. 42. Type 2 mycorrhizae: mantle revealed 237 FIG. 43. Type 2 mycorrhizae: cross-section of tubercle.... 238 FIG. 44. Type 2 mycorrhizae: c y s t i d i a with hypha 238 FIG. 45. Type 2 mycorrhizae: c y s t i d i a showing end 238 FIG. 46. Type 2 mycorrhizae: rhizomorph contrasted with a rhizomorph of Type 33 mycorrhizae... 239 FIG. 47. Type 2 mycorrhizae: cross-section of rhizomorph 239 FIG. 48. Type 2 mycorrhizae: plan view of rhizomorph 239 FIG. 49. Type 2 mycorrhizae: outer mantle pattern 24 0 FIG. 50. Type 2 mycorrhizae: inner mantle pattern 240 FIG. 51. Type 2 mycorrhizae: cross-section 241 FIG. 52. Type 2 mycorrhizae: Hartig net 241 FIG. 53. Type 3 (Mycelium radicis atrovirens) mycorrhizae: habit 244 FIG. 54. Type 3 mycorrhizae: emanating hyphae 244 FIG. 55. Type 3 mycorrhizae: net prosenchymous mantle 24 5 FIG. 56. Type 3 mycorrhizae: regular synenchymous mantle 245 FIG. 57. Type 3 mycorrhizae: textura epidermoidia mantle 246 ix FIG. 58. Type 3 mycorrhizae: Hartig net 246 FIG. 59. Type 4 (Cenococcum geophilum) mycorrhizae: habit 248 FIG. 60. Type 4 mycorrhizae: emanating hypha 248 FIG. 61. Type 4 mycorrhizae: mantle pattern 248 FIG. 62. Type 4 mycorrhizae: Hartig net around epidermal c e l l s 249 FIG. 63. Type 4 mycorrhizae: Hartig net around c o r t i c a l c e l l s 249 FIG. 64. Type 5 (Endc-gone-like) mycorrhizae: showing lack of mantle and patchy Hartig net development 251 FIG. 65. Type 5 mycorrhiza: narrow hyphae 251 FIG. 66. Type 5 mycorrhiza: Hartig net view 1 2 52 FIG. 67. Type 5 mycorrhiza: Hartig net view 2 252 FIG. 68. Type 6 (Tuber-like) mycorrhizae: habit 254 FIG. 69. Type 6 mycorrhizae: habit a f t e r s t a i n i n g procedure 254 FIG. 70. Type 6 mycorrhizae: mantle 254 FIG. 71. Type 6 mycorrhizae: cystidium 2 55 FIG. 72. Type 6 mycorrhizae: rhizomorph 255 FIG. 73. Type 6 mycorrhizae: mantle pattern 256 FIG. 74. Type 6 mycorrhizae: cross-section... 256 FIG. 75. Type 10 mycorrhizae: mantle and Hartig net 258 FIG. 76. Type 10 mycorrhizae: outer mantle hyphae 258 FIG. 77. Type 11 mycorrhizae: habit 260 FIG. 78. Type 11 mycorrhizae: outer mantle and emanating hyphae 260 x FIG. 79. Type 11 mycorrhizae: noninvaginated hyphae around epidermal c e l l s 2 60 FIG. 80. Type 12 mycorrhizae: emanating hyphae 262 FIG. 81. Type 12 mycorrhizae: rhizomorph 2 62 FIG. 82. Type 12 mycorrhizae: cross-section 262 FIG. 83. Type 13 mycorrhizae: c y s t i d i a view 1 264 FIG. 84. Type 13 mycorrhizae: c y s t i d i a view 2 2 64 FIG. 85. Type 13 mycorrhizae: inner mantle hyphae 265 FIG. 86. Type 13 mycorrhizae: Hartig net 2 65 FIG. 87. Type 14 mycorrhizae: c y s t i d i a 2 67 FIG. 88. Type 14 mycorrhizae: mantle pattern 267 FIG. 89. Type 23 mycorrhizae: mantle and emanating hyphae 274 FIG. 90. Type 23 mycorrhizae: mantle pattern 274 FIG. 91. Type 24 mycorrhizae: Douglas-fir seedling and mushroom formed by Laccaria laccata 276 FIG. 92. Type 33 mycorrhizae: habit 283 FIG. 93. Type 33 mycorrhizae: rhizomorph .283 FIG. 94. Type 33 mycorrhizae: emanating hyphae 284 FIG. 95. Type 33 mycorrhizae: emanating hyphae after staining procedure 284 FIG. 96. Type 33 mycorrhizae: mantle pattern 285 FIG. 97. Type 33 mycorrhizae: cross-section 285 x i ACKNOWLEDGEMENT Major funding for t h i s study was a G.R.E.A.T. award [#87(Gc-9)] from the Science Council of B r i t i s h Columbia. I thank Shannon Berch for writing the o r i g i n a l proposal, for making contacts with MacMillan Bloedel Limited, for her supervision throughout the project, and for use of her equipment. I thank B i l l Beese, Dave Clark, Ralph Bower and the other s t a f f at MacMillan Bloedel Limited for the i r f r i e n d l y cooperation and f i n a n c i a l and technical assistance. Thanks to Steve Grossnickle for attending committee meetings and for giving helpful advice. Thanks to Bob Danielson, Tim Ballard, and Les Lavkulich for reading and commenting on the Thesis, and to Shaobing Yu and Tim Williams for the f i e l d assistance. 1 INTRODUCTION 1.1 Mycorrhizal symbiosis Harley and Smith (1983) give a comprehensive review of mycorrhizal symbiosis. The term mycorrhiza ( l i t e r a l l y , "fungus-root") refers to the symbiosis ( l i v i n g together) that exists between fungus (mycobiont) and plant root (phytobiont or host plant) and that can be characterized by a particular morphology. With respect to the ectomycorrhizae t h i s morphology consists of a Hartig net. The Hartig net i s composed of hyphae surrounding the epidermal and/or c o r t i c a l c e l l s usually with no i n t r a c e l l u l a r penetration. A sheath or mantle of t i g h t l y woven fungal hyphae around these fine roots i s also usually present. There are thousands of fungi that may form ectomycorrhizae, including below-ground f r u i t i n g t r u f f l e - l i k e fungi and most mushrooms belonging to the Ascomycotina and the Basidiomycotina. Many ectomycorrhizal fungi have no known sexual phase ("fungi imperfecti") and a few others belong to the Zygomycotina. Many woody temperate and boreal plant species form ectomycorrhizae including a l l conifers belonging to the Pinaceae. The Cupressaceae form vesicular-arbuscular (VA) mycorrhizae which involve fungi belonging to the Zygomycotina. These endomycorrhizal fungi form arbuscules within the c o r t i c a l c e l l s and vesicles in the cortex. The arbuscules (li k e the Hartig net of ectomycorrhizae) are the si t e s of nutrient transfer between the symbionts and resemble tiny 1 trees because of the repeated branching of the hyphae. The vesicles are storage organs (li k e the mantle of ectomycorrhizae) that may also serve i n vegetative reproduction. Starr (1975) proposed several continua for c l a s s i f y i n g symbiotic associations that may be summarized and applied to our understanding of mycorrhizae as follows: A) Spatial (extrahabitational to intrahabitational) - the amount of fungal tissue within the cortex versus emanating hyphae i n the s o i l . B) Size (equal to considerably unequal) - the biomass each biont contributes to the mycorrhiza. C) Temporal (persistant to transient) - there may be a succession of early, late, or multi-stage fungal species involved in colonizing a seedling (Dighton and Mason 1985). Some fungi may only form mycorrhizae i n the spring or f a l l under pa r t i c u l a r s o i l conditions (Slankis 1974). D) Need (obligate to facultative) - mycorrhizal symbiosis i s an obligate association for conifers growing in s o i l and a l l mycorrhizal fungi depend entire l y on photosynthate derived from the host for growth. However, there are reports of strains of some mycorrhizal fungi which show limited growth using c e l l u l o s e or l i g n i n (Harley and Smith 1983). E. Nutrition (biotrophic to necrotrophic) - mycorrhizal symbiosis i s strongly biotrophic in that each biont i s l i v i n g for extended periods without one d i r e c t l y causing the death of the other. 2 F) S p e c i f i c i t y (highly s p e c i f i c to non-specific) - although some ectomycorrhizal fungi are host s p e c i f i c , most mycorrhizal fungi are nonspecific except as being on either ecto- or endomycorrhizal hosts (Harley and Smith 1983). G) Harm-benefit (harmful to beneficial) - mycorrhizal symbiosis i s mutualistic implying that i t i s b e n e f i c i a l to each symbiont i n terms of species fitness or increasing the probability that each w i l l produce f e r t i l e offspring. Mycorrhizal benefit or effectiveness has also been used to describe the degree to which d i f f e r e n t mycobionts contribute to increased plant growth and survival. H) Integration (highly integrated to separate) - mycorrhizal symbiosis involve fungi within the root tissue and/or within root c e l l s and therefore a l l appear highly integrated using the l i g h t microscope. At the l e v e l of the electron microscope, the degree of integration of root-fungus interface d i f f e r s from symbiont to symbiont which may be an indication of s p e c i f i c i t y and compatibility (Massicotte et al. 1987). Starr's (1975) continua for c l a s s i f y i n g symbiotic associations remind us of the myriad of possible combinations that exist for mycorrhizal symbiosis. Each fungus-plant combination i s l i k e l y to vary in position along one or more of the continua depending on s i t e conditions. The influence d i f f e r e n t species and isolates of fungi have on the growth of the host plant can also vary greatly. With forest industry objectives of managing mycorrhizae to improve seedling growth and survival, finding mycobionts that convey the greatest 3 benefit to the host becomes a l l important. A good deal of time has been spent inoculating mycorrhizal fungi onto tree roots without a r r i v i n g at an understanding of the often mixed res u l t s . It i s well accepted that mycorrhizal fungi are needed for adequate nutrient and/or water uptake by most vascular plants, and have several important roles in ecosystem processes (Harley and Smith 1983). However, rarely has the contribution of mycorrhizal fungi to seedling growth and survival been detected because they are but one of the factors involved. Signifi c a n t growth responses due to mycorrhizal fungi are more l i k e l y to be demonstrated on severely disturbed s o i l , dry areas, or s i t e s that have lacked mycorrhizal hosts for several years (Perry et al. 1987). In such areas there may be a lack of either mycorrhizal fungi or suitable conditions for mycorrhiza formation. Perry et al. (1987) and Linderman (1987) have reviewed stategies for mycorrhiza research in the P a c i f i c Northwest. One of the approaches they suggested, and the one taken here, was to document the mycorrhizal status of containerized nursery seedlings before and after outplanting. In conjunction with MacMillan Bloedel Limited, the mycorrhizal status of Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco.), western hemlock (Tsuga heterophylla (Raf.) Sarg.), and western red cedar (Thuja plicata Donn.) were examined in the spring from MacBean Nursery near Nanaimo, B r i t i s h Columbia. The selected seedlots were sampled again in the 4 f a l l a fter one growing season on a range of s i t e conditions on eastern Vancouver Island. A r t i f i c a l l y inoculated Douglas-fir from an Oregon nursery was also examined. 1.2 Objectives of the Study 1) To determine i f selected seedlots i n MacBean Nursery were mycorrhizal and whether prior methyl bromide fumigation of a potting mixture had an effect on nlycorrhizal colonization of Douglas-fir. 2) To determine the mycorrhizal status of new roots of selected seedlots within the f i r s t growing season under a variety of s i t e conditions. 3) To determine i f the new roots were colonized primarily by nursery fungi or f i e l d fungi by characterizing the types present so that they can be recognized again. 4) To determine i f Douglas-fir seedlings grown in an Oregon nursery inoculated with Rhizopogon vinicolor A.H. Smith showed greater weight gain after one season of being outplanted than uninoculated controls. 5 2 LITERATURE REVIEW To guide the research direction taken a good deal of background information was reviewed. F i r s t , the importance and function of mycorrhizal symbiosis and how i t relates to the forest industry i s discussed. Second, a section on the factors which may affect mycorrhiza formation in forest clearcuts of the P a c i f i c Northwest i s followed by a review of studies which have assessed the impact of clearcutting and slash-burning on mycorrhizal fungi. Third, attempts to manage mycorrhizae in the nursery and f i e l d are discussed including the use of Rhizopogon vinicolor. The la s t section deals with the characterization of ectomycorrhizae which i s necessary in order to distinguish types involving d i f f e r e n t species of fungi. 2.1 The Importance of Mycorrhizae in Forestry The function of mycorrhizae Most vascular plant species have evolved with mycorrhizal symbiosis and most woody plants need i t to survive (Trappe and Fogel 1977). Harley and Smith (p. 387, 1983) state, "Mycorrhizae are active l i v i n g components of the s o i l population having some properties l i k e those of roots and some l i k e those of microorganisms." Some authors prefer not to c a l l mycorrhizae associations, but instead anatomical organs that carry out important physiological functions (Dominik 1969) . A l l conifers form mycorrhizae. The symbiosis i s 6 necessary for the survival of a l l ectomycorrhizal host plants (Trappe and Fogel 1977). This i s c l e a r l y indicated by the dependence on mycorrhizae for adequate phosphorus supply in bare root nurseries (Trappe and Strand 1969; Wright 1971), and the need to a r t i f i c i a l l y inoculate seedlings outplanted onto s i t e s that have not previously supported ectomycorrhizae (Mikola 1973; Marx 1980). Fogel and Hunt (1983) estimated that 73% of the t o t a l net primary production in a second-growth Douglas-fir stand was invested in growth and maintenance of roots and mycorrhizae. The degree of nutrient or water stress largely determines the proportion of photosynthate allocated to the roots and mycobionts. Under nursery conditions t h i s stress i s minimal to allow for maximum shoot size. Seedlings with a r e l a t i v e l y small root system may have to acclimatize quickly once outplanted to avoid nutrient and water stress. Amaranthus and Perry (1987) found adequate mycorrhiza formation was c r i t i c a l to seedling growth and survival on cold, droughty si t e s in southwest Oregon. L i t t l e work has beeen done on the dependency of cedar on vesicular-arbuscular (VA) mycorrhizae. However, Parke et al. (1983c) found that although less than one-third of the root length was mycorrhizal, shoot weight of mycorrhizal western red cedar seedlings was about four times greater after 12 weeks than the nonmycorrhizal seedlings. Mycorrhizae have been shown to increase water and nutrient uptake of a variety of tree species. Hyphae emanate from the mycorrhiza and form an extensive network of mycelium 7 that greatly increases the volume of s o i l exploited by roots (Bowen 1973) . More than 2000 hyphae may emanate from the surface of a single Cenococcum mycorrhiza and one m i l l i l i t e r of s o i l can have as much as 4 m of hyphae (Trappe and Fogel 1977) . I t i s estimated that i t takes 100 times as much material to produce an equal surface area by root growth as by hyphal growth (Harley 1989). That i s to say, the metabolic cost of obtaining nutrients v i a hyphae i s much less than with roots alone. Furthermore, mycorrhizal feeder roots may have reduced maintenance costs because they p e r s i s t longer than nonmycorrhizal roots (Slankis 1973; Trappe and Fogel 1977; Marshall and Perry 1987). The small diameter and great length of fungal hyphae greatly increase water and nutrient absorption over that possible for roots or root hairs because surface area and access to s o i l micropore space i s much greater (Reid 1979) . Schramm (1966) noted that the external mycelium of pine mycorrhizae extended more than 1 m into the s o i l . Hyphae or mycelial strands may also bridge the a i r gap that arises as the s o i l and roots dry and shrink (Harley and Smith 1983). Rhizomorphs are mycelial strands produced by many ectomycorrhizal fungi that have larger diameter core hyphae which may function much l i k e the xylem of a root. Rhizomorphs can extend several centimeters or meters from the mycorrhiza into the s o i l and decrease water flow resistance from s o i l to roots (Reid 1979). Duddridge et al. (1980) reported movement of t r i t i a t e d water through rhizomorphs to the mycorrhizae of 8 pine. Mycorrhizal seedlings may be more drought tolerant as indicated by a more negative permanent w i l t i n g point and increased growth under drought (Reid 1979; S a f i r and Nelsen 1985). Parke et al. (1983a) found that Douglas-fir seedlings with Rhizopogon vinicolor mycorrhizae recovered faster from drought than did nonmycorrhizal seedlings or seedlings forming mycorrhizae with other fungi that are not strong rhizomorph producers. The drought-stressed mycorrhizal Douglas-fir seedlings fixed C 0 2 at a rate lOx that of nonmycorrhizal seedlings. Mycorrhizae may affect stomatal physiology as shown by decreased stomatal resistances to water and C O 2 movement and by increased transpirational fluxes and rates of photosynthesis (Harley and Smith 1983). Higher hydraulic conductivity, especially when phosphorus a v a i l a b i l i t y i s low, may make mycorrhizal seedlings more tolerant to other environmental extremes such as large temperature fluctuations (Harley and Smith 1983; Smith and Gianinazzi-Pearson 1988). The fungal mantle may have the capacity to store nutrients and water and help prevent desication of the feeder roots (Harley and Smith 1983). Improved uptake of soluble nutrients and reduced nutrient leaching accompany increases in water uptake by mycorrhizal fungi (Harley and Smith 1983). VA mycorrhizal fungi may increase the uptake of phosphorus, zinc, and copper (Harley and Smith 1983; Smith and Gianinazzi-Pearson 1988). Ectomycorrhizal fungi may increase the uptake of phosphorus, nitrogen, and potassium as well as several micronutrients 9 (Bowen 1973; Trappe and Fogel 1977; Harley and .Smith 1983). The translocation of phosphorus through the rhizomorphs of Thelephora terrestris Fr., Rhizopogon luteolus Fr. & Nordh., and Pisolithus tinctorius (Pers.) Coker & Couch toward the mycorrhizae has also been shown (Skinner and Bowen 1974a; Kammerbauer et a l . 1988). There i s evidence ectomycorrhizal fungi may regulate forest f l o o r turnover rates. When tested i n pure culture, some mycobionts have the a b i l i t y to u t i l i z e organic nitrogen and phosphorus which may allow for d i r e c t recycling of nutrients (Bowen 1973; Harley and Smith 1983; Cromack 1985; G r i f f i t h s et al. 1987; Sangwanit and Bledsoe 1987). Gadgil and Gadgil (1971, 1974) indicated that mycorrhizae may suppress decomposition by al t e r i n g carbon to nitrogen (C/N) ratios of the resource to levels saprobic fungi cannot u t i l i s e or otherwise antagonizing saprobic fungi. This may lead to the accumulation of forest floor material making mycorrhizae a major factor in the formation of raw humus. However, Dighton and Mason (1985) point out that there may be alternative explanations for the observed effect that the experimental design did not account for. Amaranthus et al. (1987a) found nitrogen-fixing bacteria associated with the mycorrhizae of Douglas-fir seedlings. Trappe and Fogel (1977) found chlamydospores and hyphae of VA mycorrhizal fungi that were encrusted with such bacteria. Hyphae may have also have greater access than roots to the atmospheric nitrogen fixed by f r e e - l i v i n g bacteria. Nitrogen-10 f i x i n g bacteria may also occur i n fungal sporocarps (Li and Castellano 1987), mycorrhizae (Amaranthus et al. 1987a), bryophyte t h a l l i , and root nodules (Harley and Smith 1983). There may be s i g n i f i c a n t transfers of nutrients and carbon between plants of the same or di f f e r e n t species that share mycorrhizal fungi (Read et al. 1985; Finlay and Read 1986ab). Hyphal bridging may nurse seedlings in the shade of trees, reduce competitive interactions between plants, and allow for greater f l o r i s t i c d i v e r s i t y (Grime et al. 1987; Perry et al. 1987). Exudates from fungal hyphae help to build water stable aggregates that improve s o i l structure and f e r t i l i t y (Bowen 1973; T i s d a l l and Oades 1979; Perry et al. 1987), and decrease s o i l erosion (Harley and Smith 1983). Mycorrhizae can affect s o i l formation by producing humic compounds and accelerating the decomposition of primary minerals (Perry et al. 1987). Bowen and Theodorou (1967, in Harley and Smith 1983) demonstrated in culture the a b i l i t y of four ectomycorrhizal fungi to bring phosphate into solution from rock phosphate. Certain ectomycorrhizal fungi produce oxalic acid some of which can precipitate with calcium forming crystals of calcium oxalate. This can resu l t in increases in s o i l a c i d i t y , chemical weathering, and phosphorus a v a i l a b i l i t y (Cromack et a l . 1979; Cromack 1985). Some mycorrhizal fungi produce siderophores that enhance the s o l u b i l i t y of iron (Graustein et al. 1977; Entry et al. 1987). Others may have the a b i l i t y to keep toxic amounts of metals, such as iron, aluminium, manganese, and sulphur from entering the plant 11 (Trappe 1977; Marx and Artman 1979). Mycorrhizal fungi may also decrease s o i l t o x i c i t y associated with the production of allelochemicals or s o i l s t e r i l i z a t i o n (Perry et al. 1987). Slankis (1973) reviewed the role of hormones in ectomycorrhiza formation, development, and longevity. Mycorrhizal fungi produce vitamins and many of the plant hormones including the gas ethylene (Harley and Smith 1983). Graham and Linderman (1981) showed that very low levels of ethylene i n the root zone stimulate l a t e r a l root formation. The mycobionts may also influence seedling height or a l t e r root to shoot ratios due largely to t h e i r influence on the cytokinin to auxin r a t i o (Trappe 1977; Bledsoe et al. 1982; Harley and Smith 1983; Ho 1987; Ho and Trappe 1987). Ectomycorrhizal seedlings are l i k e l y to have more feeder roots due to increased branching that could lead to substantial increases in root surface area and reduce the root resistance to water without weight changes (Alexander 1981; Harley and Smith 1983; Hung and Molina 1986). Through greater seedling vigor, production of a n t i b i o t i c substances, stimulation of rhizosphere and mantle surface organisms that may compete with the pathogens, or actual physical protection of the feeder roots with a mantle, mycorrhize may decrease the occurrence of several root diseases (Zak 1971; Marx 1973; Harley and Smith 1983; Duchesne et a l . 1987; Kope and Fortin 1988). Hyphae of mycorrhizal fungi are important in the diet of several s o i l animals including collembola, insect larvae, and 12 nematodes which have the i r own role in nutrient cycling. Sporocarps are eaten by many animals but are an especially important food source for several species of small mammals (Maser et al. 1978). Most of the mushrooms and a l l the t r u f f l e and t r u f f l e - l i k e fungi eaten by people are ectomycorrhizal with trees (Harley and Smith 1983). The concept of symbiotic effectiveness Mutualism was defined by De Bary i n 1884 as, "parasitism...in which parasite and host mutually and permanently further and support one another" (Harley 1989). In short, the mycobiont helps the tree take up water and nutrients and in return the fungus i s supplied with soluble carbohydrates that may give i t some advantage in competing with other s o i l microorganisms. Many think of mycobiont effectiveness in terms of increased plant growth. This i s esse n t i a l l y the difference between these two competing a c t i v i t i e s and i s therefore a very coarse measure of effectiveness that i s very sensitive to nutrient a v a i l a b i l i t y (see discussion p. 378, Harley and Smith 1983). Although strains or species of mycorrhizal fungi can d i f f e r greatly in the i r influence on host growth, other factors should also be considered when selecting fungi for a r t i f i c i a l inoculation (Mikola 1973; Trappe 1977). Mycorrhizae have a much larger role i n ecosystem processes than just enhancing tree growth. They are "key li n k s " in below-ground nutrient and energy cycling (Trappe and 13 Fogel 1977). Therefore, when talking about cost/benefit or the effectiveness of mycorrhizae i n enhancing seedling growth, i t does not imply a mycobiont i s useless i f the seedling f a i l s to show a growth reponse under certain growing conditions (Smith 1985) . In fact, even i f a mycobiont i s shown to produce larger seedlings i n the nursery or on a variety of s i t e conditions, t h i s may not have any r e l a t i o n to the wood-producing potential of the adult (Harley and Smith 1983). The fungus may have s p e c i f i c adaptions that allow for higher stand productivity in the long run and convey benefits to the host that were not measured. Harley (1989) states that, "we can only judge benefit to the host or to the fungus by determining the selective advantage to them of any t r a i t s or characters that we measure." Because th i s i s very d i f f i c u l t or impossible to do, we continue to think in terms of stand productivity, not of producing trees or mycorrhizal fungi with high capacities to produce f e r t i l e offspring. Plant species fitness may be more related to long-term changes i n more indi r e c t factors such as the influence hyphal exudates of some mycobionts have on s o i l c haracteristics. This may be one of the factors, governed by economic p r o f i t or production ecology thinking, that may lead to declines in mycorrhizal inoculum potential and s i t e degradation: we want to see immediate benefits from a simple management technique without f i r s t t r ying to understand the complexities of the processes involved. But s t i l l , in the following discussion, an e f f i c i e n t symbiosis i s given the r e s t r i c t e d meaning as one in 14 which the mycobiont helps to improve growth and survival of recently outplanted seedlings. 2.2 Mycorrhizal colonization potential Mycorrhizal colonization potential broadly involves two factors: f i r s t , proximity of growing roots and viable fungal inocula referred to here as mycorrhizal inoculum potential (MIP); second, environmental conditions that can determine the timing and the extent of mycorrhiza formation, and the species of fungi involved. Root growth capacity The a b i l i t y to produce new root i s needed for seedlings to establish after outplanting (Linderman 1987). In dry areas there may only be a narrow planting window in which environmental conditions are favorable for root growth. Moisture stress can develop in conifer seedlings even when the weather i s cool and s o i l moisture i s at f i e l d capacity (Burdett 1987) . Root growth capacity (RGC) i s one of the physiological indicators of vigor of seedlings leaving the nursery and of the a b i l i t y of outplanted seedlings to avoid moisture stress. RGC i s affected by seedlot, l i f t i n g date, nursery climate, nursery c u l t u r a l practices, and cold storage duration (Lavender 1964; Sutton 1980). RGC usually refers to the a b i l i t y to produce new roots, not to add g i r t h to old roots although t h i s i s involved. New roots are less than 1 mm in diameter and are ca l l e d the fine 15 roots or the feeder roots because they are most active in nutrient absorption. Some of the fine roots of many conifers (e.g., pines, spruce, Douglas-fir) have determinant growth, that i s , they only grow and function so long before abscission. Such roots are ca l l e d short roots (Bogar and Smith 1965; Danielson et al. 1984b; Brundrett et al. 1990). A l l the fine roots of other species (e.g., hemlock, cedar) continue to grow, for perhaps the entire l i f e of the plant, and do not form diffe r e n t i a t e d short roots. Although i t i s the fine roots that become mycorrhizal, studies on RGC usually do not mention mycorrhizal status. The presence of soluble carbohydrates such as sucrose in the roots i s important for mycorrhizal colonization to occur (Bjorkman 1970). Marx et al. (1977) found that variation in the sucrose content of short roots could account for 85% of the variation in the intensity of mycorrhiza formation. Although new root growth of transplanted Douglas-fir seedlings uses primarily current photosythate and i s related to l i g h t intensity, under harsh s i t e conditions the le v e l of stored reserves becomes more important to the production of new roots (van den Driessche 1987). Seedlings with higher RGC may have higher mycorrhizal colonization potential because soluble carbohydrates fuel the production of new root tissue. High levels of phosphorus and nitrogen may decrease the sucrose content of the fine roots and lower mycorrhizal colonization levels (Hatch 1937; Harley and Smith 1983). Endogenous levels of ethylene and/or auxin are also important in new root 16 formation. Mycorrhizal fungi that produce these hormones may increase root growth capacity (Linderman 1987). It i s possible that high RGC without mycorrhizal colonization may cause nutrient deficiencies (Burdett 1987). Mycorrhizal colonization level The a b i l i t y of roots to explore large volumes of s o i l soon after outplanting greatly increases the chances of coming into the v i c i n i t y of fungal inocula (Amaranthus and Perry 1987) . The number of propagules in the s o i l may affect the rate and the extent of i n i t i a l colonization of the root system. Patterns of mycorrhizal colonization usually follow a sigmoidal curve with an i n i t i a l lag phase followed by rapid colonization of most of the fine roots, and then an attenuation phase where most of the new roots are colonized as soon as they are produced. Thus, once mycorrhizal colonization has begun i t may soon spread over the entire root system at a rate largely dependent on environmental conditions. Therefore, the mycorrhizal colonization level several months after i n i t i a l colonization i s not related to the density of fungal inoculum in the s o i l (Harley and Smith 1983) . Kropp (1982) found mycorrhizal colonization of western hemlock increased gradually through the season from a few in the f i r s t 2 to 3 months to nearly t o t a l colonization by f a l l (5 1/2 months). Beese (1987) found colonization of Douglas-f i r was 90 to 95% after the f i r s t growing season (after about 17 7 months) on a variety of s i t e s varying in burn intensity. Although fine roots of ectomycorrhizal plants eventually become f u l l y colonized, the fine roots of western red cedar may only become about one-third colonized by VA mycorrhizal fungi after 3 to 5 months in s o i l (Parke et al. 1983c; Beese 1987) . Thus, i t may take more than one growing season in the f i e l d before the attenuation phase i s reached for western red cedar. Measurement of mycorrhizal colonization l e v e l at a single point i n time i s generally inadequate for assessing response to environmental variables (Parke 1985). For example, i f sampling occurs at the attenuation phase of mycorrhizal colonization of a l l treatments then no s i g n i f i c a n t differences are l i k e l y to be detected. If the rate of mycorrhizal colonization i s different between two s i t e s i t may be possible to f i n d differences in colonization levels i f sampling occurs before the attenuation phase on one of the s i t e s . But another reason for a lack of s i g n i f i c a n t differences between treatments might be because measures of mycorrhizal colonization are usually highly variable p a r t i c u l a r l y in the rapid colonization phase (e.g., Beese 1987; Wilson et al. 1987) . Mycorrhizal colonization level i s usually not correlated with seedling perfomance because there are many other factors involved (Wright 1964; Beese 1987). The main problem with comparing mycorrhizal colonization level with seedling growth i s that any factor that affects root growth w i l l almost 18 c e r t a i n l y affect the measured level of colonization. It i s very d i f f i c u l t to separate t h i s from factors that d i r e c t l y af f e c t fungal colonization and growth (Harley and Smith 1983). Furthermore, the formation of mycorrhizae can a f f e c t root morphology and allow for increased shoot growth without differences i n percentage colonization. For example, Alexander (1981) found weight of mycorrhizal plants was not related to colonization l e v e l , but was correlated with the t o t a l number of short roots. However, mycorrhizal colonization level i s sometimes correlated with seedling growth (see references in Wilson et al. 1987). Gessner and Zare-Maivan (1985) found a correlation between the number of mycorrhizal t i p s and root biomass. Holden et a l . (1983) found a strong positive relationship between plant size and mycorrhizal colonization i f over 40 to 60% of the root t i p s were colonized. Grossnickle and Reid (1982) found that even low levels of mycorrhizal colonization by some mycobionts produced a good growth response. Perry et al. (1987) suggest that the rate of mycorrhiza formation i s at least as important as l e v e l of colonization and that performance i s probably related to mycobiont d i v e r s i t y . The r e l a t i v e e f f i c i e n c y of the mycobionts in nutrient uptake and water transport and the extent of t h e i r hyphal network in s o i l may be more important than mycorrhizal colonization level (Parke et al. 1984). 19 Environmental conditions and mycorrhiza formation The leading cause of f i r s t - y e a r mortality of outplanted conifer seedlings i s usually moisture stress (Livingston and Black 1987; Parke et al. 1983a). The period following outplanting in which seedlings have to form mycorrhizae may be short i n dry climates or microclimates of the outplanting s i t e . However, lack of s o i l moisture in the spring i s usually not a problem. Livingston and Black (1987) found shade cards increased seedling survival, but i r r i g a t i o n had no effect indicating transpiration rates of the shoot could not be maintained by the r e l a t i v e l y small root system. Nambiar et al. (1979) found the water stress experienced by pine seedlings for several weeks after outplanting was due primarily to the suppressive effect of low s o i l temperature on root regeneration. There i s increasing interest in those factors that influence the formation and e f f i c i e n c y of mycorrhizae. S o i l parameters such as aeration, moisture content, temperature, pH, f e r t i l i t y , and organic matter have an important role. Many, i f not a l l these factors are affected by clearcutting and s i t e preparation (Parke et al. 1983c; Amaranthus and Perry 1987; Perry et al. 1987). Because many other authors have reviewed the environmental factors affecting mycorrhiza formation (e.g., Bowen and Theodorou 1973; Slankis 1974; Harley and Smith 1983), they w i l l only be mentioned here and b r i e f l y discussed in r e l a t i o n to clearcutting and s i t e preparation. 20 S o i l s in clearcuts experience greater diurnal temperature fluctuations than in forests and may dry faster i n the spring and between r a i n f a l l s (Pritchett and Fisher 1987) . Dry s o i l may l i m i t the a b i l i t y of mycorrhizal fungi to germinate and grow which would lower the chance that planted seedlings w i l l become quickly colonized (Slankis 1974). Mycorrhizal fungi vary greatly in the i r a b i l i t y to survive or grow at low s o i l water potentials (Harley and Smith 1983). Although some fungi, such as Cenococcum geophilum Fr. are able to colonize roots under extreme conditions, dry conditions discourage the formation of mycorrhizae by most fungi (Bowen and Theodorou 1973; Harley and Smith 1983). However, once mycorrhizae are formed, many mycobionts can grow at water potentials well below the permanent wil t i n g point of t h e i r host and t h i s may contribute to increased drought tolerance of mycorrhizal seedlings (Bowen 1973; Theodorou 1978). Slash burning may greatly reduce the amount of organic matter on a s i t e and the high temperatures may k i l l some forms of inoculum (Wright 1971; Lamb and Richards 1974). With blackened s o i l surface, more solar energy i s absorbed. With the insulating layer of organic matter gone, heat i s conducted deeper into the mineral s o i l which has a higher thermal conductivity than does duff or organic s o i l (Pritchett and Fisher 1987). Higher s o i l temperatures may allow for increased root growth e a r l i e r in the spring and higher mycorrhizal colonization at greater s o i l depth (Wright and Tarrant 1958; Mikola et al. 1964; Wright 1971). However, 21 Harvey et a l . (1980) found clearcutting reduced the numbers of active mycorrhizae 7.5 m into an adjacent stand. Mycorrhizal fungi, at least in recent clearcuts, do not appear to have increased tolerance to high temperatures compared to fungi in undisturbed forest s o i l (Parke et a l . 1983b). The mycelial death point for many fungi i s around 41°C (Harley and Smith 1983) . On a hot, sunny day, the surface s o i l temperature in clearcuts with southwest aspect may well exceed 55°C which i s the l i m i t for protein denaturation and w i l l probably destroy many forms of fungal inoculum (Bowen and Theodorou 1973). However, basidiospores of some ectomycorrhizal fungi can tolerate temperatures as high as 60°C and then are able to germinate over wide pH, r e l a t i v e humidity, and temperature ranges (Schramm 1966; Lamb and Richards 1974). Parke et al. (1983b) found heat treatment of s o i l s at 35°C for one week did not affect mycorrhizal inoculum potential (MIP) suggesting not a l l inoculum forms would be destroyed at deeper rooting depths by solar heating. The optimum growth temperature of many ectomycorrhizal fungi from temperate regions grown in culture i s between 15 and 27°C (Harley and Smith 1983; Cline et al. 1987). Although Harley and Smith (1983) state that mycorrhizal colonization by VA mycorrhizal fungi generally increases up to about 30°C, Parke et al. (1983b) found mycorrhiza formation by both VA and ectomycorrhizal fungi to be severely r e s t r i c t e d at 29.5°C. Maximum mycorrhiza formation occured on Douglas-fir and VA mycorrhizal clover under greenhouse conditions at 18.5°C in 22 s o i l s from clearcut and undisturbed forests i n southwest Oregon. Parke et al. (1983b) found both VA and ectomycorrhizae could form at 7.5°C. The lack of mycorrhizae at lower temperatures may be due to lack of root or fungal growth (Parke 1985). Root growth can decrease substantially at temperatures below 10°C for a number of boreal species (Grossnickle 1987). Conifer root growth i n southwest Oregon begins i n early spring when temperatures reach about 5.5°C (Cleary et al. 1978), and optimal root growth of Douglas-fir occurs at about 20°C (Lavender and Overton 1972). Summer drought i n Oregon l i m i t s root growth to cooler, wetter periods of the year (Parke et al. 1983b); however, root elongation of Douglas-fir seedlings decreases sharply with the lowering of s o i l temperature. Poor f i e l d survival at high temperatures could be caused by suppressed root growth e a r l i e r i n the year at low s o i l temperature resulting in increased s u s c e p t i b i l i t y to summer drought (Lopushinsky and Kaufmann 1984). Parke et al. (1983b) suggest seasonal drought tolerance could be achieved by mycorrhizal fungi able to grow and colonize roots at low s o i l temperatures when moisture i s not l i m i t i n g . Mycorrhizal fungi have high oxygen demands (Harley and Smith 1983). S o i l compaction and water-logging may reduce s o i l aeration (Pritchett and Fisher 1987). Oxygen di f f u s i o n through water i s about 10,000 times slower than through s o i l a i r . Gadgil (1972) found that even intermittent water-logging may have a deleterious effect on the a c t i v i t i e s of Douglas-fir 23 mycorrhizae. Skinner and Bowen (1974b) found compaction of an podzolized forest sand reduced rhizomorph growth from ectomycorrhizae by up to 80%. The effects of s o i l compaction and poor s o i l aeration on root growth are similar and may combine to give lower root weight and mycorrhizal colonization levels. Organic matter and some root exudates are known to stimulate, and sometimes i n h i b i t mycorrhiza formation (Slankis 1974; Harvey et al. 1976; Alvarez et al. 1979; Schoenberger and Perry 1982; Parke et al. 1983c; Rose et al. 1983; Lindeberg 1986). Parke et al. (1983c) found l i t t e r did not affec t mycorrhizal colonization of Douglas-fir or western red cedar, but suggested i t may stimulate seedling growth by containing microorganisms stimulatory to seedling growth or mycobiont metabolic a c t i v i t y and growth. Slash-burning may increase the proportion of s o i l microorganisms that can negatively af f e c t seedling growth (Perry et al. 1987). Mycorrhiza formation can either be stimulated or inhibited by rhizosphere organisms (Slankis 1974; Bowen and Theodorou 1979; Chakraborty et al. 1985; Amaranthus and Perry 1987a; McAfee and Fortin 1988). Mycorrhizal fungi need vitamins and some may use certain amino acids that organic matter may provide (Harley and Smith 1983), but high concentrations of certain l i t t e r extracts may have an inhibitory effect (Rose et al. 1983). Some of the l i t t e r treatment effects observed may relate to mineral nitrogen leaching from the l i t t e r . 24 Optimal pH for most mycorrhizal fungi tested l i e s between 3.5 and 6.5 and there are often large differences between strains (Harley and Smith 1983; Hung and Trappe 1983). Slash-burning may cause a pH increase but i t i s not clear i f the change i s great enough to affect mycorrhizal colonization potential (Wright 1971; Harvey et al. 198 0; Beese 1987; Pr i t c h e t t and Fisher 1987) though i t may af f e c t species of fungi which can only grow within narrow pH l i m i t s . Mycorrhiza formation i s encouraged by low s o i l f e r t i l i t y : there are reports of an inverse relationship between mycorrhizal colonization level and r e l a t i v e phosphorus and nitrogen levels (Hatch 1937; Meyer 1973; Harley and Smith 1983; Black 1985). Burning may cause increases in phosphorus, potassium, calcium, and magnesium which may then be lost through leaching (Grier 1975). Although nitrogen losses due to ammonia v o l a t i l i z a t i o n may be substantial, there may be increases in mineral nitrogen following a burn (Pritchett and Fisher 1987). Nitrate increases following slashburning can depress mycorrhizal colonization (Bowen and Theodorou 1973). The effects of slashburning on a v a i l a b i l i t y of nutrients i s strongly influenced by severity of the burn (Black 1985; Beese 1987). Sources of mycorrhizal inoculum Relatively small amounts of inocula are adequate for e f f e c t i v e l y colonizing seedlings (Pilz and Perry 1984). An important source of inoculum for the formation of mycorrhizae in the P a c i f i c Northwest are small mammals such as the red-25 backed vole (Clethrionomys gapperi). These rodents dig up and eat sporocarps (e.g., t r u f f l e s ) formed by VA and ectomycorrhizal fungi and then leave droppings of fungal inoculum wherever they t r a v e l (Maser et al. 1978). Wind-blown top s o i l , insects, birds, and other mammals are other primary vectors (Perry et al. 1987). Spores of many ectomycorrhizal mushroom species are blown into clearcuts from the surrounding forests. Some thin-walled spores may not survive for long in s o i l i f conditions are not suitable for germiniation. Resting spores may survive for long periods unless l o s t through erosion or leaching (Perry et al. 1987). These resting spores are thick walled and often have a maturation, dormant, and/or a quiescent period (Lamb and Richards 1974; Tommerup 1987). Dormancy or quiescence may break with time or i n response to s p e c i f i c stimuli such as temperature shock or a certain chemical. Spores of many mycorrhizal fungi need growing roots of a host i n order to germinate (Harley and Smith 1983; Tommerup 1987). Some spore inocula may be lo s t i f germination occurs i n response to chemicals from nonhosts. Besides spores, vegetative inoculum also exists in forest s o i l as s c l e r o t i a , dormant hyphae, or hyphae of active mycorrhizal t i p s (Parke et al. 1984). Sclerotia are firm masses of hyphae produced by some species of mycorrhizal fungi, such as Cenococcum geophilum, that can remain viable over periods of unfavorable environmental conditions. The hyphae of mycorrhizae are perennial i f they are supplied with host carbohydrates (Harley and Smith 1983). Some 26 ectomycorrhizal fungi produce cellulase suggesting that they may use cellulos e as a carbon source for maintenance or growth when a host plant i s not available (Bowen and Theodorou 1973; Harley and Smith 1983) . Such fungi may grow slowly and then form mycorrhizae when a host plant i s available depending on how well they survive s i t e disturbances. Some VA mycorrhizal fungi may be capable of limited saprobic growth on dead roots or organic debris and spread independently through s o i l in the absence of suitable host plants (Harley and Smith 1983; Parke et al. 1983b, c; Warner and Mosse 1982) . Tommerup and Abbott (1981) found that hyphae of some VA mycorrhizal species could renew growth from roots that were stored for at least 6 months at a matric potential of -50 MPa. Hyphae emanating from ectomycorrhizae on roots of conifers after clearcutting also may remain viable for several months after root death (Harvey et al. 1980). Although Hacskaylo said (1973) that most ectomycorrhizal fungi are thought not to survive in any form for extended periods in the absence of vigorous tree roots, Ferrier and Alexander (1985) found that hyphae of excised fine roots remain active as long as 2 years in the f i e l d . The invasion of noncrop species and roots from adjacent stands into a clearcut may help to maintain MIP. These VA mycorrhizal and ectomycorrhizal hosts may act as reservoirs of diverse fungal inoculum during crop development (Amaranthus et al. 1987b; Borchers and Perry 1987). Roots from an adjacent stand can also be a source of mycorrhizal inoculum in 27 clearcuts (Harvey et al. 1980). Many herbaceous plants invading clearcuts form VA mycorrhizae (Parke et a l . 1983b), but many of the plants colonizing severely disturbed s i t e s may be nonmycorrhizal (Reeves et a l . 1979; Testier et a l . 1987). Some pioneering noncrop plants such as Dryas, Arctostaphylos, Salix, Betula, and Populus may form ectomycorrhizae with some of the same fungal species as do the conifers (Zak 1976; Molina and Trappe 1982; Harley and Smith 1983). Alnus appears to form ectomycorrhizae with only a limited number of fungi and some are s p e c i f i c to t h i s host and do not form mycorrhizae with conifers (Trappe and Bowen 1979). However, M i l l e r et al. (1987) demonstated that Alnus also has several mycobionts in common with conifers including Thelephora terrestris, Cenococcum geophilum, and Laccaria laccata (Scop.: Fr.) Berk. & Br. Amaranthus and Perry (1987) suggested that interactions of other ectomycorrhizal host species and the i r associated symbionts may be c r u c i a l for the colonization of conifer seedlings in the natural forest environment. Inoculum potential in the organic layers Growing seedlings in greenhouses in s o i l collected from clearcuts and undisturbed forests i s a common means of assessing r e l a t i v e MIP. These "greenhouse bioassays" allow control over environmental conditions that may affect mycorrhizal colonization potential (Parke et al. 1984). However, they may also create a r t i f i c i a l conditions that 28 a f f e c t the r e l a t i v e abundance of mycorrhizal types and colonization levels (Pilz and Perry 1984). Mechanical s i t e preparation often involves the removal of organic layers to create a more favorable rooting medium in the exposed mineral s o i l (Alvarez et al. 1979). Mycorrhizae of older trees are most abundant in the organic layers of forest s o i l s (Mejstrik 1971; Harvey et al. 1976), and t h i s makes the organic layers inoculum r i c h . Although l i t t e r and humus may contain inoculum of both VA and ectomycorrhizal fungi, i t i s not known i f mycorrhizal colonization potential of organic and mineral s o i l r e f l e c t s differences in MIP or differences in aeration, moisture, pH, nutrient a v a i l a b i l i t y , or the a c t i v i t y of microorganisms in the l i t t e r layer. The importance of organic matter as a source of mycorrhizal inoculum probably also depends on the inoculum potential of the mineral s o i l . The l a t t e r i s affected by leaching of spores from the organic s o i l (Trappe and Bowen 1979) and the number of fine root t i p s in the mineral s o i l with viable hyphae. If mycorrhizal inoculum i s present and the factors influencing mycorrhiza formation are favorable, then root colonization may be extensive i n mineral s o i l . Alvarez et al. (1979) found the number of mycorrhizal t i p s per centimeter of root was s i g n i f i c a n t l y greater for f i r seedlings grown in mineral s o i l compared to those grown i n mineral s o i l with organic layers. Perry et al. (1982) found mycorrhizal colonization was not correlated with quantity of s o i l organic material or carbon to nitrogen r a t i o . McMinn (19 65) found 29 greater mycorrhizal colonization near root channels and decaying roots than in adjacent mineral s o i l which suggests organic matter and/or better aeration favor mycorrhiza development. Even i f the s o i l organic matter i s high in inoculum, high phosphorus or nitrogen or the presence of allelochemicals may i n h i b i t the formation of mycorrhizae (Rose et al. 1983). Harvey et al. (1976) found that s o i l organic matter, in the form of s o i l humus or decayed wood, provides an important and stimulatory substrate for the formation and a c t i v i t y of ectomycorrhizae within a mature Douglas-fir -larch timber type in western Montana. Decayed wood may also serve a c r i t i c a l role in supporting mycorrhizae in periods of dryness. Kropp (1982) suggested dead wood may be an important reservoir of mycorrhizal inoculum needed for the establishment of hemlock seedlings in these substrates. Different types of mycorrhizae were more frequent when the seedlings were planted into mineral s o i l , but the same t o t a l percentages of short roots were colonized in both substrates. In under 6 months nearly a l l the fine root t i p s of most seedlings were mycorrhizal. In contrast, Christy et al. (1982) found 61% of the naturally regenerated hemlock seedlings sampled from rotton wood were s t i l l nonmycorrhizal after the f i r s t growing season, but were smaller than those seedlings that did form mycorrhizae. They suggested that the absence of mycorrhizal fungi on rotten logs may be due to antagonism from saprobic fungi or to t h e i r i n a b i l i t y to colonize the substrate unless 30 hemlock roots are present. Different results from these two studies may indicate mycorrhiza formation on container-grown seedlings i s for some reason di f f e r e n t than on naturally regenerated seedlings: there may have been mycorrhizal inoculum in the potting mix (Danielson et al. 1984a) that allowed for high mycorrhizal colonization after the container-grown seedlings were outplanted even though the seedlings appeared nonmycorrhizal before outplanting (see section 2.3). Slash burning effects on mycorrhizal inoculum potential The main objectives of burning clearcuts are to make reforestation easier by destroying slash and ground vegetation, and to reduce the thickness of the l i t t e r layer to allow more heat to be conducted into the mineral s o i l which can improve spring root growth. However, burning greatly a l t e r s physical, chemical, and b i o l o g i c a l properties of the s o i l (Trappe and Bowen 1979; Bissett and Parkinson 1980; Pritchett and Fisher 1987). Although the effects are not necessarily permanent (Wright 1971; Amaranthus and Perry 1987) , the findings of many authors (e.g. Wright and Tarrant 1958; Mikola et al. 1964; Wright 1971; Harvey et al. 1980; Schoenberger and Perry 1982; Perry et al. 1982; Parke et al. 1984; McAfee and Fortin 1986; Amaranthus et al. 1987b) generally agree that burned clearcuts have lower levels of inoculum than do unburned clearcuts. Wright and Tarrant (1958) found naturally established Douglas-fir i n burned clearcuts had lower mycorrhizal 31 colonization than those in unburned clearcuts. Harvey et a l . (1980) found the numbers of "active" mycorrhizal t i p s to be zero by the summer after a f a l l burning of a 1 year old clearcut. To take advantage of residual ectomycorrhizal mycelium as inoculum they suggested outplanting be done before July of the following season. L i t t l e i s known about how long other forms of inoculum can remain viable i n the absence of l i v i n g hosts and which are most affected by slash burning (Parke et al. 1984). Unfortunately, studies on the effects of clearcutting and slash burning on MIP usually only involve greenhouse bioassays with no follow-up f i e l d confirmation of the trends observed. Those studies that have involved, both pot and f i e l d bioassays frequently have c o n f l i c t i n g results. For example, P i l z and Perry (1984) found certain types of mycorrhizae in greater abundance in burned clearcuts than unburned clearcuts; however, t h e i r greenhouse test of MIP of s o i l s from these same areas showed the opposite. Although greenhouse bioassays may provide a crude measure of MIP, the factors affecting mycorrhizal colonization potential by di f f e r e n t types of mycobionts probably change. Parke et al. (1983b) found no s i g n i f i c a n t differences in MIP between 1.5 year old clearcuts and a undisturbed s i t e . Greco (1978, in Beese 1987) found higher mycorrhizal colonization of bare root Douglas-fir seedlings planted in burned s i t e s than unburned s i t e s . In a greenhouse bioassay Black (1985) found a l l the seedlings formed mycorrhizae but 32 percentage colonization increased with burn intensity. This may have been related to phosphorus deficiency of the hard burn s o i l : the symbiosis appeared to benefit Douglas-fir seedlings under P l i m i t i n g , but not under N or N and P l i m i t i n g conditions. Parke et al. (1984) found 20% fewer mycorrhizae on seedlings in s o i l collected from unburned clearcuts than on seedlings grown in s o i l from adjacent stands. Seedlings grown in s o i l from burned clearcuts formed 40% fewer mycorrhizae. Formation was most rapid on seedlings grown in s o i l from the adjacent stands. The average age of the 36 " d i f f i c u l t to regenerate" s i t e s sampled was 9.4 years and colonization levels were not correlated with clearcut age. In contrast, P i l z and Perry (1984) found the diminished MIP in older clearcuts was not noticed in recently logged s i t e s , and suggested prompt regeneration may be important to ensure adequate formation of indigenous mycorrhizae. P i l z and Perry (1984) also suggested that mycorrhizal nursery stock may be more useful on older or severely burned clearcuts. The speed of mycorrhiza formation may be important on d i f f i c u l t to regenerate si t e s because there may only be a few weeks in which conditions are favorable for both root and fungal growth (Perry et al. 1987). Although many nonmycorrhizal fungi are often seen f r u i t i n g after a f i r e (Bissett and Parkinson 1980), the effects of burning on species richness of mycorrhizal fungi remains a mystery. Several studies have indicated that there are s h i f t s in the proportions of ectomycorrhizal types formed on seedlings after 33 a disturbance (Wright 1971; Schoenberger and Perry 1982; Parke et al. 1983b; P i l z and Perry 1984; Perry et al. 1987). This may be due to changes in environmental factors that allow mycorrhiza formation involving some fungi but not others (Perry et al. 1987). Clearcutting and burning may also influence the frequency and types of VA mycorrhizae. In many studies VA mycorrhizae types are recognized as either coarse (about 200 species') or fine (Glomus tenue) endophyte (Harley and Smith 1983) . In greenhouse bioassay using s o i l from an unburned clearcut and clearcuts that varied in burn intensity, the highest VA mycorrhizal colonization levels occurred on western red cedar that was grown in the low intensity burn s o i l . However, fine endophyte was r e l a t i v e l y more abundant i n severely burned s o i l (M. Curran, in Beese 1987) . The second part of th i s study involved a f i e l d bioassay of western red cedar planted into actual s i t e s varying in burn intensity. Although several of the environmental factors were very di f f e r e n t , the f i e l d and the greenhouse bioassay showed similar trends in mycorrhizal colonization among f i r e treatments, but more seedlings formed mycorrhizae in the f i e l d . The formation of VA mycorrhizae was not s i g n i f i c a n t l y affected by severity of burn but trends suggest a s l i g h t decrease in the percentage of seedlings and roots per seedling colonized due to increased burn intensity. In agreement with the greenhouse bioassay, the percentage of seedlings with fine endophyte nonsignificantly increased with severity of burn (Beese 1987). Colonization of roots and the 34 percentage of seedlings with VA mycorrhizae decreased with burn intensity and was attributed to the loss of organic layers that may have contained most of the inoculum. In another f i e l d study (Parke et al. 1983a), fine endophyte was the only type that colonized the roots of western red cedar in s o i l s with organic layers removed. Parke et al. (1983b) found fine endophyte was by far the dominant mycobiont present on VA mycorrhizal hosts invading 1.5 year old clearcuts and in undisturbed forest s o i l . Fine endophyte was also the only type of VA mycorrhiza on western red cedar in the greenhouse using s o i l from clearcut and undisturbed forest (Parke et al. 1983c). Glomus tenue appears to be important in higher altitudes and most rigorous or pioneer conditions that often involve quite low phosphorus conditions (Parke et al. 1983b; Harley and Smith 1983). Wang et al. (1985) found that coarse endophytes occur in pH of 5.5 to 7.5, but only fine endophyte could colonize roots at s o i l pH 4.5. If there i s a r i s e in s o i l pH following burning then one would expect the r e l a t i v e abundance of fine endophyte to decrease. Beese (1987) stressed that most studies give l i t t l e or no information about the nature of the prescribed f i r e treatments and that differences in s o i l c haracteristics between two burned s i t e s can be greater than differences between a burned and unburned s i t e . Ectomycorrhizal d i v e r s i t y in southwest Oregon i s correlated with s i t e productivity and e f f i c i e n t mycorrhiza formation after disturbance (Perry 1985). Tree age, season, 35 climate, s o i l conditions, and human disturbance may af f e c t the kinds and r e l a t i v e abundance of mycorrhizal types (Chu-Chou and Grace 1987) . Mycorrhizal fungi can be early, late, or multi-stage i n the succession of types that may occur as a seedling ages (Dighton and Mason 1985; Chu-Chou and Grace 1987; Danielson and Pruden 1989). This suggests that adjacent forests are important as an inoculum source for the clearcut plantation as i t matures. Studies of ectomycorrhizal d i v e r s i t y changes with respect to forestry practices are needed. For example, Browning and Whitney (1987) applied the Shannon-Wiener di v e r s i t y index to relate the d i v e r s i t y of ectomycorrhizal types on pine with differences between 6 si t e s in Ontario. Mycobionts d i f f e r in a c t i v i t i e s such as nutrient uptake, disease suppression, and decomposition a b i l i t y (Harley and Smith 1983). Each mycobiont may function in i t s own manner under a given set of environmental conditions to influence seedling growth and survival and ecosystem processes (Perry et al. 1987). Harsh or d i f f i c u l t to regenerate s i t e s are those where the available period for seedling establishment and favorable growth i s r e l a t i v e l y short (Amaranthus and Perry 1987). On such s i t e s , the rate of mycorrhiza formation and d i v e r s i t y of mycobionts present i s l i k e l y to be as important as the numbers of mycorrhizae that form. Although organic matter may contain abundant inoculum, factors influencing the formation of mycorrhizae on outplanted seedlings may not be favorable in t h i s rooting medium on some s i t e s . Perry et al. (1987) 36 suggests one of three scenarios may exist after clearcutting and s i t e preparation: MIP i s not reduced and therefore reforestation f a i l u r e or success i s not related to i t ; MIP i s reduced but outplanted seedlings survive and MIP i s restored; MIP i s reduced, mycorrhizal hosts are absent, and planted seedlings do not survive (due to low MIP or harsh s i t e conditions) which allows for further reductions i n MIP with time and makes reforestation more d i f f i c u l t . Older, burned clearcuts that have been successfully reforested have abundant inocula (Amaranthus and Perry 1987). Conclusion P r a i r i e and steppe s o i l s may be d e f i c i e n t in ectomycorrhizal inoculum (Harley and Smith 1983), but i t i s hard to imagine that i f mycorrhizal inoculum can be abundant on highly disturbed s i t e s such as coal spoils (Schramm 1966; Daft and Hacskaylo 1977; Grossnickle and Reid 1982), that there could be any deficiency on clearcuts in the P a c i f i c Northwest which has a diverse array of fungal species. From the few studies that have assessed MIP in the P a c i f i c Northwest, there appears to be adequate inoculum on recently logged s i t e s that were not severely disturbed. The statement made by Mikola in 1973 that clearcutting does not seriously harm mycorrhizal fungi unless the area remains treeless for a long time generally holds true. Although in a greenhouse bioassay Parke et al. (1984) found clearcut age i s not related to MIP, other authors found that clearcuts lacking 37 ectomycorrhizal hosts for several years are more l i k e l y to have lower MIP than recent clearcuts with organic matter removed by scalping or burning (Perry et al. 1987). However, some reports do suggest MIP i s affected by s i t e preparation (e.g., Amaranthus et al. 1987b) as shown by decreases in i n i t i a l mycorrhizal colonization levels of seedlings planted in these s o i l s . Bledsoe et al. (1982) suggested preplanting surveys of s i t e inoculum potential may be useful because the s i t e s they studied i n eastern Washington had plenty of natural inoculum even though they were burned and r e l a t i v e l y dry. However, even i f the inoculum potential i s adequate, container grown seedlings are switching from a situation where they r e a l l y do not need mycorrhizae to conditions encountered after outplanting where they need to rapidly form mycorrhizae with suitable fungi in order to survive. In general, the mycorrhizal colonization potential of outplanted seedlings i s better on recently logged clearcuts that were not severely burned, but d i f f i c u l t to regenerate s i t e s may require seedlings pre-equipped with s p e c i f i c mycorrhizae before being outplanted. 2.3 Sylviculture and the management of mycorrhizae. After reviewing the functions of mycorrhizae, Trappe (1977) states that there i s "no room to doubt that the establishment and maintainance of good populations of 38 appropriate mycorrhizal fungi must become an integral part of good nursery management." Mycorrhizal nursery stock It i s well accepted that trees grown i n s o i l need mycorrhizae but very few nurseries ensure that t h e i r stock i s mycorrhizal before outplanting or soon afterwards. Although other factors affecting seedling growth often obscure the mycorrhizal component, high levels of mycorrhizal colonization involving proven b e n e f i c i a l fungi can greatly improve reforestation success even on routine reforestation s i t e s (e.g., Trappe 1977; Marx 1980). Several authors (e.g., Mikola 1973; Molina 1977, 1981a; Pregent and Hawey 1988; Kropp and Langlois 1990) have reviewed the use of mycorrhizal seedlings for reforestation. Techniques for mycorrhizal inoculation in nurseries, and sources of commercially-produced inoculum are found elsewhere (e.g., Molina 1977; Trappe 1977; Marx 1980; Molina and Palmer 1982; Castellano et a l . 1985; Hung and Molina 1986; Perry et al. 1987). Trappe and Strand (1969) demonstrated the necessity of mycorrhiza formation for adequate phosphorus uptake by Douglas-fir seedlings i n a bare root nursery. Without a r t i f i c i a l inoculation, mycorrhiza formation i s usually slow and e r r a t i c . The use of seedlings inoculated with Pisolithus tinctorius to enhance seedling survival of Pinus in southeastern U.S. i s now well established (Marx and Cordell 1988). A Georgia is o l a t e and l o c a l strains of P. tinctorius were tested in Oregon (Molina 1979; Perry et 39 al. 1987). Although P. tinctorius strains isolated i n Oregon increased seedling survival on hot s i t e s over nonmycorrhizal controls (Molina 1981), inoculation and outplanting tests in the P a c i f i c Northwest using t h i s fungus were mostly negative or at least nonsignificant (Castellano and Trappe 1985), and other fungi collected l o c a l l y were t r i e d (see Castellano and Trappe 1985 for species and references). Emphasis switched from trying to find a s p e c i f i c fungus for a l l s i t e conditions to selecting fungal species that might enhance acclimation of seedlings to t h e i r destined planting s i t e . A system using strains or ecotypes of fungi each known to be suited to grow and benefit seedlings under particular s i t e conditions was envisioned (Trappe 1977). I n i t i a l l y , t h i s might involve the c o l l e c t i o n from the s i t e of fungi that are known to be common on young seedlings with exceptional growth. The need for s i t e s p e c i f i c i t y i s not certain for a l l species, but there are reports that d i f f e r e n t strains of the same fungus d i f f e r in t h e i r effects on the host (Mikola 1973; Trappe 1977; Harley and Smith 1983). In bare root nurseries, inoculated Douglas-fir seedlings with Laccaria laccata had greater dry weight gain than noninoculated seedlings ( S i n c l a i r 1971; Molina 1982), but the mycobionts f a i l e d to enhance growth once outplanted. Castellano and Trappe (1985) inoculated two seedlots of Douglas-fir with spores of Rhizopogon vinicolor. After being outplanted for 2 years on dry s i t e s in southwestern Oregon mycorrhizal seedlings had s i g n i f i c a n t l y greater survival, stem 40 height, root c o l l a r diameter, and biomass than uninoculated seedlings. They could not explain why the two seedlots studied had d i f f e r e n t responses to the various inoculation le v e l s . Marx and Bryan (197 0) showed that genotype of the host could affect the l e v e l of mycorrhizal colonization of pine by single isolates of Thelephora terrestris and Pisolithus tinctorius. In a styroblock container nurseries, the formation of mycorrhize may not be necessary to grow seedlings with healthy looking shoots because the roots are p e r i o d i c a l l y flushed with soluble f e r t i l i z e r s and are well watered. High rates of soluble f e r t i l i z e r may l i m i t mycorrhiza development, and there i s concern that seedlings produced t h i s way may have poor growth and survival on some sit e s because they do not have an adequate root system. Some mycobionts w i l l extensively colonize the roots but only under reduced f e r t i l i t y which can res u l t i n smaller seedlings that are unacceptable for planting after only 1 year nursery growth. Managers can learn to monitor seedling roots and mycorrhiza development after a l t e r i n g nursery practices to promote spontaneous formation of mycorrhizae by several mycobionts and improve root morphology without decreased shoot growth. Gary Hunt, Heffley Reforestation Center (personal communication), i n the dry i n t e r i o r of B.C. found mycorrhizae involving a number of dif f e r e n t fungi w i l l form in a container nursery by lowering nutrient application rates and improving the quality of peat used. Seedling size was maintained or sometimes even 41 increased and the root quality was substantially improved: the degree of root branching and number of fine roots greatly increased following mycorrhizal colonization by some fungi. Hyphae helped to bind roots together and make a more s o l i d plug that better retained the potting mix when l i f t e d and was easier to plant. Although these fungi may not spread to new roots once outplanted, they improved seedling quality with minimal cost input. S i n c l a i r (1974) found that Douglas-fir seedlings in nursery beds grew better with two mycobionts than just one. Perry et al. (1987) suggests that seedlings with many mycorrhizal types before outplanting may have greater survival potential than those with one or a few dominant fungi. Danielson et al. (1984a) found that peat collected from the f o o t h i l l s of eastern Rocky Mountains contains ectomycorrhizal and VA mycorrhizal inoculum. Prior fumigation of the potting mix and the use of fungicides destroys mycorrhizal inoculum (Molina 1977b); however, spores blown in from surrounding forests may s t i l l allow for mycorrhiza formation (Marx and Davey 1969). Benomyl, a selective fungicide may protect roots against root-parasitic ascomycetes in the nursery while allowing mycorrhizal basidiomycetes to grow and sometimes improve seedling growth (Alvarez and Linderman 1983; de l a Bastide and Kendrick 1987). Mycorrhizal fungi are an additional photosynthate sink for the seedling that may result in decreased seedling size. The idea of comparing different mycobionts on the basis of 42 cost/benefit to the seedling arose. Some species l i k e Laccaria laccata may not affect seedling size and not improve the survival and growth when outplanted under the conditions tested. Rhizopogon vinicolor may benefit Douglas-fir by producing abundant rhizomorphs with large core hyphae that may improve water relations (Parke et a i . 1983a), but with a cost to the seedling i n terms of lower shoot weight. The presence of other species and particular environmental conditions may af f e c t the results (Perry et al. 1987). Bledsoe et al. (1982) produced 2 year old container-grown Douglas-fir seedlings inoculated with Hebeloma crustuliniforme (Bull.rSt. Am.) or Laccaria laccata. Within 5 months of outplanting i n eastern Washington the inoculated species were "out-competed" by indigenous mycobionts (new roots rapidly became colonized by native mycorrhizal fungi) and seedling weight was lower for inoculated seedlings than for controls after 1 year. They suggested the inoculated fungi may not have competed well with indigenous fungi because they were isolated from wetter si t e s west of the Cascades and stressed that sources of fungal inoculum should be suited to planting s i t e conditions. Commercially produced inoculum of L. laccata i s available and tested (Molina 1982; Molina and Chamard 1983; Hung and Molina 1986). Rhizopogon luteolus Fr. & Nordh. inoculations have enhanced seedling growth in the southern hemispere (see Castellano et al. 1985 for references). In southwestern Oregon, Castellano et a l . (1985) inoculated container-grown 43 Douglas-fir seedlings with basidiospores of R. v i n i c o l o r and R. colossus Smith and found slow release f e r t i l i z e r (Osmocote) suppressed mycorrhiza formation by both fungi to less than 10%. R. v i n i c o l o r formed mycorrhizae on more than 88% of the feeder roots at a l l basidiospore application rates with both high ("near operational rate") and low levels of soluble f e r t i l i z e r s . Mycorrhizal seedlings were t a l l e r except at high levels of soluble f e r t i l i z e r . Caliper and shoot and root weights (excluding most of the mycobiont) were generally lower than controls. Results d i f f e r e d for the R. colossus inoculated seedlings. Because several studies by other authors have shown that vegetative inoculum and the use of slow release f e r t i l i z e r allow abundant mycorrhiza formation, they suggest i t did not allow for nutrient levels to drop low enough for spore germination. Several other studies have also shown that when substrate f e r t i l i t y increases, mycorrhizal seedlings are frequently smaller than uninoculated seedlings (see Pregent and Hawey 1988). Containerized Douglas-fir seedlings grown in Cottage Grove Nursery and outplanted onto dry s i t e s i n Oregon are now routinely inoculated with spores of Rhizopogon v i n i c o l o r . There was good mycorrhizal colonization in the nursery and inoculated seedlings had greater basal area growth and 4 to 7 times more root t i p s after one year of being outplanted. After two f i e l d seasons, inoculated seedlings also showed higher survival and height growth (P. Hahn, personal communication). 44 Spores of VA fungi are not wind dispersed except perhaps when t o p s o i l i s blown (Perry et al. 1987). Cedar i s generally nonmycorrhizal in container nurseries, but there i s some indication that low levels of inoculum may be present in the peat (Danielson et al. 1984a; Beese 1987). Morgan (1985) suggested chopped cedar roots added to the potting mix could be used as inoculum. S t e r i l i z e d peat enriched with VA mycorrhizal inoculum added i s commercially available (Premier Peat Moss, Quebec). However, most fungicides, including benomyl, w i l l distroy the inoculum (Trappe et al. 1984). Mycorrhiza management in the field L i t t e r containing ectomycorrhizal fungi has been used as inoculum for over a century (Trappe 1977). Amaranthus and Perry (1987) have demonstrated that a small amount of s o i l transferred from an established plantation on a clearcut that was burned to the planting holes of a hard to regenerate clearcut in southwest Oregon can greatly improve the survival and growth of outplanted Douglas-fir seedlings. S o i l tranferred from a mature forest did not enhance seedling growth. In the dry i n t e r i o r of B.C., s o i l transfer studies also look promising (Gary Hunt, personal communication). The chance taken with s o i l transfer studies i s that the s o i l tranferred may contain pathogens (Marx 1980), and may or may not be more ben e f i c i a l to seedlings than inoculation with known strains of fungi. Although pathogens have not been a problem with s o i l transfers in Oregon, there i s concern over 45 dispersing propagules of undesirable plant species which may grow and compete with conifers (Molina 1981). 2.4 C l a s s i f i c a t i o n of Ectomycorrhizae The need for classification Many studies only deal with estimates of mycorrhizal colonization. Perhaps more important are the species of fungi involved and the contribution each mycobiont makes to the estimate of percentage colonization. Many studies have shown seedling responses to di f f e r e n t mycobionts can d i f f e r greatly (see references in Trappe 1977) , and t h i s i s an economic reason why recognition of the fungi i s desirable. The strong interest i n managing mycorrhizae for improved growth and survival of outplanted seedlings i s limited by our poor understanding of the taxonomy and ecology of the mycobionts. Two of the basic requirements of any study are to know what organisms are being worked with and to be able to recognize them again. For example, to assess the benefits of a fungus a r t i f i c i a l l y inoculated onto seedlings in the nursery one must be able to recognize the mycorrhiza amongst the other types formed in the nursery and after some period i n the f i e l d . Description, c l a s s i f i c a t i o n , and i d e n t i f i c a t i o n i s an early stage in the study of nature. The study of form i s closely related to physiological functions (Dominik 1969), and i s essential to our understanding of species adaptations to the environment. 46 VA mycorrhizae are usually simply recognized as either fine or coarse endophyte in ecological studies. This i s because of the lack of other morphologically d i s t i n c t features and the d i f f i c u l t i e s involved in ide n t i f y i n g the species. Ectomycorrhizae have high morphological d i v e r s i t y due to the many families of fungi involved and the variety of structures produced. Sometimes i t i s possible to i d e n t i f y mycorrhizae by tracing rhizomorphs between the sporocarp and the mycorrhizae. More often t h i s i s not possible or at best very time consuming. Furthermore, not a l l fungi produce sporocarps, and those that do may only produce them at certain times of the year, may skip years, or may not f r u i t u n t i l the tree i s several years old. So i t i s very desirable to be able to recognize which species of fungi are involved in colonizing the tree roots using only the mycorrhizae collected when i t i s convenient for the researcher. The main d i f f i c u l t y in c l a s s i f y i n g ectomycorrhizae i s the low number of d i s t i n c t i v e morphological characters and the large number of potential mycobionts. Trappe (1977) estimated there are some 2 000 potential mycorrhizal associates of Douglas-fir. Some mycorrhizae, such as Cenococcum geophilum on various hosts, are quite d i s t i n c t i v e and can be easily recognized based even on short descriptions written by other authors. The uncommon or less d i s t i n c t i v e ectomycorrhizae are mostly undescribed and the identity of the mycobionts at best known only to genus. With considerable practice, d i f f e r e n t ectomycorrhizal types, involving one or more species of fungi, 47 can be distinguished on the basis of form, hyphal morphology and arrangement, color, and color changes in certain chemical reagents. The hope i s that some day each mycorrhizal type w i l l be i d e n t i f i e d (fungus plus plant Latin binomials) and well described with a long l i s t of characteristics that w i l l distinguish i t from a l l other types. So far, r e l a t i v e l y few ectomycorrhizae are i d e n t i f i e d and described i n s u f f i c i e n t d e t a i l . Approaches to classification Trappe (1967a), Zak (1973), Godbout and Fortin (1985), and Agerer (1986) reviewed many publications that include attempts to characterize and c l a s s i f y ectomycorrhizae. Relative to the number of potential mycobionts, very few publications have actually i d e n t i f i e d the fungus and characterized the mycorrhizal type i t forms i n s u f f i c i e n t d e t a i l that i t would not be mistaken for another type on the same or d i f f e r e n t host. The main arguments for current approaches to ectomycorrhizal c l a s s i f i c a t i o n are examined h i s t o r i c a l l y so the reader w i l l have a better understanding why a p a r t i c u l a r approach i s taken in the Thesis. Melin (1925, in Trappe 1962) noted that fungi d i f f e r greatly i n t h e i r a b i l i t y to form mycorrhizae and in their effects on plant growth. Melin c l a s s i f i e d t h i s a b i l i t y by a c t i v i t y and virulence l e v e l of the fungus. For example, active fungi of low virulence only form Hartig net. With medium virulence both Hartig net and mantle are formed. High 48 virulence fungi with strong fungal a c t i v i t y leads to one-sided parasitism p a r t i c u l a r l y when host i s weak. Such a c l a s s i f i c a t i o n was not in i t s e l f adequate because of the changes that occur as the symbiosis develops and the potential for a large number of morphologically d i s t i n c t mycorrhizae in each category. Later, Melin (1927, in Zak 1973) divided mycorrhizae into four types and two subtypes based on a few morphological characters. Melin (1923, i n Agerer 1986) f e l t that i d e n t i f i c a t i o n of mycobionts by finding hyphal connections between sporocarps and mycorrhizae i s too uncertain because of the d i f f i c u l t i e s involved in tracing the path. After about three decades of examining mycorrhizae of natural plant associations, Dominik (1959) created one of the f i r s t keys to the types of ectomycorrhizae. The i n i t i a l d i visions of his dichotomous key were based on mantle structure: simple mantles were either f e l t - l i k e (prosenchymous) or pseudoparenchymous (synenchymous) i n cross-section (Appendix 6). Although he did not define these terms (they are defined elsewhere e.g., Dominik 1969), each pattern was shown in diagrams of cross-sections of mycorrhizae. Two layered mantles were those with a simple mantle d i r e c t l y over the r o o t l e t and another external to t h i s that encloses the entire short root. These key divisions lead to subtypes that are distinguished primarily on the basis of mantle structure and the hyphae emanating from the mantle. Each subtype i s then further divided into "genera" based primarily on color 49 and/or further characteristics of the emanating hyphae. Dominik noted that some colors disappear in alcohol and cautioned of changes as the mycorrhizae age. This l a t e r point i s the main c r i t i q u e others (e.g., Zak 1973; Agerer 1986) had of Dominik's c l a s s i f i c a t i o n : as the roots age or grow in dif f e r e n t s o i l conditions a genus designation may change i f i t s color i s influenced by that of the root. Thus, the same fungus may take part in forming the mycorrhizal associations of d i f f e r e n t subtypes and genera. Agerer (1986) considers that establishment of a key based largely on color characters seen in cross-section ignores many other p o t e n t i a l l y useful characters. Unless the user of the key has years of experience with mycorrhizae, Dominik said his key should only be applied to mature mycorrhizae. Early stages of mycorrhizal colonization may lack important characteristics which l i m i t s even the simplest c l a s s i f i c a t i o n scheme (e.g., Kropp 1982; Parke et al. 1983b; P i l z and Perry 1984). In 1969, Dominik enlarged his key based on similar divisions, but expanded on de t a i l s of the c y s t i d i a . Although the main problems of the e a r l i e r key remain, such as lack of symbiont identity, the key i s often used to designate particular mycorrhiza types collected in the f i e l d (e.g., Marks 1965; Mejstrik 1971). Trappe (1965) described morphological and anatomical features of a mycorrhizal type on Douglas-fir that forms tubercles which corresponds to .Dominik's two layered mantle type. Notes were made on the size, color, texture, and bruising of the tubercle and the fine roots contained within. 50 Stained sections revealed that two very d i f f e r e n t hyphal types were involved. Mantle hyphae were hyaline, r e l a t i v e l y wide, and of basidiomycete a f f i n i t y . Those hyphae emanating from the mantle and forming the rind or tubercle were pigmented, nonsepate, and reached only 3 um in diameter. Notes on the development and occurrence were also made but no fungus was i d e n t i f i e d or grown in culture. Zak (1971) examined the tuberculate mycorrhiza further and shown that one fungus, Rhizopogon vinicolor, formed both hyphal types. The characterization was more detailed and included color under u l t r a v i o l e t l i g h t , color changes in several chemical reagents, and descriptions of the fungus isolated from the mycorrhiza when grown on various media. Mycobiont i d e n t i f i c a t i o n was suggested by three clues: f i r s t , the presence of R. vinicolor sporocarps found close to the mycorrhizae; second, the s i m i l a r i t y of cultures grown from sporocarp and mycorrhiza fragments in terms of morphology and color even under u l t r a v i o l e t l i g h t and in response to chemicals; t h i r d , the similar mantle char a c t e r i s t i c s of f i e l d c ollected and synthesized mycorrhizae. It may also be possible to trace rhizomorphs d i r e c t l y from the mycorrhiza to the sporocarp, but in most instances t h i s i s very time consuming and tedious (Chilvers 1968). Trappe (1967b) synthesized and then described different types of Douglas-fir mycorrhizae using four species of fungi in four genera. Detailed notes included: 1) gross morphology (color, texture due to emanating hyphae, occurrence of 51 rhizomorphs, branching pattern, s i z e ) , 2) mantle structure (hyphal organization, thickness), 3) mantle hyphae (at dif f e r e n t depths and emanating from the surface: wall thickness, hyphal diameter, pigmentation, septation, presence of clamp connections, granules, and H-shaped fusions between hyphae), 4) rhizomorphs (color, branching pattern, diameter, hyphal characteristics as above), 5) Hartig net (hyphal charact e r i s t i c s , depth into cortex penetrated, root c e l l separation due to presence), 6) i n t r a c e l l u l a r penetration, 7) root anatomy (arches of xylem, c e l l sizes in cortex), and 8) color changes due to chemical reagents. Thus, pure culture sythesis under standard conditions could be used to accurately characterize mycorrhizae. Assuming the morphology of mycorrhizae grown i n pure culture i s the same or very similar to mycorrhizae formed under f i e l d conditions, f i e l d collected mycorrhizae can be compared and possibly i d e n t i f i e d . There i s a general uniformity of mycorrhiza morphology on different hosts due to one mycobiont (Alexander 1981), but host identity might be important because d i f f e r e n t plant species may induce formation of st r u c t u r a l l y d i f f e r e n t mantles by the same fungus (Zak 1973; Molina and Trappe 1982). Although di f f e r e n t strains of well defined species of fungus tend to produce morphologically i d e n t i c a l mycorrhizae (Marx and Bryan 1970; Alexander 1981), chemically and physiologically the mycorrhizae may be quite d i f f e r e n t (Zak 1969; Trappe 1977; Zak and Larsen 1978). Microscopic characters tend to remain constant regardless of s o i l 52 compaction or moisture (Godbout and Fortin 1985) , but s o i l type and chemistry may be responsible for minor changes in mycorrhiza morphology (Zak 197 3). Pure culture synthesis of mycorrhizae can confirm s p e c i f i c host/fungus associations, which can give insights into the ecology of both symbionts; i t provides material for physiological studies and permits the comparison of cost/benefit of diff e r e n t mycobionts and fungal strains to host growth. Growth characteristics of mycobionts on dif f e r e n t media may prove very useful in the c l a s s i f i c a t i o n of mycorrhizae (Trappe 1967b). Agerer (1986) provided many references for detailed descriptions of synthesized mycorrhizae and mentioned some l i m i t i t i o n s of such an approach. These include the fact that d i f f e r e n t fungi can have very similar characteristics when grown in pure culture, and many ectomycorrhizal fungi cannot be cultured. According to Trappe (1967a) the construction of a key used to identif y fungi forming mycorrhizae should be based primarily on stable hyphal characteristics, such as de t a i l s of clamp connections (when present) and septal pores. Less constant features, such as color and form of the mycorrhizae should be used only after stable characteristics are a l l used. Trappe (1967b) states, "Morphology and anatomy of mycorrhizae of specific ectotrophs should be described in detail i f c l a s s i f i c a t i o n s of and relationships between d i f f e r e n t types are ever to be systematically defined." However, Dominik (1969) defended his approach as being more useful to 53 s y l v i c u l t u r e and forest ecology d i s c i p l i n e s : time spent producing keys to ectomycorrhizal types based on morphological and anatomical characteristics f i r s t , with eventual mycobiont i n d e n t i f i c a t i o n (e.g., through association of sporocarps with s p e c i f i c mycorrhizae) should be given p r i o r i t y . The alternate approach i s to work f i r s t on the i d e n t i f i c a t i o n of the symbionts (through methods such as pure culture synthesis involving spores of i d e n t i f i e d fungi). This requires that a great deal of time be spent culturing fungi i n the laboratory, and in Dominik's opinion, gives few insights into the ecology of mycorrhizae. To identify, culture, synthesize, make comparisons between synthesized mycorrhizae and f i e l d collected mycorrhizae, and then write a detailed decription that covers a l l morphological variants may take months or years for each fungal species. With the large number of fungal species and the vary small number of mycologists working on the problem, Dominik may be partly r i g h t . Zak (197 3) proposed a compromise to the problems of both approaches. This i s to describe ectomycorrhizae as f u l l y as possible and then name i t preferably after identifying the fungus. But i f the mycobiont i s not known then the mycorrhiza can be given a temporary name in quotes that relates to i t s habit. Many detailed descriptions of mycorrhizae exist whose mycobiont could not be i d e n t i f i e d , and some have been given temporary names (e.g., Chilvers 1968; Agerer 1988). Chilvers (1968) found i t i s possible to identi f y and c l a s s i f y d i s t i n c t i v e mycorrhizae using as many c r i t e r i a as 54 possible when making decisions. Eight d i s t i n c t i v e types of Eucalyptus mycorrhizae were described with the aid of very informative drawings of habit, mantle in plan view, emanating hyphae, rhizomorphs, cross-section and longitudinal section. The roots were cleared of t h e i r c e l l contents (not necessary for subsequent identifications) by heating in lactophenol and whole mounts were made for microscopic examination and characterization. Importance was placed on differences in hyphal structure, pigmentation, and organization i n the mantle. He c l e a r l y defined the terms regular and irregular synenchyma, and net and f e l t prosenchyma (Appendix 5) . These terms were then applied to how the mantle appeared in plan view. More s p e c i f i c a l l y , descriptions included: 1) appearance under dissecting scope (color, branching, number of apices, abundance of emanating hyphae), 2) mantle structure (thickness, and hyphal arrangement and characteristics at various depths), 3) Hartig net (hyphal width and wall thickness), 4) outgrowing hyphae (cystidia separately described i f also present), and 5) rhizomorph anatomy. The main impetus for Chilvers (1968) approach to mycorrhiza characterization came from Dominik's work, but only the mycorrhizae of Eucalyptus from one province of Australia was examined to l i m i t the number of types to be dealt with. Chilvers f e l t mycobiont i d e n t i f i c a t i o n i s desirable but not c r u c i a l before useful conclusions can be made about ecology of mycorrhizae provided each type can be recognized with i t s own set of d i s t i n c t i v e and stable characteristics. Six of the 55 mycorrhizae could not be i d e n t i f i e d and were simply given a number designation. Although the type designations may contain more than one species, Chilvers thought the type designations probably were of closely related fungi possibly belonging to the same genus. This approach allows for r e l a t i v e large numbers of mycorrhizae to be examined and categorized thereby allowing qu a l i t a t i v e as well as quantitative ecological surveys of mycorrhizae. The main drawbacks include not being able to identify the fungus as can be done with sporocarps, and, again, not being sure whether more than one fungus forms a mycorrhizal type. Chilvers suggested "c a l i b r a t i n g " t h i s approach by using i t in conjunction with the more time-consuming methods that involve mycobiont i d e n t i f i c a t i o n and then judge t h e i r uniqueness. This would help to determine important characteristics of each morphological type of mycorrhizae, i f there i s morhological consistancy for di f f e r e n t hosts growing on a variety of s i t e conditions, and how many species of fungi are involved. Alexander (1981) f e l t that rapid assessment of types i s essential i f advances are to be made in the quantitative assessment of mycorrhizal populations. Following Chilvers, Alexander concentrated on mantle surface features of cleared roots as an aid in the characterization of a basidiomycete mycobiont, but a method developed for VA mycorrhizae ( P h i l l i p s and Hayman 1970) was used for clearing and staining the ectomycorrhizal roots. 56 Although most members of the Endogonaceae form VA mycorrhizae, several authors have characterized ectomycorrhizae formed by at least three Endogone species and one Glomus species on ectomycorrhizal hosts (Fassi et al. 1969; Chu Chou and Grace 1987; Walker 1985; Warcup 1985). The mycorrhizae formed may appear nonmycorrhizal because the mantle i s usually absent or poorly developed. The roots must be sectioned or cleared of t h e i r contents and stained to f a c i l i t a t e Hartig net characterization. For E. lactiflua Berk. & Br., staining with Lugol blue causes the Hartig net to become an intense v i o l e t while hyphae of other fungi are l i g h t e r blue (Fassi et al. 1969; Walker 1985). In 1973, Zak reviewed the topic of ectomycorrhizal c l a s s i f i c a t i o n and discussed many macroscopic and microscopic characters that may be used to describe ectomycorrhizae. These included habitat, color, form, taste and odor, u l t r a v i o l e t l i g h t fluoresence, color changes to chemical reagents, emanating hyphae, rhizomorphs, mantle texture and textura type. However, few authors characterize mycorrhizae to the extent Zak (1973) suggested or did (Zak 1969, 1971). Chu-Chou and Grace (1983a, 1987) characterized several types of d i s t i n c t i v e mycorrhizae on Douglas-fir aged 9 months to 75 years i n New Zealand. Most of the types were i d e n t i f i e d by comparing cultures of fungi isolated from the mycorrhizae with cultures derived from i d e n t i f i e d sporocarps associated with Douglas-fir. The fungi Schramm (1966) studied were i d e n t i f i e d by selecting tree species with only a few 57 mycorrhizae and comparing mycelia or rhizomorphs of the mycorrhizae with sporocarps. Agerer (1986) noted that such comparisons are much more d i f f i c u l t in forests when many fungi may be involved i n forming the mycorrhizae of a single tree. Chu-Chou and Grace (1983b) produced a dichotomous key to the 13 types of mycorrhizae encountered on pine based on color and emanating hyphae. Froidevaux (1974) i d e n t i f i e d mycorrhizae under Douglas-fir in the coast range of western Oregon Using a number of chemical tests. Voiry (1981, in Godbout and Fortin 1985) presented the concept of morpho-structural series as an aid to ectomycorrhizal c l a s s i f i c a t i o n : species within a genus tend to be similar with respect to mantle compactness and emanating hyphae or rhizomorphs. Danielson (1984a) b r i e f l y described 6 mycorrhizae of pine and mycobiont c u l t u r a l c h a r a c t e r i s t i c s . Agerer (second delivery, 1988) produced the Color Atlas of Ectomycorrhiza with the main aim of showing ectomycorrhizae in t h e i r true color. However, there i s some concern that age of the mycorrhiza and certain s o i l variables, such as pH, may affec t mycelium color of some mycobionts (Zak 1973; Molina and Trappe 1982). The very detailed and lengthy descriptions include: morphological characteristics, chemical tests, anatomical characteristics of plan views of the mantle, anatomical cha r a c t e r i s t i c of emanating elements, and characteristics of cross-sections and longitudinal sections. The more complete descriptions are found i n s c i e n t i f i c journals and the atlas subscriber i s mailed these references 58 along with 10 to 15 color plates per year with t h e i r shorter description and a key to the ectomycorrhizae so far described. When complete there w i l l be 200-300 plates. Agerer (1986, 1988) set a new standard for ectomycorrhizal characterization but others concerned with mycorrhizal types may not have the time, experience, or materials needed to review the pertinent l i t e r a t u r e and then characterize the mycorrhizae in such d e t a i l before they can monitor q u a l i t a t i v e changes in abundance. After many more years when most ectomycorrhizal types have been i d e n t i f i e d and described i t i s l i k e l y that mycorrhizal experts w i l l s t i l l be needed to use the keys. Then there i s the problem of estimating the r e l a t i v e abundance of each mycorrhizal type. The r e p l i c a t i o n needed for studies of mycorrhizal dynamics may l i m i t the amount of time allocated to identifying and grouping mycorrhizal types. Researchers not s p e c i a l i z i n g in the taxonomy of mycorrhizae and more concerned with forest ecology have used Dominik's (1959, 1969) keys to arrive at a type designation for ectomycorrhizae which cannot or have not been indentified. Preferably, the type designation would be followed with t h e i r own decription. Some researchers have simply described the mycorrhizae in s u f f i c i e n t d e t a i l to distinguish between the types they encountered in t h e i r study (e.g., Kropp 1982; Parke et al. 1983b; P i l z and Perry 1984), and f e l t i t was enough to say something to the effect of "this treatment caused changes in the proportions of mycorrhizal types". This i s only one step beyond doing only percent 59 mycorrhizal colonization and yet has more meaning. For example, Parke et al. (1983b) found temperature to influence the species composition of mycorrhizae as indicated by morphological type. However, to f u l l y r elate findings with other studies and gain greater insight into the ecology of mycorrhizae requires mycobiont i d e n t i f i c a t i o n . When possible, ectomycorrhizal types are perhaps best sent to the experts. 60 3 MATERIALS AND METHODS I n t h e l i t e r a t u r e r e v i e w i t was c o n c l u d e d t h a t c l e a r c u t s many y e a r s o l d t h a t l a c k m y c o r r h i z a l h o s t s a n d t h a t were s e v e r e l y b u r n e d may be low i n m y c o r r h i z a l i n o c u l u m , b u t s u c h s i t e s were n o t a v a i l a b l e ( P e r r y e t al. 1 9 8 7 ) . H o w e v e r , a number o f s i t e s c o n s i d e r e d d i f f i c u l t t o r e g e n e r a t e were e x a m i n e d . I n t h i s s t u d y , h i g h e l e v a t i o n s i t e s w i t h s o u t h w e s t a s p e c t a n d t h i n , r a p i d l y d r a i n e d s o i l s were c o n s i d e r e d most h a r s h ( L i v i n g s t o n a n d B l a c k 1987) . 3 . 1 S p e c i e s a n d s e e d l o t s S e e d l o t s o f Pseudotsuga menziesii ( D o u g l a s - f i r , F d ) , Tsuga heterophylla ( w e s t e r n h e m l o c k , Hw) , a n d Thuja plicata ( w e s t e r n r e d c e d a r , Cw) t h a t were t o be p l a n t e d on h a r s h s i t e s were s e l e c t e d f o r s t u d y ( T a b l e s I a n d I I ) . A n a d d i t i o n a l s e e d l o t , F d 9 5 0 9 , was s a m p l e d November 19 , 1987 , t o e x a m i n e t h e e f f e c t o f m e t h y l b r o m i d e f u m i g a t e d p o t t i n g m i x t u r e on s u b s e q u e n t m y c o r r h i z a f o r m a t i o n a n d was n o t f o l l o w e d a f t e r o u t p l a n t i n g . A l l s e e d l o t s e x c e p t Fd3290 were grown i n M a c B e a n N u r s e r y ( l o c a t e d a b o u t 12 km s o u t h o f N a n a i m o , B . C . ) i n s t y r o b l o c k c o n t a i n e r s w i t h a c a v i t y s i z e o f 3 x 13 cm. S e e d s were sown i n t h e s p r i n g i n a p e a t - v e r m i c u l i t e p o t t i n g m i x t u r e r a t i o o f 2 t o 1. T h e p e a t s o u r c e was F i s o n s f r o m S e b a B e a c h , A l b e r t a . N u t r i c o t e 1 6 - 1 0 - 1 0 t y p e 360 s l o w r e l e a s e f e r t i l i z e r was i n c l u d e d i n t h e p o t t i n g m i x t u r e a t a r a t e o f 7 k g p e r m 2 . I n a d d i t i o n , s o l u b l e f e r t i l i z e r s were u s u a l l y i n c l u d e d i n t h e 61 Table I. Seedling production information for selected seedlots from MacBean Nursery SEEDLOT AND SPECIES DATE SOWN DATE LIFTED BLACK OUT SEEDLOT ELEVATION Fd9509 26MAR87 SAMPLED 19NOV87 915 m Fd9766 27MAR87 05JAN88 03AUG87 -31AUG87 650 m Fd4503 01MAY87 03FEB88 10AUG87 -07SEPT87 457 m Hw7321 16MAR87 17FEB88 NONE 915 m Cw4511 20MAR87 19FEB88 NONE 600 m watering of the seedlings. Benlate, Bravo, Captan, and Rovral was used to control Botrytis. Belmark and Diazinon were used to control weevils. The seedlings were l i f t e d early the following year before one to two months of cold storage (Table I; S. Maher, personal communication). Two thousand Fd3290 seedlings were grown by Rex Timber Incorporated, Cottage Grove, Oregon and lat e r transported to MacBean Nursery for cold storage. Styroblocks of cavity size 3 x 15 cm f i l l e d with vermiculite and steam-sterilized peat was used. Half of the seedlings were a r t i f i c i a l l y inoculated with spores of Rhizopogon vinicolor from sporocarps collected in Oregon (Castellano and Trappe 1985). Other d e t a i l s about growing conditions were not available. The seedlings were examined before and after outplanting on Site 1 to determine i f any improvement in seedling growth, as a resul t of fungal inoculation, could be detected after one f i e l d season. 62 Table II. Selected seedlots and s i t e information. SPECIES MAP BIOGEO- ELEVATION, SITE & COORD- ROAD OPENING CLIMATIC ASPECT, AND PREPAR SITE SEEDLOT INATES ACCESS NUMBER ZONE* SLOPE (APPROX) -ATION IC Fd3290 48' 39" M i l l Bay 92B.063. CDFa 180 m 0% NONE 123' 34" Benko Rd. 13.02 11 Fd3290 180 m SW 2% 2F Fd9766 48' 41" Shawnigan 92B.061. CWHal 450 m SW 20% LIGHT 123' 48" H8A 4430 BURN 2H Fd9766 470 m SW 30% 2R Fd9766 490 m s 2% 3A Fd9766 48' 41" Shawnigan 92B.062. CWHal 660 m NW 15% LIGHT 123' 48" L4000 3318 BURN 3B Fd4503 640 m NW 10% 4 Fd4503 49' 14" N.W. Bay F028. CWHb4 800 m SW 45% NONE 124' 30" 143A-41 1402 5 Fd4503 49' 15" N.W. Bay F029. CWHal 200 m 0% NONE 124' 20" 155K 1404 6 Fd4503 49 ' 16" N.W. Bay F029. CDFa 60 m SE 2% LEV-124' 16" Kaye P i t 4304 ELED 7H Hw7321 48' 45" Shawnigan CO80. CWHb4 64D m NE 5% NONE 124' 03" R5000 42.08 7C Cw4511 670 m — 0% LIGHT BURN * As mapped by Nuszdorfer, Kassey, and Scagel (1985). continued... 3.2 Study s i t e s and s o i l sampling Description of study sites A l l s i t e s were clearcut 1 to 3 years before being replanted with the selected seedlots in March and A p r i l , 1988. The s o i l s on most sit e s were rapidly to well drained sandy loams. Site 1 s o i l c l a s s i f i c a t i o n was Duric Dystric Brunisol but a l l the others were Humo-Ferric Podzols as determined from s o i l maps of the region (Jungen 1985). 63 Table II. Selected seedlots and s i t e information (continued). PERCENT DEPTH OF PERCENT SOIL SEEDLING ORGANIC SAND/ - DRAINAGE CLASSIFICATION IITE SURVIVAL HORIZON (cm) SILT/CLAY CLASS FOR AREA* 1C 100 1 58/26/16 WELL DURIC DYSTRIC (SANDY LOAM) DRAINED BRUNISOL 11 98 1 59/27/14 (SANDY LOAM) 2F 93 2.1 60/29/11 RAPIDLY ORTHIC HUMO-(SANDY LOAM) DRAINED FERRIC PODZOL 2H 91 1.5 68/25/7 (SANDY LOAM) 2R 100 0 59/30/11 (SANDY LOAM) 3A 96 0 57/29/14 WELL DURIC HUMO-(SANDY LOAM) DRAINED FERRIC PODZOL 3B 96 3 48/40/12 (LOAM) 4 96 0 72/14/14 RAPIDLY ORTHIC HUMO-(SANDY LOAM) DRAINED FERRIC PODZOL 5 98 1.8 80/12/8 RAPIDLY DURIC HUMO-(SANDY LOAM) DRAINED FERRIC PODZOL 6 0 97/1/2 RAPIDLY GRAVEL AND SAND (SAND) DRAINED 7H 9.6 64/18/18 WELL TO ORTHIC HUMO-(SANDY LOAM) RAPIDLY FERRIC PODZOL 7C 96 10.8 44/37/19 DRAINED (CLAY LOAM) ' As mapped i n Jungen 1985. continued Site 1 was located about 2 km east of the town of Bay on eastern Vancouver Island in the Coastal Douglas-fir Biogeoclimatic Zone (Table I I ) . The area was replanted within 1 year of harvest and was not burned or s c a r i f i e d . Of a l l the study s i t e s , s o i l disturbance was probably the lowest in Site 1. Unlike the other s i t e s , water was observed in depressions near the study plots in June and November, 1988, indicating water a v a i l a b i l i t y may not have been a problem. Water 64 Table II. Selected seedlots and s i t e information (continued). SITE % c* % O.M. * % N* C:N* P (ppm)* pH(H20)* pH(CaC12) * IC 2.64 4.6 0.148 18 34 5.8 5. 0 11 2.83 4.9 0.15 19 36 5.4 4. 7 2F 2.09 3.6 0.041 51 41 5.4 4. 4 2H 2.07 3.6 0.045 46 98 5.2 4. 4 2R 0.65 1.1 0.02 33 40 5.8 5. 2 3A 2.6 4.5 0.055 47 34 5.1 4. 4 3B 3.4 5.9 0.064 53 41 5.4 4. 5 4 1.26 2.2 0.098 13 98 5.1 4. 1 5 4.27 7.4 0.091 47 135 5.3 4. 3 6 0.19 0.3 0.011 17 203 6.6 5. 4 7H 3. 57 6.2 0.107 33 3 5.0 4. 1 7C 7.44 12.8 0.177 42 6 4.6 3. 8 * % c = percent t o t a l carbon; % O.M. = percent < organic matter; % N = percent t o t a l nitrogen; C:N = carbon to nitrogen r a t i o ; P = Mehlich 3 available phosphorus; pH(H20) = pH with 2 :1 water to s o i l ; pH(CaCl2) = pH with 2:1 0.01M calcium chloride to s o i l (see t e x t ) . a v a i l a b i l i t y , minimal s o i l disturbance, low elevation, and f l a t topography probably made Site 1 one of the most favorable s i t e s studied for seedling establishment. Site IC received the control seedlings and Site II was planted with seedlings inoculated with R. vinicolor. The two plots were side by side and very similar in a l l respects (Figs. 1 and 2). 65 Sites 2 and 3 were located about 12 km south-west of Duncan i n the Coastal western Hemlock Biogeoclimatic Zone. Slopes in Site 2 were steeper and probably warmer and drie r than Site 3 because of the south-west aspect and lower elevation (Figs. 3 and 4) . Site 2F (more "favorable") was a lower elevation plot with less slope and non crop vegetation than Site 2H (more "harsh"). Site 2R was a logging roadway (Fig. 5) . Site 3A received seedlot Fd9677 which originated from a similar elevation (Table II) . Site 3B was d i r e c t l y downhill from Site 3A across a logging road. The dust from the road may have accounted for the higher s i l t content in Site 3B. Site 4 was located about 2 3 km east of Nanoose Bay not far from Rowbotham Lake in the Coastal western Hemlock Biogeoclimatic Zone (Table I I ) . Conditions for seedling establishment were probably the most harsh of a l l the study s i t e s because of the high elevation, steep southwest slope, and the shallow s o i l with few planting s i t e s (Fig. 6). Site 5 was located near Englishman River F a l l s Park about 11 km east of Nanoose Bay (Table II) . The s i t e was f l a t and rapidly drained with no standing water in the depressions when v i s i t e d i n July and November, 1988 (Fig. 7). Site 6 was located about 2 km south-west of the Island and Alberni highway junction. The s i t e was once a gravel p i t that was leveled, f e r t i l i z e d , and sown with clover (Fig. 8) . The s o i l consisted primarily of sand and gravel with 66 r e l a t i v e l y high pH, high phosphorus, and low nitrogen (Table II) . Site 7 was about 25 km east of Duncan i n the Coastal western Hemlock Biogeoclimatic Zone and had s o i l s with thick organic horizons and low phosphorus a v a i l a b i l i t y in the mineral s o i l (Table II) . Site 7H (hemlock) was near the bottom of a valley and Site 7C (cedar) was up out of the valley on l e v e l ground (Figs. 9 and 10). 67 Non-crop species within study sites For Sites 2 through 7 the percent cover of non-crop plant species and naturally regenerated conifers was v i s u a l l y estimated (Appendix 1). Most of these plants had low percent cover. Gautheria shallon Pursh was high i n percent cover on Sites 2F, 2H, 3, 4 and 5 but was s t i l l young and low to the ground. Senecio sylvaticus L. had the next highest percent cover ranging from 2 to 3 0% on Sites 2 through 7. Epilobium minutum L i n d l . , Hypochaeris radicata L. , Lactuca muralis (L.) Fresen. , and Rubus leucodermis Dougl. a l l had over 40% cover on Site 4. Epilobium angustifolium L. was the most common non-crop species on Site 7. However, i t was not l i k e l y that any of the non-crop species on any of the s i t e s were seriously competing with the outplanted seedlings within the f i r s t f i e l d season (Figs. 1-10). A few individuals from each non-crop plant species were removed from plots in Sites 2 through 7. The plants were i d e n t i f i e d to species i f possible and the roots of most were examined for mycorrhizal status (see Section 3.5 for methods). Non-crop plants were either nonmycorrhizal or formed varying levels of vesicular-arbuscular (VA) or e r i c o i d mycorrhizae. Members of the Pinaceae formed high levels of ectomycorrhizae and a naturally regenerated cedar (on Site 4) formed high levels of VA mycorrhizae (Appendix 1). 73 Soil analysis On each s i t e , mineral s o i l samples were taken beside the f i r s t 15 seedlings sampled. The s o i l samples were passed through a 2 mm mesh sieve and equal volumes (100 ml) of each were bulked for each plot. Percent t o t a l nitrogen and carbon (Page et al. 1982) was determined and C to N r a t i o calculated. Percent organic matter was estimated by multipling t o t a l carbon by 1.724, a predetermined c a l i b r a t i o n found in the manual for the Leco analyser. Available phosphorus was determined using the Mehlich 3 extractant (Mehlich 1978). S o i l pH was measured in water and in 0.01 M CaCl2 using a 2:1 l i q u i d to s o i l r a t i o . S o i l texture analysis was done using the hydrometer method (Page et al. 1982). The depth of organic horizon was determined by measuring the length of plug on sampled seedlings lacking signs of mineral s o i l contact to a maximum of 15 cm per plug divided by the number of seedlings examined. A l l si t e s except 2R and 6 had some organic horizon but the seedlings were usually planted deep enough so that the plugs were only in contact with mineral s o i l . 3.3 Sampling of seedlings The selected seedlots were outplanted by MacMillan Bloedel employed treeplanters in March and A p r i l , 1988. Twenty-five seedlings (or 14 inoculated and 16 control seedlings i n the case of Fd3290) were not outplanted and kept in cold storage u n t i l May 19, 1988 when they were taken to the University of B r i t i s h Columbia for the determination of 74 seedling status before outplanting. Fd9509C and Fd9509F were randomly sampled from several styroblocks. The other selected seedlots were removed from one box i n cold storage and were therefore were sampled a l l from a s p e c i f i c area of the nursery within each seedlot ( i . e . from the same styroblock or from a few adjacent styroblocks; S. Maher, personal communication). The higher elevation s i t e s (2, 3, 4, and 7) were sampled in late September and the lower elevation s i t e s (1, 5, and 6) were sampled in late November, 1988 (Table II) . Seedlings were sampled along transects in Sites 2R and 6. Within each of the other s i t e s , 20 x 30 m plots were marked using 5 x 5 cm wooden stakes. Seedlings were randomly sampled from plots using a coordinate system and random numbers. Roots were excavated using a shovel to l i f t and loosen and hands to car e f u l l y retrieve most or a l l of the new roots that grew away from the plug since outplanting. This was not d i f f i c u l t for most samples because of the short time spent i n the f i e l d and because of the sandy s o i l texture (Table II) . Samples were kept in p l a s t i c bags in coolers for transporting to U.B.C. Shoot height, caliper (root c o l l a r diameter), and oven dried-weight were measured. Distinctive mycorrhizal types were removed and partly described (see section 3.6). Roots of nursery seedlings were oven dried and weighed. The roots of f i e l d samples were frozen or washed (see below) and then frozen u n t i l ready to process. Dry root weight of f i e l d collected samples was estimated from fresh weights because i t 75 was thought drying might hinder mycorrhiza characterization (Appendix 2). 3.4 Root preparation The roots were thawed and gently washed under running water over a 3 55 um mesh sieve. The washings collected in the sieve of three samples from Site 2 were examined and found to contain 5 to 10% of the sample's t i p s . But i t was not determined i f the proportion being nonmycorrhizal or dead was dif f e r e n t than that for the t i p s remaining on the roots. For f i e l d collected samples the new roots were removed and treated as separate samples. The plug roots were cut to f a c i l i t a t e peat removal. In order to accurately determine percentage mycorrhizal colonization in a reasonable amount of time i t was necessary to make the roots transparent and then use a stain that binds to c h i t i n in hyphae. A modification of P h i l l i p s and Hayman's (1970) method was used: the roots were put into beakers and covered with 10% KOH and heated at 60-90°C for 3 h with periodic r e f i l l i n g of the beakers with water. Beaker contents were poured over a 106 um sieve in order to retrieve broken root t i p s , rinsed with water, and then returned to the beaker to soak i n water overnight. The roots were rinsed again and then bleached using 30 ml of 30% hydrogen peroxide plus 10 ml of concentrated ammonium hydroxide in 17 00 ml. water u n t i l l i g h t brown or white (1 to 2 h). After r i n s i n g the roots were stained with 0.01% (wt.: vol.) Trypan Blue in 85% l a c t i c acid, 76 glycerol, and water in a r a t i o of 1:1:1. The stain was reused many times after passing through a 53 um mesh sieve, adding 0.001% more stain and evaporating off excess water. Mostly low (x5-16) magnification was adequate i n assessing mycorrhizal colonization and allowed for a large number of roots to be counted. 3.5 Determination of percentage colonization Douglas-fir and western hemlock form ectomycorrhizae. Mycorrhizal t i p s , especially in the nursery, often lacked a mantle and would have appeared nonmycorrhizal at low magnification i f not f i r s t cleared and stained. The cortex of nonmycorrhizal t i p s was transparent so that xylem traces were cl e a r l y v i s i b l e . The cortex of mycorrhizal t i p s appeared darker blue because of the a f f i n i t y of Trypan blue for fungal hyphae (Figs. 11 and 12). A l l root t i p s greater than 1.5 times longer than wide were counted as either mycorrhizal or not using mostly low (5-16x) magnification. These observations were confirmed with frequent examination of questionable root t i p s at high (400-1250X) magnification. Percent colonization equals the number of mycorrhizal t i p s divided by the t o t a l number of t i p s times 100%. Only about 500 t i p s were counted for Hw plugs because of the very large number of t i p s present (often greater than 2500). Western red cedar forms VA mycorrhizae (Fig. 13) . For these roots percent colonization was determined using the g r i d l i n e intersect method (Giovannetti and Mosse 1980). 77 FIGS. 11-13. Fine roots after staining procedure. FIG. 11. Nonmycorrhizal (*) and ectomycorrhizal Douglas-fir short roots from S i t e 1. x52. FIG. 12. Nonmycorrhizal hemlock root t i p from MacBean Nursery. x538. FIG. 13. Fine (f) and coarse (c) endophyte forming vesicular-arbuscular mycorrhizae on western red cedar. x2150. ?0 3 . 6 Types* of mycorrhizae Western red cedar After determining percent colonization, about 3 0 mycorrhizal root segments were randomly selected and mounted on a s l i d e i n Hoyer's medium (Anderson 1954) along a l i n e drawn on the bottom of the s l i d e with a f e l t pen. Using 400x magnification, the s l i d e was scanned along the l i n e for roots. Each root was recorded as either VA (fine) i f the hyphae were less than 2.5 um wide or as VA (coarse) i f most hyphae were greater than 2.5 um wide (Fig. 13). The presence of arbuscules associated with the hyphae was confirmed using xlOOO or X 1 2 5 0 magnification. Douglas-fir and western hemlock Soon after sampling the seedlings the roots were examined using a dissecting scope. Before staining, morphologically d i s t i n c t mycorrhizal types were p a r t i a l l y characterized and then frozen in v i a l s in water or mounted on s l i d e s in Hoyer's medium for further characterization at a later date. After staining and while determining percent colonization, t i p s of morphologically d i s t i n c t types of mycorrhizae were again mounted on slides or stored in v i a l s in l a c t i c acid. A characterization form was used to record observations on each type of mycorrhiza encountered (Table I I I ) . Emanating hyphae of d e f i n i t e length (i.e. intact ends seen) were called c y s t i d i a . Only the more common and d i s t i n c t i v e types of mycorrhizae were f u l l y described and photographed. Many types 79 were not encountered u n t i l after the staining procedure but the color probably was that of the root because i f the color of these t i p s was d i s t i n c t i v e some would have been spotted and preserved before staining. Once stained, most of the types could only be distinguished using high magnification based on stable characteristics of hyphae within and a r i s i n g from the mantle. Mycorrhizal characteristics seen at 5-125x magnification (habit) were generally no longer useful after staining and mounting of the t i p s on s l i d e s . A dichotomous key to the types of ectomycorrhizae was constructed with an attempt to use the most stable and d i s t i n c t i v e hyphal chara c t e r i s t i c s of the stained t i p s . Detailed descriptions of the ectomycorrhizal types are in Appendix 5. A glossary of the technical terms used i s included in Appendix 6. 80 Table I I I . Form used to record characteristics of each type of ectomycorrhiza encountered.+ Source: (sample # ) Mycorrhizal TYPE: Most d i s t i n c t i v e characteristics: Emanating hyphae Cystidia Abundance Length Clamps/septa Pigmentation Ornamentation Branching Strands on whole outer hyphae core hyphae Abundance Length Diameter Clamps/septa Pigmentation Ornamentation Branching Mantle Compactness Textura type Thickness Hyphal diameter Clamps/septa Pigmentation Intersections Hartig net Invaginations Hyphal diameter Pigmentation Clamps/septa Beading Habit Color Mantle texture Diameter Branching pattern Plots found in Typical abundances Additional characteristics: + A glossary of the terms used in the characterization of ectomycorrhizae i s included in Appendix 6. 81 3.7 Data analysis Data on the samples collected are in Appendix 4. Data for each seedlot were summarized in Tables showing mean values and t h e i r standard errors. Two-tailed t-tests were done to compare two means. One-way analysis of variance was used to compare three or more means and i f a l l means were not equal then s i g n i f i c a n t differences between them were separated by Tukey's test (P =.05). Pearson's r - c o e f f i c i e n t s and t h e i r significance (P =.05) was calculated for a l l variable combinations for each seedlot to determine which variables were correlated. Although the number of mycorrhizal t i p s of each type could not be determined, the percentage of seedlings sampled with each ectomycorrhizal type was calculated. In addition, the r e l a t i v e abundance of each ectomycorrhizal type was computed as follows: w = mycorrhizal type t w = number of seedlings with mycorrhizal type w on plug and/or new root T = sum t w for a l l w P = t w / T = proportion of mycorrhizal type w P X 100 = r e l a t i v e abundance of type w. This was computed for the whole root system for seedlings from each s i t e , and for the plug and new root of Site 1 seedlings. 82 4 RESULTS 4.1 Seedling status before and after outplanting Fd9509: fumigation of potting mixture Seedlings grown in the previously methyl bromide fumigated potting mix were s i g n i f i c a n t l y larger in terms of weight, height and caliper than control seedlings (Table IV) . Mycorrhizal colonization was not s i g n i f i c a n t l y d i f f e r e n t (P < 0.10) due to the high v a r i a b i l i t y in colonization levels between seedlings (see section 4.4). Table IV. Fd9509: mycorrhizal colonization and seedling growth in potting mixture previously fumigated with methyl bromide or nonfumigated (control).+ CONTROL METHYL BROMIDE n= 23 23 % MYCORRHIZAE OF PLUG 46 a 31 a (6) (6) SHOOT HEIGHT (mm) 258 a 295 b (9) (4) CALIPER (mm) 2.7 a 3 b (0.1) (0.07) SHOOT WEIGHT (mg) 1404 a 1691 b (82) (78) + Row wise: two-tailed t-tests were done to test differences between mean values. Means with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P =.05). Values in parentheses are the standard errors of the means given above. 83 Comparison of Fd9766 and Fd4503 Before outplanting, Fd4503 had greater seedling weight, height, and caliper than did Fd9766 but root weight and the t o t a l number of t i p s were not s i g n i f i c a n t l y d i f f e r e n t . However, Fd9766 l e f t MacBean Nursery with much higher levels of mycorrhizal colonization than did Fd4503 (Table V). This suggested that greater photosynthate drain caused by higher mycorrhizal colonization of Fd9766 was a factor in determining seedling size. However, mycorrhizal colonization l e v e l was not correlated with shoot weight (see section 4.2). Therefore, differences in seedling size were either due to differences in growing conditions and dates sown, or to genetic differences: Fd9766 originated from higher elevation, and therefore may have been slower growing r e l a t i v e to Fd4503 under similar conditions (Table I ) . After one f i e l d season on Site 3, Fd9766 and Fd4503 were not d i f f e r e n t in terms of shoot weight, height, caliper, new root production, and mycorrhizal colonization. However, plug root weight of Fd4503 exceeded that of Fd9766 (Table V) . Colonization levels of Fd4503 may have simply caught up to that of Fd9766, but the probability that the Fd4503 seedlings were sampled from an area of MacBean Nursery with low mycorrhizal colonization i s a more l i k e l y explanation, and Fd4503 seedlings from the f i e l d (from a dif f e r e n t area of the nursery) may have l e f t the nursery with a colonization pattern not unlike Fd9766. Similarly, the colonization l e v e l of the plug roots of outplanted seedlings from other s i t e s did not 84 Table V. Comparison of Fd9766 and Fd4503 from MacBean Nursery and Site 3.+ NURSERY SITE 3 Fd9766 Fd4503 Fd9766 Fd4503 n= 25 25 25 25 PLUG: % MYCORRHIZAE 86 b 12 a 76 y 82 y • (3) (3) (4) (5) MYCORRHIZAL TIPS 660 b 94 a 506 y 852 z (42) (27) (57) (81) TOTAL TIPS 763 a 719 a 658 y 1030 z (34) (41) (55) (67) WEIGHT (mg) 912 a 933 a 1479 y 1815 z (40) (39) (78) (89) NEW ROOT: % MYCORRHIZAE 89 y 87 y (2) (4) MYCORRHIZAL TIPS 348 y 300 y TOTAL TIPS 388 y 320 y (39) (60) WEIGHT (mg) 282 y 246 y (31) (30) SHOOT: HEIGHT (mm) 173 a 264 b 255 y 268 y (3) (5) (12) (12) CALIPER (mm) 2.8 a 3.1 b 4.1 y 4.1 y (0.08) (0.07) (0.15) (0.15) WEIGHT (mg) 868 a 1568 b 2994 y 2707 y (40) (60) (256) (238) ROOT/SHOOT RATIO 1.06 b 0.6 a 0.66 y 0.85 z (0.02) (0.02) (0.05) (0.05) % BROWSE DAMAGE 62 76 % STEM DAMAGE 13 24 + Row wise: separate two-tailed t-tes t s were done to test differences between mean values taken for nursery and f i e l d sampling. Means with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P =.05). Values i n parentheses are the standard errors of the means given above. 85 correspond to the lev e l found in the nursery for that seedlot (Figs. 14 and 15). For example, mycorrhizal colonization of the plug roots was lower for Sites 2H and 2R than i n MacBean Nursery indicating one of two things: f i r s t , new roots t i p s formed i n the plug but were not colonized. Mycorrhizal colonization of new roots was often greater than that of the plug root (Figs. 14 and 15), and plug weight usually increased for seedlings from most sitds indicating that complete separation of plug and new roots was not possible (Figs. 16 and 17) . Second, mycorrhizal colonization i n the nursery was highly variable and the samples counted were from an area in the nursery where colonization levels were high. The second p o s s i b i l i t y i s more l i k e l y the case for reasons that w i l l become more apparent later (see section 4.4). Fd9766 and Fd4503: Seedling weight on other s i t e s Seedling weight did not d i f f e r for any of the Fd9766 planted s i t e s (Fig. 16) . Larger sampling size and diff e r e n t sampling months may have allowed for more differences to be detected in Fd4503 seedlings. Sites 3B and 4 were sampled in late September. Shoot and plug root weight of Site 3 seedlings s i g n i f i c a n t l y increased since outplanting unlike Site 4 seedlings. Of a l l the site s sampled, Site 4 was considered potentially most d i f f i c u l t to regenerate because of the rapidly drained poorly developed s o i l s , the high elevation, and the steep south-west aspect. However, there was s t i l l 96% survival after one growing season (Table I I ) . 86 PERCENT COLONIZATION PERCENT COLONIZATION NURSERY SITE 3B SITE 4 SITE 5 SITE 6 FIGS. 14 and 15. Mycorrhizal colonization of Douglas-fir from MacBean Nursery before and after outplanting. Same pattern bars with same l e t t e r above are not s i g n i f i c a n t l y d i f f e r e n t and the asterisk (*) indicates s i g n i f i c a n t difference between plug root and new root colonization l e v e l (P = .05). FIG. 14. Fd9766. FIG. 15. Fd4503. 87 WEIGHT (gm) • SHOOT HH PLUG ROOT EE! NEW ROOT b b 3 -2 -b a a b • b NURSERY SITE 2F SITE 2H SITE 2R SITE 3A 16 WEIGHT (gm) 3 -SHOOT EE) PLUG ROOT NEW ROOT C a b Isi?— I NURSERY SITE 3B SITE 4 SITE 5 17 b e l l SITE 6 FIGS. 16 and 17. Seedling weight of Douglas-fir from MacBean Nursery before and after outplanting. Same pattern bars with same l e t t e r above are not s i g n i f i c a n t l y d i f f e r e n t (P = .05). FIG. 16. Fd9766. FIG. 17. Fd4503. 88 Sites 5 and 6 were lower in elevation and sampled two months lat e r . Shoot weight and new root weights of these seedlings were greater than those from Site 3B. New root weight of Site 6 seedlings grown in the f e r t i l i z e d sand-gravel s o i l (Table II) was greater than Site 5 seedlings (Fig. 17). Fd9766 and Fd4503: Other variables measured As with percent colonization, there were no differences in the number of mycorrhizal t i p s , the t o t a l number of t i p s , or caliper of the outplanted Fd9766 seedlings. Shoot height was greatest on Site 2H but only s i g n i f i c a n t l y d i f f e r e n t from Site 3A seedlings (Table VI). Of the outplanted Fd4503 seedlings, those from Site 3B had the greatest t o t a l number of root t i p s and mycorrhizal t i p s in the plug. However, the number of new root t i p s , l i k e new root weight, was greatest for the Site 6 seedlings. Caliper was greatest for Site 5 seedlings but t h i s was not s i g n i f i c a n t l y d i f f e r e n t from Site 6 seedlings. Although they were sampled two months e a r l i e r and had lower browse damage, Site 5 and 6 seedlings were not s i g n i f i c a n t l y t a l l e r than Site 4 seedlings (Table VII). 89 Table VI. Fd9766: mycorrhizal colonization and seedling growth.+ NURSERY SITE 2F SITE 2H SITE 2R SITE 3A MONTH SAMPLED: MAY SEPT. SEPT. SEPT. SEPT. n= 25 15 15 16 25 PLUG: % MYCORRHIZAE 86 b 73 ab 61 a 63 a 76 ab (3) (7) (7) (5) (4) MYCORRHIZAL TIPS 660 a 524 a 447 a 511 a 506 a (42) (103) (86) (86) (57) TOTAL TIPS 763 a 708 a 670 a 785 a 658 a (34) (104) (72) (89) (55) WEIGHT (mg) 912 a 1340 b 1297 b 1365 b 1479 b (40) (90) (60) (69) (78) NEW ROOT: % MYCORRHIZAE 86 a 80 a 80 a 89 a (4) (4) (3) (2) MYCORRHIZAL TIPS 312 a 360 a 364 a 348 a (53) (76) (59) (38) TOTAL TIPS 343 a 437 a 462 a 388 a (52) (88) (74) (39) WEIGHT (mg) 314 a 391 a 381 a 282 a (40) (72) (55) (31) SHOOT: HEIGHT (mm) 173 a 291 be 304 c 281 be 255 b (3) (9) (12) (7) (12) CALIPER (mm) 2.8 a 4.1 b 4.1 b 4 b 4.1 b (0.08) (0.2) (0.16) (0.15) (0.15) WEIGHT (mg) 868 a 2611 b 3329 b 3488 b 2994 b (40) (188) (249) (252) (256) ROOT/SHOOT RATIO 1.06 c 0.66 ab 0.52 ab 0.52 a 0.66 b (0.02) (0.05) (0.03) (0.02) (0.05) % BROWSE DAMAGE 47 40 19 62 % STEM DAMAGE 0 0 0 13 + Row wise: means with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at P=0.05 as tested by ANOVA (Appendix 3). Values i n parentheses are the standard errors of the means given above. 90 Table VII. Fd4503: mycorrhizal colonization and seedling growth.+ MONTH SAMPLED: NURSERY MAY SITE 3B SEPT. SITE 4 SEPT. SITE 5 NOV. SITE 6 NOV. n= 25 25 25 28 15 PLUG: % MYCORRHIZAE 12 a 82 b 79 b 87 b 75 b (3) (5) (5) (3) (4) MYCORRHIZAL TIPS 94 a 852 c 475 b 640 be 421 b (27) (81) (42) (68) (44) TOTAL TIPS 719 a 1030 b 604 a 713 a 574 a (41) (67) (37) (69) (56) WEIGHT (mg) 933 a 1815 c 1156 a 1540 b 1803 be (39) (89) (51) (65) (107) NEW ROOT: % MYCORRHIZAE 88 b 72 a 93 b 88 ab (4) (5) (3) (5) MYCORRHIZAL TIPS 300 a 138 a 693 b 1201 c (60) (23) (78) (271) TOTAL TIPS 320 a 177 a 729 b 1349 c (60) (23) (79) (268) WEIGHT (mg) 246 a 166 a 624 b 1414 c (30) (17) (68) (230) SHOOT: HEIGHT (mm) 264 a 268 a 291 ab 324 b 307 ab (5) (12) (12) (11) (10) CALIPER (mm) 3.1 a 4.1 be 3.8 b 4.8 d 4.6 cd (0.07) (0.15) (0.12) (0.15) (0.18) WEIGHT (mg) 1568 a 2707 b 2372 ab 3943 c 4217 c (60) (238) (160) (261) (462) ROOT/SHOOT RATIO 0.6 a 0.85 b 0. 59 a 0.58 a 0.83 b (0.02) (0.05) (0.03) (0.03) (0.10) % BROWSE DAMAGE 76 72 29 0 % STEM DAMAGE 24 44 21 0 + Row wise: means with the same l e t t e r are not significantly-d i f f e r e n t at P=0.05 as tested by ANOVA (Appendix 3). Values i n parentheses are the standard errors of the means given above. 91 HW7321 The western hemlock seedlings sampled from the MacBean Nursery had 30% of the i r root t i p s mycorrhizal, but again the v a r i a b i l i t y about th i s mean was very high (Table VIII) . As seen with most of the Douglas-fir s i t e s , new roots had higher levels of colonization than did the plug roots. The number of root t i p s produced per gram root was greater for western Table VIII. Hw7321: mycorrhizal colonization and seedling growth.+ NURSERY SITE 7H n= 25 24 PLUG: % MYCORRHIZAE 30 84 (7) (3) WEIGHT (mg) 970 1481 (80) (108) NEW ROOT: % MYCORRHIZAE 92 (2) MYCORRHIZAL TIPS 1046 (158) TOTAL TIPS 1095 (158) WEIGHT (mg) 593 (75) SHOOT: HEIGHT (mm) 212 353 (6) (19) CALIPER (mm) 3.3 5.5 (0.09) (0.17) WEIGHT (mg) 1607 6012 (95) (516) ROOT/SHOOT RATIO 0.63 0.35 (0.06) (0.02) % BROWSE DAMAGE 0 „% STEM DAMAGE 25 + Values i n parentheses are the standard errors of the means given above. 92 hemlock than for Douglas-fir (Tables VI and VII). Ninety-two percent of the new root t i p s were colonized on Site 7H which was the second highest mycorrhizal colonization l e v e l found on the outplanted seedlings (Site 5 had 93% of new root mycorrhizal for Douglas-fir). CW4511 Western red cedar did not form mycorrhizae i n MacBean Nursery (Table IX). The seedlings from Site 7C had 8% of the plug roots mycorrhizal and 15% of new roots mycorrhizal. Fine Table IX. Cw4511: mycorrhizal colonization and seedling growth.+ NURSERY SITE 7C PLUG: n= 25 25 % MYCORRHIZAE 0 8 (3) WEIGHT (mg) 633 1324 NEW ROOT: (33) (101) % MYCORRHIZAE — — —. 15 (4) WEIGHT (mg) 400 SHOOT: (52) HEIGHT (mm) 278 (8) 325 (10) CALIPER (mm) 2.6 (0.07) 3.9 (0.13) WEIGHT (mg) 1311 (59) 2942 (192) ROOT/SHOOT RATIO 0.5 (0.03) 0.6 (0.04) % BROWSE DAMAGE 0 % STEM DAMAGE 25 + Values i n parentheses are the above. standard errors of the means given 93 or coarse endophyte or both colonized the roots of 64% of the sampled seedlings (Fig. 13). For the entire fine root system, 40% of the seedlings had both fine and coarse endophyte, 4% (or 1 seedling) only had fine endophyte, and 20% had only coarse endophyte. Despite the low levels of colonization by vesicular-arbuscular mycorrhizal fungi, the chlorosis displayed by many of these seedlings, and the very low s o i l phosphorus levels (Table I I ) , the seedlings s t i l l more than doubled in weight on average after one f i e l d season on Site 7C (Table IX). Fd3290: A r t i f i c i a l l y inoculated seedlings from Oregon A l l of the inoculated seedlings had formed Rhizopogon vinicolor mycorrhizae varying from 2 to 85% of root t i p s colonized and a mean value of 47%. Control seedlings had low levels of colonization by other fungi (see section 4.3). By June following outplanting there was an indication that percent colonization was increasing (Fig. 18) . By the f u l l November sampling, colonization of new roots was 90-91%. The plug roots of control seedlings had lower (77%) mycorrhizal colonization than new roots and both root types of the inoculated seedlings. Shoot and root weights did not d i f f e r between inoculated and noninoculated seedlings (Fig. 19). The inoculated seedlings were t a l l e r than noninoculated seedlings before outplanting but a l l but one or two of the seedlings were deer browsed thereby eliminating any height difference when sampled from Site 1. There was no difference in caliper 94 P E R C E N T COLONIZATION WEIGHT (gm) 6 5 -4 -3 -2 1 H 0 'I~ SHOOT LZL; PLUG ROOT C • CONTROL Rv = INOCULATED be NEW ROOT a be cd 1 NURS.- C NURS.- Rv JUNE - C JUNE - Rv NOV.- C NOV.- Rv 19 FIGS. 18 and 19. Douglas-fir seedlings a r t i f i c i a l l y inoculated with Rhizopogon vinicolor (Rv) versus control seedlings (C) from the Oregon nursery before and after outplanting on Site 1. Same pattern bars with same l e t t e r above are not s i g n i f i c a n t l y d i f f e r e n t (P = .05). FIG. 18. Mycorrhizal colonization l e v e l . The asterisk (*) indicates s i g n i f i c a n t difference between plug root and new root colonization level (P = .05). FIG. 19. Seedling weight. 95 between treatments before or after outplanting (Table X). Table X. Fd3290C (control) versus Fd3290I (inoculated with Rhizopogon vinicolor): mycorrhizal colonization and seedling growth.+ NURSERY c - MAY I SITE c : l - JUNE I SITE c : l - NOV. I n= 16 14 5 5 25 25 PLUG: % MYCORRHIZAE 15 a 47 b 39 ab 68 be 77 cd 92 e (7) (8) (14) (14) (4) (1) MYCORRHIZAL TIPS 102 a 350 b 432 be 551 c (49) (70) (51) (46) TOTAL TIPS 703 ab 783 be 552 a 596 a (65) (70) (51) (48) WEIGHT (mg) 739 a 806 a 1453 be 1265 b 1894 be 1841 cd (57) (67) (313) (123) (144) (104) NEW ROOT: % MYCORRHIZAE 50 a 52 a 90 b 91 b (15) (14) (4) (3) MYCORRHIZAL TIPS 393 a 431 a (47) (54) TOTAL TIPS 418 a 455 a (45) (53) WEIGHT (mg) 77 a 103 a 596 b 675 b (25) (33) (72) (63) SHOOT: HEIGHT (mm) 284 a 318 bd 297 abc 269 ab 329 cd 335 cd (11) (8) (26) (35) (10) (9) CALIPER (mm) 2.5 a 2.6 a 4 b 4.2 b 5.7 c 5.7 c (0.08) (0.1) (0.34) (0.29) (0.24) (0.23) WEIGHT (mg) 1279 a 1512 a 2852 b 2371 b 6455 c 5651 c (78) (98) (531) (211) (515) (446) ROOT/SHOOT RATIO 0.57 c 0.53 c 0.57 c 0.58 c 0.41 a 0.48 be (0.02) (0.03) (0.09) (0.06) (0.02) (0.03) % BROWSE DAMAGE 80 100 96 91 % STEM DAMAGE 80 60 48 17 + Row wise: multiple two-tailed t-tes t s were done to test differences between a l l mean value combinations. Means with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P =.05). Values in parentheses are the standard errors of the means given above. 96 4.2 Variable correlations A l l variable combinations showing a s i g n i f i c a n t correlation were with a positive Pearson r - c o e f f i c i e n t indicating a dire c t relationship. Under nursery conditions the d i f f e r e n t seedlots and treatments did not show any s i g n i f i c a n t correlations between mycorrhizal colonization le v e l and other seedling measurements. Therefore, i t cannot be stated for certain that lower colonization levels of Fd9509F and Fd4503 allowed for greater shoot weights compared to Fd9509C and Fd9766 repectively. Only seedlot Fd3290C had a si g n i f i c a n t correlation between the number of mycorrhizal t i p s and shoot weight. Mycorrhizal colonization did not increase the t o t a l number of root t i p s (Table XI) . Shoot parameters were more l i k e l y to be correlated with the t o t a l number of root t i p s or root weight than with mycorrhizal status. Similarly, seedling shoot measurements taken after outplanting were more l i k e l y to be correlated to root weight or t o t a l t i p number rather than to mycorrhizal status (Table XII). However, there were some exceptions: for seedlings from Sites 2H and 3B, percent mycorrhizae of the plug (and new roots of Site 2H) was correlated with ca l i p e r . The number of mycorrhizal t i p s of plug and new roots was correlated with shoot weight and caliper of Site 6 seedlings, but th i s was also the case for t o t a l number of t i p s on new roots, an effect of the high f e r t i l i z a t i o n levels. Similarly, for other s i t e s the number of mycorrhizal and t o t a l t i p s on plug roots was correlated with shoot weight or caliper or both. The high 97 colonization levels resulted in both correlations being s i g n i f i c a n t . Percentage mycorrhizae was usually correlated with the number of mycorrhizal t i p s but was only correlated with the t o t a l number of t i p s on Sites II and 2F. New root weight was always correlated with the t o t a l number and the number of mycorrhizal t i p s found on new roots. However, mycorrhizal colonization l e v e l of new roots was correlated with the number of mycorrhizal t i p s in the plug on seedlings from Sites 3A, 3B, 4, and 5, but not with the t o t a l number of ti p s in the plug (Table XII) . This may indicate that nursery fungi were involved in colonizing new roots. + Notes for Tables XI and XII on the following three pages: No data collected indicated with a dash (-). Significance levels for Pearson r - c o e f f i c i e n t s . A l l s i g n i f i c a n t correlations were positive. Nonsignificant probability levels are given. Significance eguals *, **, ***, for P < .05, P < .01, and P < .001 respectively. F u l l variable names as follows: SWT shoot weight SHT shoot height CAL caliper ROS root over shoot mass r a t i o RWP root weight plug RWN root weight new MTP mycorrhizal t i p s plug MTN mycorrhizal t i p s new TTP t o t a l t i p s plug TTN t o t a l t i p s new Table XI: C = control seedlings; F = potting mixture previously fumigated; I = inoculated with Rhizopogon v i n i c o l o r . Table XII: IC = control seedlings; II = R. v i n i c o l o r inoculated seedlings; 2F = favorable; 2H = harsh; 2R = roadway; 7H = hemlock; 7C = cedar. 98 Table XI. Significance of variable correlations under nursery conditions (see note p. 98).+ SPECIES & SEEDLOT Fd9509 Fd3290 Fd9766 Fd4503 Hw7321 Cw4511 C F C I SWT X SHT * * * .43 * * * * * * * * * * .05 * * * SWT X CAL * * * * * * * * * * * * * * * * * .05 * * * SWT X RWP - - * * * * * * * * * * * * * * .05 SWT X MTP - - * .26 .81 .49 - -SWT X TTP - - * * * * * * .97 .18 - -SWT X PMP .84 .78 .07 .57 .46 .35 .41 -SHT X CAL * * * .27 .12 * * * * * .30 .54 * * SHT X RWP - - * * * * * * .80 .09 .39 SHT X MTP - - .32 .38 .93 .27 ~ -SHT X TTP - - * * * .98 . 58 - -SHT X PMP .92 .94 .30 .61 .95 .28 .30 -CAL X RWP - - * * * * * * * * * .50 CAL X MTP - - .12 .64 .67 . 60 - -CAL X TTP - * * .80 .81 - -CAL X PMP .39 - .13 .45 .53 .57 . 50 -RWP X MTP - - .09 .87 .89 .68 - -RWP X TTP - - ** * * * .63 * - -RWP X PMP - - .18 .17 .25 .80 .27 -MTP X TTP - - .91 .28 * * * * - -MTP X PMP - - * * * * * * * * * * * * - -TTP X PMP - - .81 .46 .32 .07 - -n = 23 23 16 14 25 25 25 25 99 Table XII. Significance of variable correlations under f i e l d conditions (see note p. 98).+ IC 11 2F 2H 2R 3A 3B 4 5 6 7H 7C SWT X RWP * * * * * * .32 * * * * * * * * * * * * *** * * * * * * * SWT X RWN ** ** .63 .17 * .86 .31 .80 ** .06 * * * .13 SWT X MTP * * .88 .50 .13 .08 .35 .35 .47 * * - -SWT X MTN .06 .11 .89 .27 .24 .08 .56 .95 .54 * * * -SWT X TTP * * * .94 .44 .16 .09 * .23 . 67 .08 - -SWT X TTN .07 .10 .99 .24 .13 .10 .49 .65 .57 * * ~ SWT X PMP .34 .38 .39 .53 .56 .59 .24 .89 .34 .31 .92 .47 SWT X PMN .10 .14 .07 .14 .30 .27 .18 . 53 .90 .18 .50 .56 CAL X RWP * * * * .17 * * * .06 * * * * * * * * * * * * .06 * * * * * * CAL X RWN .43 * * * .76 .47 .29 .39 .36 .13 * .07 * * .15 CAL X MTP .05 * * .41 * .16 * .91 .69 . 56 .54 - -CAL X MTN . 14 * .70 .67 .59 * * .80 .69 .82 * * -CAL X TTP * * * .53 * .60 * * .06 .22 .79 .99 - -CAL X TTN .20 * .64 .68 .52 * * .71 .78 .87 * * * -CAL X PMP .97 .37 .35 * .11 .73 * .38 .66 .30 .72 .68 CAL X PMN .08 .26 .19 * .87 .68 .07 .57 .52 .18 .79 .76 RWP X RWN * * * * * .46 .53 .36 .20 .56 .07 * * * * * * * .21 RWP X MTP . 16 * * * * * * .06 * .31 .97 * * * . 12 - -RWP X MTN .07 * * . 12 .65 .65 .08 .92 .74 .14 * * .08 -RWP X TTP .09 * * * * * * * * * *** * .12 * * * * - -RWP X TTN .07 * * .16 .73 .63 * .82 .73 .16 * * .06 -RWP X PMP .92 .78 .61 * .80 .35 .22 .10 . 10 .21 .75 . 15 RWP X PMN .21 .25 .07 * .69 .74 .14 .99 .28 .69 . 53 . 39 c o n t i n u e d . . . 100 Table XII. Significance of variable correlations under f i e l d conditions (continued; see note p. 98).+ SITE 1C 11 2F 2H 2R 3A 3B 4 5 6 7H 7C RWN X MTP .77 * .62 .61 .82 .16 .94 .43 .09 .73 - -RWN X MTN * * * * * * * * * * * * * * * * * * * * * * * * * ** * * -RWN X TTP .93 * * .67 .36 .67 .08 .67 .49 .18 .70 - -RWN X TTN * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * -RWN X PMP .42 .87 .51 .57 .71 .51 .87 .08 * .51 .56 .38 RWN X PMN .16 * * .40 .47 .67 .45 .45 .10 .59 .39 .18 MTP X MTN .07 * * .09 .63 .73 * * * .30 .39 .10 16 - -MTP X TTP * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * - -MTP X TTN .11 * * .11 .47 .94 * * * .32 .88 .14 .38 - -MTP X PMP * * * * * * * * * * * * * * * * * * * .44 - -MTP X PMN .10 .13 .07 .07 .32 * * * * .08 - -MTN X TTP .12 * * .15 .68 .95 * * .86 .22 .12 .32 - -MTN X TTN * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * -MTN X PMP .28 * .24 .85 .97 * * .20 .95 .10 .58 .26 -MTN X PMN * * * ** .20 .58 * * ** .08 .20 ** -TTP X TTN .16 * * .16 . 53 .85 * * .79 .29 . 15 .45 - -TTP X PMP .58 .18 .89 * .33 .65 .84 .60 * .30 - -TTP X PMN .08 .17 .24 * .86 .34 .51 .28 .06 .30 - -TTN X PMP .39 * .35 .91 .80 * .26 .45 .15 .92 .39 -TTN X PMN .06 * * * . 55 .71 .41 .08 . 11 .13 .76 * * -PMP X PMN .70 * * . 10 .13 * * * * * * * * * * . 12 .09 * * * n = 25 25 15 15 16 25 25 25 28 15 24 25 101 4.3 Ectomycorrhizal types in the nursery Over 99% of the mycorrhizal t i p s encountered on seedlings from the MacBean Nursery were of Type 1 (see Apeendix 5 for type descriptions) i d e n t i f i e d as being formed by Thelephora terrestris. Type 3 mycorrhizae formed by Mycelium radicis atrovirens accounted for less than 1% of the mycorrhizae on most seedlots examined. Type 24 mycorrhizae formed by Laccaria laccata did not occur on any of the sampled seedlings, but mushrooms of th i s fungus were seen f r u i t i n g amongst other seedlings in the nursery. T. terrestris mycorrhizae also formed on seedlings from the Oregon nursery; however, no sporocarps were seen and strands were not common. Over 99% of the mycorrhizal t i p s encountered on the control seedlings were of th i s type. Less than 1% were of Type 18, the mycobiont of which could not be i d e n t i f i e d . A l l of the inoculated seedlings formed Type 2 mycorrhizae involving Rhizopogon vinicolor. One of these seedlings with low colonization levels also had a few t i p s of T. terrestris mycorrhizae. Colonization pattern of Thelephora terrestris in the nursery The l e v e l of root colonization by T. terrestris in the nursery was highly variable. Not only was v a r i a b i l i t y high within the sampled seedlings from a small area of one shade house ( i . e . a selected seedlot) but also between shade houses (i. e . between seedlots). Fd9766 seedlings had mostly high levels of T. terrestris mycorrhizae while most Fd4503 102 seedlings had low percentage colonization or were nonmycorrhizal (Fig. 20) . This suggests T. terrestris mycorrhizae probably had a clumped d i s t r i b u t i o n pattern in MacBean Nursery and was sensitive to seedling growing conditions or genotype. Fd9509F seedlings (prior fumigation of potting mix) had a similar colonization pattern as nonfumigated controls but more of the seedlings lacked high levels of colonization and more had low levels or lacked T. terrestris mycorrhizae (Fig. 20). The use of fumigated potting mix appeared to delay i n i t i a l colonization or slow the rate that T. terrestris spread to root t i p s as they formed. Shoot growth was probably higher than the controls before extensive mycorrhizal development occurred and redirected more photosynthates from the shoot. However, at the one sampling point, there was no indication T. terrestris colonization influenced shoot growth (Table XI). Fd3290 seedlings grown in the Oregon nursery mostly lacked T. terrestris mycorrhizae. A r t i f i c i a l inoculation with R. vinicolor may have decreased the spread of T. terrestris on Fd3290I compared to Fd3290C (Fig. 20). 4.4 Ectomycorrhizal types formed after outplanting Occurrence of Thelephora terrestris on field samples It was not determined i f the T. terrestris mycorrhizae which formed on new roots involved strains that originated in the nursery. The percentage of seedlings from the Oregon nursery with T. terrestris mycorrhizae was low and remained 103 PERCENTAGE OF SEEDLINGS SAMPLED 100% 75% 50% 25% 0% Fd9509C Fd9509F Fd3290C Fd3290l Fd9766 SPECIES & SEEDLOT Fd4503 Hw7321 J ABSENT (0%) LOW (1-15%) i MED-LOW (16-50%) 1 MED-HIGH (51-85%) HI HIGH (86-100%) FIG. 20. Colonization pattern of Thelephora terrestris (Type 1) mycorrhizae on nursery seedlings. low after one season in the f i e l d . This pattern suggests T. t e r r e s t r i s mainly originated in the nursery and not from f i e l d s o i l : T. t e r r e s t r i s mycorrhizae occurred on r e l a t i v e l y few of the Fd3290C seedlings yet the colonization l e v e l of the plug was not as high as Fd3 2 9 0I seedlings indicating that there was "room" for T. t e r r e s t r i s from the s o i l ( i f present) to colonize these t i p s without dir e c t competition from other mycorrhizal types (Figs. 21 and 22). But th i s did not happen. Instead the r e l a t i v e abundance of T. terrestris mycorrhizae was low in re l a t i o n to a number of other types that colonized the roots (Fig. 22). Conversely, most seedlings from MacBean Nursery had T. t e r r e s t r i s mycorrhizae before and after outplanting (Figs. 2 0 and 21). Sporocarps of T. t e r r e s t r i s were common i n the nursery. Therefore, even nonmycorrhizal seedlings probably had spores of T. t e r r e s t r i s in the potting mix that were able to germinate after conditions changed following outplanting and colonize new roots. T. t e r r e s t r i s spores involved in forming Type 1 mycorrhizae in MacBean Nursery were probably blown in from nearby forests and therefore more l i k e l y adapted to eastern Vancouver Island compared to the variety of T. t e r r e s t r i s on the seedlings from Oregon. As a result T. t e r r e s t r i s mycorrhizae had the greatest r e l a t i v e abundance on loca l seedlings (Fig. 22), and was on almost a l l the seedlings sampled (Fig. 21). No MacBean Nursery seedlings were sampled from Site 1. This would have helped to confirm T. t e r r e s t r i s could grow on Site 1 s o i l s . 105 PERCENTAGE OF SEEDLINGS S A M P L E D 100 T " 1C 11 2F 2H 2R 3A 3B 4 5 6 7H SITE OCCURRENCE ON SEEDLINGS: PLUG & NEW ROOT • PLUG ROOT ONLY iH NEW ROOT ONLY FIG. 21. Occurrence of Thelephora terrestris (Type 1) mycorrhizae on f i e l d collected seedlings. SITE: 1C 11 2F 2H 2R 3A 3B 4 5 6 7H TYPE: N i LZD2 » 3 H 4 BB 5 ^ 6 • M INOR FIG. 22. Relative abundance of the major types and combined re la t ive abundance of the minor types of ectomycorrhizae encountered on f i e l d col lected seedlings. Type 1 = Thelephora t e r r e s t r i s ; Type 2 = Rhizopogon v i n i c o l o r ; Type 3 = Mycelium r a d i c i s a t r o v i r i n s ; Type 4 = Cenococcum geophilum; Type 5 = £ n d o g o n e - l i k e ; Type 6 = Tuber- l ike; minor types see F i g . 25. Occurrence of Rhizopoaon vinicolor on field samples The Type 2 mycorrhizae that formed on f i e l d samples were morphologically i d e n t i c a l to R. vinicolor mycorrhizae formed on Douglas-fir in the Oregon nursery following a r t i f i c i a l inoculation. On the basis of c y s t i d i a characteristics (see Appendix 5) , i t i s assumed that a l l Type 2 mycorrhizae were formed by R. vinicolor. Of the control seedlings, 72% formed R. vinicolor mycorrhizae and 16% of these formed i t only on the new roots. This suggests the fungus originated i n the s o i l and colonized new roots before the plug roots (Fig. 23). Two of the inoculated seedlings lacked R. vinicolor mycorrhizae i n the plug but one of these had R. vinicolor mycorrhizae on the new roots. Therefore, i t i s possible that the plot that should have contained only inoculated seedlings (according to the map drawn by the tree planters) actually overlapped the area containing control seedlings. R. vinicolor mycorrhizae were also present on a large percentage of the Douglas-fir seedlings from Sites 2 through 6 (Fig. 23). Next to T. terrestris mycorrhizae, R. vinicolor had the greatest r e l a t i v e abundance on the Douglas-fir grown in MacBean Nursery and outplanted onto these s i t e s (Fig. 22). Overall, more seedlings from the f i e l d had R. vinicolor mycorrhizae on the new roots than on the plug roots. The reverse was true for T. terrestris mycorrhizae (Table XIII). This may further support the hypothesis that most of the T. terrestris mycorrhizae formed on the new roots involved strains of t h i s fungus that originated in MacBean Nursery and 108 PERCENTAGE OF SEEDLINGS S A M P L E D 100 i FIG. 23. Occurrence of Rhizopogon v i n i c o l o r (Type 2) mycorrhizae on f i e l d collected seedlings. was l e s s a b l e t o compete w i t h s i t e a d a p t e d f u n g i i n s o i l . W e s t e r n h e m l o c k d i d n o t f o r m R. vinicolor m y c o r r h i z a e . R. vinicolor i s one o f t h e r e l a t i v e l y few e c t o m y c o r r h i z a l f u n g i t h a t i s s p e c i f i c t o D o u g l a s - f i r . T h e a b s e n c e o f R. vinicolor m y c o r r h i z a e on w e s t e r n h e m l o c k s u p p o r t s t h e s u g g e s t i o n t h a t T y p e 2 m y c o r r h i z a e on f i e l d c o l l e c t e d s a m p l e s was R. vinicolor. Other types of mycorrhizae in the field T h e D o u g l a s - f i r f r o m O r e g o n o u t p l a n t e d o n t o S i t e 1 f o r m e d many o t h e r t y p e s o f m y c o r r h i z a e b u t R. vinicolor h a d t h e g r e a t e s t r e l a t i v e a b u n d a n c e on t h e p l u g a n d t h e new r o o t s o f b o t h t h e i n o c u l a t e d and t h e c o n t r o l s e e d l i n g s . T h e r e l a t i v e a b u n d a n c e o f R. vinicolor m y c o r r h i z a e were g r e a t e r on new r o o t s t h a n on p l u g r o o t s o f t h e c o n t r o l s e e d l i n g s ( F i g . 24) . T u b e r - l i k e ( T y p e 6) m y c o r r h i z a e were r e l a t i v e l y more a b u n d a n t on t h e c o n t r o l s e e d l i n g s . T y p e 43 m y c o r r h i z a e were p r o b a b l y i m m a t u r e T u b e r - l i k e m y c o r r h i z a e i n most c a s e s on S i t e 1. I n t h e f i e l d t h e r e was a t o t a l o f 17 t y p e s on t h e c o n t r o l s e e d l i n g s a n d 15 t y p e s on t h e i n o c u l a t e d s e e d l i n g s ( F i g s . 22 a n d 25) . T h i s f u r t h e r c o m p l i c a t e s t h e i n t e r p r e t a t i o n o f t h e e f f e c t o f a r t i f i c i a l i n o c u l a t i o n on t h e s u r v i v a l a n d g r o w t h o f t h e s e o u t p l a n t e d s e e d l i n g s . T y p e s o t h e r t h a n T . terrestris a n d R. vinicolor were u s u a l l y on l e s s t h a n 50% o f t h e s e e d l i n g s s a m p l e d f r o m S i t e s 2 t h r o u g h 7H ( T a b l e X I I I ) . Endogone-like a n d T u b e r - l i k e m y c o r r h i z a e o c c u r r e d on some s e e d l i n g s f r o m some o f t h e o t h e r R E L A T I V E A B U N D A N C E 100% i 1 i — P L U G ROOT N E W ROOT P L U G R O O T N E W R O O T C O N T R O L I N O C U L A T E D T Y P E : H 1 1 E Z ] 2 H 3 WWMA H e • 43 • M I N O R FIG. 24. Relative abundances of ectomycorrhizal types on plug roots and new roots of Douglas-fir from Site 1. Type 1 = Thelephora terrestris; Type 2 = Rhizopogon vinicolor; Type 3 = Mycelium radicis atrovirins; Type 4 = Cenococcum geophilum; Type 5 = Endogone-like; Type 6 = Tuber-like; Type 4 3 was prob-ably immature Type 6 in most cases; minor types see Fig. 25. s i t e s but with lower r e l a t i v e abundances than on Site 1. Tuber-like mycorrhizae occurred on Sites 2R and 5 but were much less common than on Site 1 indicating a possible preference for Brunisols over Humo-Ferric Podzols (Table I I ) . 5ndogone-like (Type 5) mycorrhizae were absent from the seedlings on Sites 2R, 3B, and 4 (Fig. 22) . Mycelium radicis atrovirens (Type 3) mycorrhizae occurred on some of the seedlings from a l l the plots but when present i t accounted for less than 1% of the mycorrhizal t i p s on average. Cenococcum geophilum (Type 4) mycorrhizae were on most of the Douglas-fir seedlings, but l i k e M. radicis atrovirens mycorrhizae, i t usually accounted for less than 1% of the mycorrhizal t i p s . C. geophilum mycorrhizae accounted for a s l i g h t l y greater proportion of the mycorrhizal t i p s of Hw: on the new roots C. geophilum mycorrhizae averaged about 2% and about 1% of the mycorrhizal t i p s in the plug. Type numbers 7-9 were reserved in case additional major types of mycorrhizae were encountered but none were. A t o t a l of 38 minor types of mycorrhizae, numbered 10 through 47, were encountered (Fig. 25). Between 1 and 11 minor types of mycorrhizae were present on f i e l d - c o l l e c t e d seedlings. Site 4, which had the lowest mycorrhizal colonization and was probably the most harsh of the sit e s sampled, had f i v e mycorrhizal types present. Site 1, which was probably one of the more favorable s i t e s for seedling establishment (Table I I ) , had 22 types of mycorrhizae present. Seedlings from Site 5 had the highest fungal d i v e r s i t y rating 112 RELAT IVE A B U N D A N C E 100% 10. 75% 50% 25% 14 27 35 45 33 17 32 43 16 21 29 35 40 46 20 42 43 20 13 15 17 31 40 29 25 43 33 20 22 26 36 |44 38 32 25 43 33 25 32 37 39 36 35 28 36 4 l 1 19 17 ~14~ 16 29 40 36 43 36 12 33 23 34 28 14 43 0% 1 — 1 1 — 1 1 — 1 1 — 1 1 — 1 1 — 1 1 — 1 1 — 1 1 — 1 1 — 1 1 — S I T E : 1 C 11 2F 2H 2R 3A 3B 4 5 6 7H FIG. 25. Relative abundances of the minor types of ectomycorrhizae encountered on f i e l d collected seedlings. Numbers refer to types of mycorrhizae and types grouped together in a bar have the same relative abundance. (Table XIII). Although most of Type 4 3 mycorrhizae from Site 1 were probably immature Tuber-like mycorrhizae, i t may not have been on s i t e s where Tuber-like was absent (e.g. Sites 3A and 7H) . Type 4 3 was a general grouping for a l l wide, closely septate hyphae forming mycorrhizae without any other d i s t i n c t i v e c h a r a c t e r i s t i c s . Other minor types such as Type 13 were so d i s t i n c t i v e that probably only one fungal species was involved. Type 13 only occurred on Site 2F. Type 3 3 mycorrhizae were also d i s t i n c t i v e but sometimes could only be distinguished from R. vinicolor mycorrhizae by the lack of c y s t i d i a when stained because R. vinicolor mycorrhizae occassionally produced hyphal exudates. Type 3 3 was most common on the sand and gravel of Site 6 but i t also occurred on Site 2R ("roadway", Table XIII and Fig. 25). No other relationships between mycorrhizal type and s o i l or s i t e conditions were observed (Tables III and XIII). Western hemlock had nine mycorrhizal types. Two of the minor types (Types 23 and 34) were not found on Douglas-fir. 114 Table XIII. Percentage of seedlings sampled from each plot with each type of ectomycorrhiza i n plug (p) and new roots (n).+ SITE 1C II 2F 2H 2R 3A 3B 4 5 6 7H n= 25 25 15 15 16 25 25 25 28 16 24 MAIN TYPES: 1 P 20 8 80 80 88 76 88 100 79 88 79 n 24 8 93 67 88 76 80 96 79 75 71 2 P 56 92 73 60 69 68 72 52 43 50 0 n 72 96 73 73 75 92 76 44 39 56 0 3 P 20 20 33 27 13 36 48 28 54 6 25 n 8 4 7 13 0 4 12 8 36 19 21 4 P 12 4 60 40 31 32 16 24 43 0 46 n 28 16 53 53 38 48 28 32 54 6 58 5 P 36 48 7 13 0 4 0 0 7 0 13 n 24 40 0 7 0 0 0 0 11 6 13 6 P 60 24 0 0 0 0 0 0 4 0 0 n 64 20 0 0 6 0 0 0 0 0 0 MINOR TYPES: 10 4n 11 (occurred on one sample from Site 1C in June sampling) 12 — — — — — — — — — 19p 19n 13 27n 14 4p — — — — — — — 7p — 4p 8n 15 — — — 7p v 16 — — — — — — — — 7p 4n — — — — — — 4n 17 12p — — — — — — — 4p 12n — — — — — — — 4n 18 (occurred on one Fd3290C sample in the Oregon nursery) 19 — — — — — — — — 4p 4n 20 — 4p 7p — — 4p 12n 7n continued. . . 115 Table XIII continued. S I T E  1U II 2F 2H 2R 3A 3B 4 5 6 7H MINOR TYPES: 21 — 4p 22 — — — — — 4p 23 4n 24 (occurred i n MacBean Nursery but was not on samples) 25 — — — 7p — — 4 p 20n — 8n 4n 26 27 4n 28 — — — — — — 8p — — — 4p 8n — — — 8n 29 — 4p — 7p — — — — l i p 7n — — — — 7n 30 — — — — — — — — 4p 31 7n 32 16p — — — — 4p 12n — — — — 4n 8n 33 4p — — — 13p — — — — 50p 4n — — — 13n — 4n — — 50n 34 — — — — — — — — — — 4 p 4n 35 4 p — — — — - - 4 p 4n — — — — 8n 36 — — — — — 4p 8p 4p 21p 6p — — — — — — 4n 4n 14n 6n 37 — — — — -- — 8p 38 — — — — — 4p 4n 39 — — __ - - — — 4 p 4n 40 — — — 7p — — — — 14p 7n — — — — 18n 41 4n 42 — 4p 20n continued... 116 Table XIII continued. SITE IU II 2F 2H 2R 3A 3B 4 5 6 7H MINOR TYPES: 43 28p 32p — — 6p 32p — — — 6p 21p 24n 36n — — 13n 12n — — — 6n 25n 44 — — — — — 4p 45 4 p 4n — — — — — — — 4n 46 4p 47 4n 4n TOTAL TYPES: 17 15 7 11 7 13 12 5 16 9 9 Dl: 3.56 3.28 3.27 3.4 2.56 3.6 3.12 2.48 3.75 2.73 2.54 P 4p 12p — 7p — — 4p 4p 4p 13p 4n — — — — 4n — 4n + Double dash (—) indicates type was not present on any of the sampled seedlings from the s i t e . Dl = fungal d i v e r s i t y index = the average number of mycorrhizal types per seedling sampled from each s i t e . P = p a r a s i t i c fungi (hyphae invading stele or producing spores i n root c e l l s ) . 4.5 Key to the types of ectomycorrhizae encountered l a . Walls of hyphae brown after staining procedure (clearing roots and staining with Trypan blue) 2 2a. Emanating hyphae absent, inner mantle and Hartig net blue-staining Type 10 2b. Emanating hyphae present 3 3a. Clamps present on emanating hyphae Type 11 3b. Clamps absent 4 4a. Mantle and Hartig net blue-staining; only emanating hyphae with brown pigment Type 12 4b. Mantle and/or Hartig net also with brown pigment 5 5a. Mantle hyphae forming s t e l l a t e pattern, with hyphae 4-5 um wide Type 4 117 5b. Mantle hyphae (when present) forming "jigsaw" or s t e l l a t e pattern, with hyphae 1-2 um wide Type 3 lb. Walls of hyphae blue-staining or colorless 6 6a. Hyphae without septa at regular intervals. Mantle absent Type 5 6b. Hyphae with regular septation. Mantle present or absent 7 7a. Emanating hyphal ends of d i s t i n c t i v e size and shape (cystidia) present 8 8a. Cystidia with clamp where attached to mantle, (25-)75-150(-340) x (1.5-)2-3 um (width at base) Type 1 8b. Cystidia without clamp 9 9a. Cystidia b r i s t l e - l i k e , 70-120(-140) x (2-)4-5(-6) um (width at base). Mantle textura "jigsaw". . . .Type 6 9b. Cystidia not as above 10 10a. Length of c y s t i d i a usually < 50 um 11 11a. Cystidia 25-30 um long, some with 1 to 3 round spore-like structures produced at t i p Type 13 l i b . Cystidia 35-50(-70) x 3-5 um (width at center) Type 14 10b. Length of c y s t i d i a mostly > 50 um 12 12a. Cystidia 100-650 x 1.5-2(-3) um, often with one (or two) kinks; attachment to other hyphae very hard to see; changing from brown to colorless after staining procedure. Brownish exudates sometimes present Type 2 118 12b. Cystidia 40-105 x 0.5-1 um along most of length to 1-3 um wide at base; blue-staining Type 15 7b. Emanating hyphae ends rarely seen or are not intact...13 13a. Emanating hyphae clamped 14 14a. Hyphae very narrow (1-1.5 um) Type 16 14b. Hyphae often 2 um or greater 15 15a. Mantle absent, emanating hyphae very abundant, 2-3 um wide, clamps on a l l septa Type 17 15b. Mantle present 16 16a. Keyhole clamps present Type 18 16b. Keyhole clamps absent or closed clamps predominate 17 17a. Outer mantle hyphae synenchymous 18 18a. Textura not discernable Type 19 18b. Textura angular i s Type 2 0 17b. Outer mantle hyphae prosenchymous 19 19a. Mantle hyphae with swellings > 7 um wide Type 21 19b. Mantle hyphae < 7 um wide 20 2 0a. Clamps absent in mantle Type 22 20b. Clamps present in mantle 21 21a. Width of emanating hyphae highly variable along short lengths Type 23 21b. Width of emanating hyphae not variable along short lengths 2 2 22a. Emanating hyphae extremely thin walled, ribbon-like and staining poorly Type 24 119 22b. Emanating hyphae sometimes ribbon-like but walls generally staining blue 23 23a. Clamps on a l l septa of a l l emanating hyphae Type 25 2 3b. Clamps not on a l l septa of a l l emanating hyphae 2 4 2 4a. Emanating hyphae 1-4 um wide with only the larger hyphae clamped . .Type 2 6 24b. Emanating hyphae otherwise 25 25a. Mantle very compact, hyphae 3-6 um wide. Emanating hyphae common, 3.5-4 um wide Type 27 2 5b. Mantle moderately compact, hyphae 2-5 um wide. Emanating hyphae abundant, 1.5-4 um wide Type 28 13b. Emanating hyphae not clamped 2 6 26a. Emanating hyphae 2 um wide or narrower 27 2 7a. Mantle < 5 um thick with very narrow hyphae l i k e emanating hyphae Type 2 9 27b. Mantle 5-25 um thick with hyphae appearing dif f e r e n t than emanating hyphae 28 2 8a. Hyphae in mantle 3-6 um wide, textura epidermoidia Type 3 0 28b. Hyphae in mantle 1-1.5 um wide to 3 um wide near epidermis; net prosenchymous with abundant 3-way intersections Type 31 26b. Emanating hyphae often > 2 um wide 29 120 2 9a. Width of emanating hyphae < 3 um wide 3 0 3 0a. Mantle absent Type 32 30b. Mantle present 31 31a. Red-brown exudates present on emanating hyphae and rhizomorphs. Cystidia absent Type 33 31b. Red-brown exudates absent 3 2 32a. Mantle f e l t prosenchymous; hyphae infrequently branched and not forming a network 33 33a. Cystidia present, strands absent Type 14 33b. Cystidia absent, strands present Type 34 3 2b. Mantle net prosenchymous; hyphae frequently branched, forming a network 34 34a. Septa in mantle hyphae 3-20 um apart Type 35 34b. Septa in mantle hyphae mostly > 20 um apart...35 35a. Mantle 20-80 (-150) um thick, hyphae 1-3 um wide and branching to form 3-way intersections Type 3 6 35b. Mantle < 20 um thick, hyphae 2-4 um wide and branching to form 3 or 4-way intersections Type 37 29b. Width of hyphae often > 3 um wide 36 3 6a. Emanating hyphae 4 um wide or narrower 37 37a. Mantle absent Type 38 37b. Mantle present 38 3 8a. Net prosenchymous mantle with 3-way intersections Type 39 121 3 8b. Net prosenchymous mantle with 3- to multi-way intersections Type 40 36b. Emanating hyphae often or mostly > 4 um wide 39 39a. Outer mantle hyphae synenchymous 40 4 0a. Hyphae in outer mantle appearing glued together with blue-staining substance Type 41 4 0b. Hyphae of outer mantle without blue-staining substance 41 41a. Textura "jigsaw" Type 42 41b. Textura angular i s Type 2 0 3 9b. Outer mantle hyphae prosenchymous 4 2 42a. Septation in mantle and emanating hyphae < 2 0 um apart Type 43 42b. Septation in mantle (when present) and emanating hyphae mostly > 20 um apart 4 3 43a. Mantle absent Type 44 43b. Mantle present 44 44a. Mantle hyphae mostly 3-4 um wide Type 45 44b. Mantle hyphae 3-9 um wide 45 4 5a. Hartig net beaded Type 44 45b. Hartig net not beaded 46 4 6a. Mantle moderately compact; hyphae 3-9 um wide, branching infrequently to form 3-or 4-way intersections Type 46 46b. Mantle very compact; hyphae 4.5-9 um wide, branching frequently to form 3-way intersections Type 47 122 5 DISCUSSION 5.1 Mycorrhiza formation before outplanting Douglas-fir and western hemlock in MacBean Nursery Ectomycorrhizae involving Thelephora terrestris formed on most of the Douglas-fir and western hemlock seedlings. T. terrestris i s often the most common mycobiont i n nurseries that do not manage mycorrhizae (Marx et al. 1970; S i n c l a i r 1974; Hung and Molina 1986). Spores are blown in from the surrounding forest and circulated within the nursery as indicated by the presence of sporocarps on the underside of styroblocks. In southern United States, T. terrestris i s one of the primary mycobionts that colonize fumigated or s t e r i l e s o i l (Marx et a l . 1970). Regardless of what i s done to purify potting mix, spores of T. terrestris are usually abundant in a i r and soon find t h e i r way back into the potting mix. The resu l t usually i s that well f e r t i l i z e d seedlings grown in fumigated potting mix have a high proportion of T. terrestris mycorrhizae and a low di v e r s i t y of other fungi (Marx et al. 1970; Perry et al. 1987). In agreement, prior fumigation of potting mix with methyl bromide did not discourage subsequent mycorrhiza formation in the present study. The l e v e l of root colonization by T. terrestris was not correlated to shoot weight, height, caliper, or the number of root t i p s in the nursery. In a bare root nursery, Marx et al. (1978) found a positive growth response of pine seedlings to T. terrestris inoculation. Beese (1987) found cold storage does not affect 123 colonization levels. Alvarez and Linderman (1983) report T. terrestris survives at least 4 months in cold storage and then i s able to colonize new roots formed on Douglas-fir 20 days after outplanting. Those seedlings which did not form mycorrhizae in MacBean Nursery probably had spores of T. terrestris present in the potting mix. Western red cedar In agreement with Beese (1987) mycorrhizae did not form on western red cedar in MacBean Nursery. Although inoculum may have been present in the peat (Danielson et al. 1984a), high f e r t i l i t y levels or the use of fungicides (Trappe et al. 1984) may have prevented growth and mycorrhizal colonization by vesicular-arbuscular mycorrhizae. 5.2 Mycorrhiza formation after outplanting Douglas-fir and western hemlock from MacBean Nursery A l l outplanted seedlings formed mycorrhizae. The average colonization level of new roots ranged from 72 to 93% per s i t e . Although many mycorrhizal types formed, Thelephora terrestris and Rhizopogon vinicolor (Douglas-fir only) had the greatest r e l a t i v e abundance on most of the outplanted seedlings. Marx and Bryan (1970) noted that isolates of T. terrestris can d i f f e r in their a b i l i t y to produce mantles, but the mycorrhizae formed are otherwise indistinguishable from each other regardless of host. Agerer and Weiss (1989) 124 reviewed other descriptions of T. terrestris and found some features are variable, but in general, isolates from many locations were very similar. The same was found here: other than mantle thickness, abundance of c y s t i d i a , and rhizomorph diameter and color, T. terrestris mycorrhizae i n the nursery was very similar to those formed on the new roots after outplanting. However, sometimes mycorrhizae with longer c y s t i d i a were found in the f i e l d and the c y s t i d i a were generally narrower than most other descriptions (Agerer and Weiss 1989). Because the morphology of T. terrestris mycorrhizae i s quite consistant from s t r a i n to s t r a i n , i t i s often not certain i f the fungus originated in the nursery or f i e l d (Thomas et al. 1983; Danielson 1985a; Beese 1987). Completely nonmycorrhizal seedlings should have been outplanted onto some of the si t e s or naturally regenerated seedlings should have been examined for T. terrestris to determine i f the fungus occurs i n s o i l and i f i t would colonizae new roots. Although i t was not determined i f T. terrestris was present or absent in the s o i l s of any of the s i t e s , several patterns i n d i r e c t l y observed suggest new roots were colonized by T. terrestris originating in the nursery. F i r s t , T. terrestris was the dominant fungus in the nursery and on the new root, and rhizomorphs were repeatedly seen extending between the plug and new roots. The mycorrhizal colonization l e v e l of new root was frequently correlated to the number of mycorrhizal t i p s in the plug. Second, R. vinicolor 125 mycorrhizae were r e l a t i v e l y more abundant on new roots than plug roots indicating T. terrestris (Type 1) was adapted to nursery conditions and less able to colonize t i p s i n s o i l . Third, the Oregon seedlings mostly lacked T. terrestris and t h i s remained the same after outplanting even though there were nonmycorrhizal t i p s in the plug. Seedlings from MacBean Nursery outplanted onto Site 1 should have been examined to determine i f T. terrestris can grow and colonize new roots i n the Brunisol s o i l s of t h i s s i t e . This would have provided stronger evidence that T. terrestris mainly originated in MacBean Nursery. There may be wide ecological adaptability within a fungal species (Trappe 1977) : spores in the potting mix that did not germinate in the nursery may have once outplanted. This would explain why Fd4503 seedlings were nonmycorrhizal or had low levels of T. terrestris mycorrhizae before outplanting while nearly a l l those sampled from the f i e l d had higher colonization. It was also possible that the seedlings sampled from the f i e l d were from an small area in MacBean Nursery where mycorrhiza formation by T. terrestris was high. Either or both situations may have existed and help explain the patterns observed. There may be competition between dif f e r e n t mycorrhizal fungi in the colonization of new root surfaces (McAfee and Fortin 1986), and those better adapted to s i t e conditions may increase in abundance (Bledsoe et al. 1982). Thelephora may colonize new roots only when not in competition with fungi 126 better adapted to f i e l d conditions (Vare 1989). In the present study, competition for new root surfaces was such that T. terrestris maintained the greatest r e l a t i v e abundance. There were frequently 10 to 3 0% or more of the new roots that were nonmycorrhizal suggesting more of a gradual replacement of types as noted by Danielson and Visser (1989) instead of dir e c t competition implied by others (Bledsoe et al. 1982; McAfee and Fortin 1986; Vare 1989). Wilson et al. (1987) inoculated spruce seedlings with various fungi while Thelephora occurred on the control seedlings. Although Thelephora was previously not observed on s i t e , i t dominated the control seedlings to the exclusion of a l l other fungi at the end of the fourth growing season. The inoculated fungi established on other seedlings were replaced by Thelephora and Hebeloma species. Others have also found T. terrestris can p e r s i s t on roots for several years after outplanting seedlings on disturbed s o i l s (Danielson et al. 1984b; Danielson and Visser 1989). Current s i l v i c u l t u r a l practices involving the production of seedlings in styroblock containers under high f e r t i l i z a t i o n levels may be enhancing f i e l d populations of T. terrestris compared to what might occur with natural regeneration. Although T. terrestris i s well adapted to nursery conditions and may show a positive growth response (Marx et a l . 1978), Marx et al. (1984) stated that i t often f a i l s on outplanting s i t e s in southeastern United States. In northern Alberta, Danielson and Visser (1989) found a positive growth 127 response of pine to T.• t e r r e s t r i s after 2 growing seasons on o i l sand t a i l i n g s . Waiting (1982) states that T. terrestris i s a proven and important mycorrhizal fungus, p a r t i c u l a r l y on acid heaths. Although Kammerbaur et al. (1988) found that rhizomorphs of T. terrestris can reach a maximum length of 12 cm and can transport phosphorus to the mycorrhizae, there are no reports of a positive growth response to mycorrhizal colonization by T. terrestris in the P a c i f i c Northwest. With over 150 species worldwide, Rhizopogon species are widespread in the P a c i f i c Northwest and are important mycorrhiza formers with members of the Pinaceae (Castellano and Trappe 1985; Ho and Trappe 1987) . R. vinicolor i s considered to be s p e c i f i c to Douglas-fir, but i n pure culture i t may s u p e r f i c i a l l y colonize a few western hemlock roots (Molina and Trappe 1982). R. vinicolor was not found on western hemlock in the present study. R. vinicolor i s common on young Douglas-fir seedlings in clearcuts (Schoenberger and Perry 1982; Parke et al. 1983a, 1984; P i l z and Perry 1984; Perry et al. 1987). Therefore, i t was not surprising to find an abundance of R. vinicolor on new roots of Douglas-fir formed after outplanting. Tubercles did not form in the Oregon nursery and were only occasionally seen on f i e l d - c o l l e c t e d samples. Trappe (1965) noted that formation of R. vinicolor mycorrhizae begins early i n spring and i s well advanced before the tubercle forms. Judging from t h i s , and the great variation in the abundance of c y s t i d i a , most of the mycorrhizae sampled i n th i s 128 study were s t i l l immature. Microscopic characteristics of the Type 2 mycorrhizae agreed closely with the description of R. vinicolor given by Zak (1971): stubs of thin walled hyphae were seen attached to the kinked c y s t i d i a which Zak referred to as terminal sickle-shaped aseptate hyphae. Mantle hyphae characteristics were also similar and used with the presence of c y s t i d i a to group t i p s into t h i s type. Each seedling sampled was examined before staining and the presence of R. vinicolor was noted based on color, form and the rhizomorphs attached. Microscopic examination of c y s t i d i a and mantle hyphae increased the certainty of mycobiont i n d e n t i f i c a t i o n and was necessary for recently colonized t i p s . Trappe (1965) noted that greatest numbers and size of tuberculate mycorrhizae are found in well-rotted stumps and logs. The mycorrhiza i s known to occur up to 1500 m in elevation in Oregon, and on northern Vancouver Island at 2 0 to 100 m elevation in stands of various ages, mostly i n mixture with western hemlock. In the present study, most of the seedlings had th e i r roots in contact with only mineral s o i l and the s i t e s ranged in elevation from 60 to 800 m. Overall, R. vinicolor mycorrhizae were second highest i n r e l a t i v e abundance on most of the s i t e s . Western red cedar Site 7C was l i g h t l y burned meaning l i t t l e mineral s o i l was exposed. Mycorrhizae formed on 64% of the seedlings sampled. Colonization varied from 0 to 64% with only 15% of 129 the new root colonized on average for a l l the seedlings sampled. No correlation between percentage mycorrhizae and seedling growth was found. In contrast, Parke et al. (1983c) found the weight of mycorrhizal western red cedar seedlings was about four times greater than nonmycorrhizal seedlings. Beese (1987) found that colonization of western red cedar outplanted on clearcuts on eastern Vancouver Island varied from 0 to 72% and averaged 17 to 32% after 7 months i n the f i e l d . Up to 95% of the seedlings sampled from the f i e l d formed VA mycorrhizae. Burn intensity did not s i g n i f i c a n t l y affect colonization levels, but mycorrhizae involving fine endophyte were r e l a t i v e l y more abundant in severely burned s o i l . Wang et al. (1985) found that low s o i l pH encouraged mycorrhiza formation by fine endophyte (Glomus tenue H a l l ) . Parke et al. (1983a) found only fine endophyte in the roots of cedar growing in s o i l with organic layers removed. In the present study, both fine and coarse endophyte were usually present on Site 7C which had organic layers present and r e l a t i v e l y low s o i l pH. Most of the cedar survived and more than doubled in weight during the f i r s t growing season in the f i e l d despite having l i t t l e or no mycorrhizal colonization. However, many of the seedlings appeared chl o r o t i c possibly due to nutrient deficency related to mycorrhizal status. F i r s t year survival may have depended strongly on nutrients remaining in the potting mix and less on an e f f i c i e n t root system. 130 Several of the noncrop species could form VA mycorrhizae on Site 7C. However, the new roots of western red cedar were nonmycorrhizal or had low levels of colonization because the internal nutrient status of the plant did not favor mycorrhiza formation, environmental conditions were not favorable for mycorrhiza colonization, or because of deficencies in MIP. Beese (1987) found greatest formation of VA mycorrhizae on western red cedar in l i g h t l y burned s o i l s . Site 7 was l i g h t l y burned and conditions did not appear harsher than for Site 4 where most of the noncrop species, including a naturally regenerated cedar, formed high levels of VA mycorrhizae. The chlor o t i c state of the seedlings and the high v a r i a b i l i t y in colonization suggest Site 7C was VA inoculum de f i c i e n t . 5.3 A r t i f i c i a l l y inoculated Douglas-fir seedlings. Rhizopogon vinicolor colonized about half of the short roots of the Douglas-fir seedlings from Oregon that were a r t i f i c a l l y inoculated. In agreement with Castellano et al. (1985), seedlings with R. vinicolor were t a l l e r than uninoculated control seedlings and did not lower seedling weight under the growing conditions used. These seedlings were only some of those grown in Cottage Grove Nursery in Oregon. The others were planted on harsh s i t e s i n Oregon where a r t i f i c i a l inoculation with R. vinicolor has resulted in s i g n i f i c a n t growth increases and improved survival of Douglas-f i r (P. Hahn, personal communication). 131 In the present study, the size of R. vinicolor-inoculated seedlings outplanted on Site 1 was not di f f e r e n t than control seedlings after one f i e l d season. There may have been no growth response to R. vinicolor for several reasons. F i r s t , most of the seedlings had browse and stem damage. This at least eliminated the height difference that existed between control and inoculated seedlings before outplanting and probably decreased growth rates in the f i e l d . Second, besides animal damage, Site 1 was probably among the least harsh of a l l the s i t e s sampled because i t was low elevation, f l a t , and recently logged with minimal s o i l disturbance. Third, there were many other types of mycorrhizae colonizing the root system including s i t e adapted strains of R. vinicolor. The problem with T. terrestris mycorrhizae repeated i t s e l f ; mycorrhizae formed by R. vinicolor in the f i e l d could not be distinguished from those formed in the nursery. T. terrestris occurred on most of the control seedlings and ranged in colonization level from less than 1 to 95%, but only one of the inoculated seedlings had a few t i p s colonized by T. terrestris. A r t i f i c i a l inoculation or some other factor may have limited the occurrence of T. terrestris. Castellano et al. (1985) grew "completely nonmycorrhizal" control seedlings (they did not check each t i p for Hartig net) but did not indicate how T. terrestris was eliminated or i f i t usually occured i n the nursery. Other researchers have found that a r t i f i c a l inoculation with other fungi can greatly decrease the occurrence of T. terrestris in nurseries (e.g., Le Tacon 132 et al. 1985; Hung and Molina 1986). The lack of T. terrestris on the inoculated seedlings from Oregon and other studies involving container-grown seedlings (Castellano et al. 1985) indicates that i t i s possible to compare the performance of seedlings inoculated with T. terrestris and R. vinicolor with nonmycorrhizal seedlings provided spores of T. terrestris, which may be present in the potting mix, do not germinate after outplanting. This may be the main d i f f i c u l t y in producing adequate control seedlings (mycorrhizal or nonmycorrhizal) that completely lack T. terrestris (Marx et al. 1970; Castellano and Trappe 1985). In the P a c i f i c Northwest, R. vinicolor i s reported to occur on Douglas-fir of a l l ages, but mycorrhizae do not spontaneously form in containerized tree nurseries (Zak 1971; Parke et al. 1983a). Rhizomorphs were noted by Trappe (1965) to extend more than 2 5 cm from the mantle of R. vinicolor mycorrhizae. Parke et al. (1983a) found that Douglas-fir seedlings inoculated with R. vinicolor had better drought tolerance than nonmycorrhizal seedlings or seedlings inoculated with Laccaria laccata, Pisolithus tinctorius, or a "native fungus". Zak (1971) also discussed the ecological significance of the morphological characteristics of R. vinicolor mycorrhizae. For example, the numerous rhizomorphs with t h e i r very large diameter central core hyphae may absorb and conduct nutrients to tree roots as found with other mycobionts (Skinner and Bowen 1974a; Duddridge et al. 1980; Kammerbaur et al. 1988). The tubercle rind may be an 133 e f f e c t i v e barrier to pathogens or aphid style penetration to the stele. Methods for spore inoculations are available and are routinely used in Oregon nurseries (Castellano et al. 1985; P h i l Hahn, personal communication). These potential benefits, the r e l a t i v e simplicity of spore inoculations, and the frequent occurrence on new roots, present a strong argument in favor of inoculating seedlings i n the nursery with R. vinicolor. However, to avoid similar results found i n t h i s study, isolates should be collected from clearcuts that are similar to those outplanted and emphasis should be placed on clearcuts which are more d i f f i c u l t to regenerate such as Site 4, s i t e s where prior afforestation attempts have f a i l e d , or areas not s u f f i c i e n t l y restocked. 5.4 Ectomycorrhizal inoculum potential This study was not designed s p e c i f i c a l l y to obtain measures of MIP in clearcuts. Such a study would . have involved more intensive sampling at d i f f e r e n t elevations and aspects within each clearcut. Instead of just one f a l l harvest, periodic sampling throughout the f i r s t growing season would give information on the rate of mycorrhiza formation. Nonmycorrhizal seedlings would be used so that nursery fungi would not be confused with new root colonization level involving f i e l d fungi. Lastly, results from a greenhouse bioassy using s o i l collected from the clearcuts and adjacent forests would help to separate MIP from other factors that influence mycorrhizal colonization potential. Although t h i s 134 was not done, some general comments on mycorrhizal status in r e l a t i o n to s i t e conditions can be made. Soil characteristics Seedlings from the gravel p i t (Site 6) and the logging roadway (Site 2R) had r e l a t i v e l y low mycorrhizal colonization and numbers of mycorrhizal types. The lack of organic matter may have reduced MIP of these s o i l s . Several researchers have found organic matter i s high in mycorrhizal inoculum but may or may not create conditions favorable for root growth and mycorrhiza formation (Slankis 1974; Harvey et al. 1976; Alverez et al. 1979; Kropp 1982; Schoenberger and Perry 1982; Parke et al. 1983c; Rose et al. 1983; Lindeberg 1986). No other relationships between the s o i l variables measured and mycorrhizal status were observed. There are reports of reduced MIP on older clearcuts lacking ectomycorrhizal hosts, p a r t i c u l a r l y those that were previously burned (Parke et al. 1984; P i l z and Perry 1984). Sites 2 and 3 were burned but there was no indication of reduced ectomycorrhizal inoculum. There may have been a number of reasons for t h i s . Seedlings were only sampled in the f a l l after rain and cooler temperatures may have allowed for high colonization levels even i f mycorrhizal colonization potential was low e a r l i e r in the year. Thelephora terrestris, probably originating from the nursery, was dominant on the new roots and i f i t had not grown after outplanting then reduced MIP may have been more 135 evident. None of the s i t e s were severely burned or l e f t without nonmycorrhizal hosts for more than three years. Aspect and elevation Seedlings outplanted on the higher elevation s i t e s with southwest aspect (Sites 2 and 4) had lower levels of mycorrhizal colonization and fewer mycorrhizal types than most other s i t e s . The extent of mycorrhiza formation was lowest on Site 4. Some seedlings may have been sampled before the attenuation phase where new root t i p s are colonized soon after they form. As a result, colonization levels were most variable on new roots of Site 4 seedlings and ranged between 6 and 99%. Although higher in elevation, Site 3 seedlings had more types of mycorrhizae than Site 2 seedlings indicating the warmer southwest aspect of Site 2 may have decreased MIP. However, th i s was not a clear indication of reduced MIP because the conditions affecting mycorrhiza formation were probably very different for each s i t e . A greenhouse bioassay would have to be done to determine i f the s i t e s d i f f e r e d in MIP; by c o l l e c t i n g s o i l from each of the s i t e s and growing seedlings in t h i s s o i l under controlled conditions, differences in r e l a t i v e MIP could be assessed by mycorrhizal colonization level (Parke r et al. 1984). However, environmental conditions may s i g n i f i c a n t l y change and affect the rate of mycorrhiza formation and the r e l a t i v e abundance of types involved (Pilz and Perry 1984). 136 There was no indication of a strong MIP deficiency as indicated by the generally high colonization levels on the new roots of Douglas-fir after one growing season. On Site 1, the June sampling indicated mycorrhizal colonization of new roots was lower than for the November sampling indicating a lag time in mycorrhiza formation even though the l e v e l of s o i l disturbance was low on t h i s s i t e . Because seedlings were only sampled once on Sites 2 through 7, there was no indication of a lag period in which a smaller proportion of new root t i p s were colonized. It i s suspected most seedlings would have had a small proportion of t h e i r new roots colonized i n the f i r s t two or three months following outplanting and t i p s were probably colonized by Thelephora terrestris in most cases. Seedling survival was greater than 90% on a l l s i t e s when sampled i n the f a l l and seedling weight usually more than doubled. If growth and survival after the f i r s t and subsequent years i s adequate (even in years with long summer droughts) then a f u l l scale a r t i f i c i a l inoculation program may not be j u s t i f i e d . However, inoculation t r i a l s with s i t e s p e c i f i c fungi and seedling growth measurements taken over several years i s strongly recommended because of the potential benefits. The present study was only a preliminary investigation that addressed the following questions: Are mycorrhizae formed in MacBean Nursery? Ectomycorrhizae formed on Douglas-fir and western hemlock. Over 99% of the ectomycorrhizae formed involved T. terrestris. Western red cedar did not form 137 mycorrhizae. Are mycorrhizae formed on the new roots after one growing season in the f i e l d ? Yes, most t i p s of most Douglas-fir and western hemlock seedlings were colonized, but there was no measure of the rate of formation. Weastern red cedar formed low levels of VA mycorrhizae. Are these new roots colonized primarily by nursery fungi or f i e l d fungi? Most t i p s were colonized by T. terrestris that probably originated i n the nursery. Rhizopogon vinicolor was also very common on new roots. The VA mycorrhizae found on western red cedar probably originated in the s o i l . Do Douglas-fir seedlings grown in an Oregon nursery inoculated with R. vinicolor show greater weight gain after one season of being outplanted than uninoculated controls? No, for reasons already explained. Now we need to ask what would happen i f lo c a l strains of R. vinicolor are inoculated onto seedlings in the nursery. This may decrease the occurrence of T. terrestris, increase short root branching, and allow for new roots to be colonized by R. vinicolor as soon as they form. The l i t e r a t u r e reviewed strongly suggests inoculation with mycobionts originating from areas near to where the seedlings are to be outplanted can increase seedling growth and survival (Mikola 1973; Trappe 1977; Marx 1980; Molina 1981; Grossnickle and Reid 1982; Linderman 1987; Perry et al. 1987; Marx and Cordell 1988; Danielson and Visser 1989) . The present study found R. vinicolor was very common on young Douglas-fir outplanted in clearcuts on eastern Vancouver Island, but other fungi were 138 also present which might also be considered i f selecting fungi for a r t i f i c i a l inoculation. 5.5 Other ectomycorrhizal types formed on new roots Zak (1969) and Zak and Larsen (1978) described Byssoporia (Poria) terrestris mycorrhizae that often occurred along the same root as Rhizopogon vinicolor. Some v a r i e t i e s of B. terrestris may also occur on western hemlock (Zak and Larsen 1978). The bright yellow rhizomorphic Piloderma bicolor mycorrhiza i s also found on older Douglas-fir (Zak and Larsen 1978), and thought to be a late-stage fungus (R.M. Danielson, personal communication). Kropp (1982) found yellow mycorrhizae on western hemlock after one growing season of being outplanted on a clearcut but the mycobiont was not id e n t i f i e d . Mycorrhizae f i t t i n g these descriptions were not encountered on the outplanted seedlings in the present study. However, a naturally regenerated western hemlock seedling, not more than 2 years old, had bright yellow rhizomorphic mycorrhizae. Parke et al. (1983b, 1984) reported that dominant type of ectomycorrhizae on young Douglas-fir in greenhouse bioassays was not R. vinicolor but a white rhizomorphic type. In contrast, P i l z and Perry (1984) found Rhizopogon species to be very common on Douglas-fir grown in s o i l from undisturbed forests and burned and unburned clearcut s i t e s . The presence of white mycorrhizae with white rhizomorphs was not mentioned ( i t may have been one of the i r minor types) . P i l z and Perry 139 also reported that Rhizopogon species plus a brown type consistently formed at least two-thirds of the ectomycorrhizae after one growing season on Douglas-fir seedlings outplanted in clearcuts of west-central Oregon. In the present study, R. vinicolor was very common on the new roots. Without using the staining procedure, Thelephora terrestris and most of the other types would have appeared brown. No white, rhizomorphic mycorrhizae were encountered. Mycelium radicis atrovirens (Type 3) mycorrhizae M. radicis atrovirens i s a name proposed by Melin (1923, in Wilcox and Wang 1985) for a coarse, brown nonmycorrhizal fungus that did not form a Hartig net or a mantle. However, any s t e r i l e fungus with narrow, brown, f i n e l y ornamented hyphae i s commonly referred to as M. radicis atrovirens even i f ectomycorrhizal (Danielson 1985a). Under monoxenic conditions, Wilcox and Wang (1987) have found varying degrees of pathogenicity to f u l l mycorrhiza-forming a b i l i t y within the form species. Pathogenic and ectomycorrhizal strains of M. radicis atrovirens are common mycobionts of pine and spruce in boreal Canada (Summerbell 1985; Danielson and Visser 1989). They are referred to as ectendomycorrhizae i f they frequently enter c e l l s of the cortex. Danielson (1985b) found M. radicis atrovirens occurred on most of the pine seedlings examined but i t only colonized a few root t i p s of each seedling. Under mono- or bixenic conditions, Richard et al. (1971) found pathogenic strains may severely stunt growth i f the roots are 140 not protected by ectomycorrhizal fungi. However, seedlings grown i n mono- or bixenic conditions may have allowed for the pathogenic habit and abundance of the fungus which may not occur on seedlings grown in s o i l (R.M. Danielson, personal communication). Wright (1971) found M. radicis atrovirens on Douglas-fir seedlings in Oregon on root t i p s also colonized by other mycorrhizal fungi. The percentage of root t i p s with M. radicis atrovirens increased with moderate levels of ammonium-N while seedling weight decreased. Richard and Fortin (1973) studied 41 strains of M. radicis atrovirens and found that 15 of them produced conidiophores and conidia i d e n t i c a l with (conspecific) Phialocephala dimorphospora Kendrick. Wilcox and Wang (1985) i d e n t i f i e d three more species within t h i s M. radicis atrovirens group with two belonging to other genera. The M. radicis atrovirens mycorrhizae that formed in the nursery usually lacked a mantle and often had poorly developed Hartig net possibly due to the high nutrient levels. Hyphae were sometimes seen within root hairs or colonizing roots along with T. terrestris. Control of M. radicis atrovirens in MacBean Nursery i s probably not necessary because i t had very low abundance. M. radicis atrovirens mycorrhizae on the new roots usually had a mantle. Mantles varied in hyphal arrangement indicating many species of fungi with narrow brown hyphae were involved. Hartig net was observed on some ti p s but i n t r a c e l l u l a r penetration was not observed on new roots. 141 Laccaria laccata (Type 24) mycorrhizae L. laccata occurred in MacBean Nursery but was not found on any of the seedlings sampled. The sporocarp examined was connected to mycorrhizae. L. laccata i s a common mycobiont in nurseries and coniferous forests of the P a c i f i c Northwest. L. laccata i s capable of protecting young Douglas-fir against Fusarium pathogens (Sinclair et al. 1982) and may increase the number of short roots or seedling size i n nurseries ( S i n c l a i r 1971; Molina 1982; Hung and Molina 1986). Although extensive colonization of roots by L. laccata at high f e r t i l i t y levels i s possible (Molina and Chamard 1983; Hung and Molina 1986), L. laccata may not compete well with T. terrestris in container nurseries (Hung and Trappe 1987). Many nonmycorrhizal seedlings were encountered in the nursery indicating that excessive f e r t i l i z a t i o n , not competition with Thelephora terrestris, was the reason for the lack of L. laccata on the sampled seedlings. Although there are reports of L. laccata in mature forests (Thomas et al. 1983), others have found i t only on younger trees (Chu-Chou and Grace 1983a, 1987) . L. laccata was not found on any of the outplanted seedlings. Although commercially-produced inoculum i s available (Hung and Molina 198 6), the lack of L. laccata on the outplanted seedlings indicates i t should be given low p r i o r i t y in inoculation t r i a l s because i t may not colonize new roots after outplanting on sit e s similar as those sampled. 142 Cenococcum geophilum (Type 4) mycorrhizae C. geophilum has a very broad host range and the s c l e r o t i a i t produces are common in most habitats (Harley and Smith 1983). It may form mycorrhizae on seedlings growing on f a l l e n logs or in mineral s o i l (Kropp 1982). The fungus can withstand drought and grow in cold or warm s o i l s of low water potentials, nutrient a v a i l a b i l i t y , and l i g h t intensity (Trappe 1977; Harley and Smith 1983). P i l z and Perry (1984) found 2-22% of the Douglas-fir root t i p s were colonized by Cenococcum after one growing season on clearcuts in west-central Oregon. They found more Cenococcum developed in burned clearcuts than unburned clearcuts or undisturbed forests. C. geophilum may extensively colonize the roots of western hemlock (Schoenberger and Perry 1982; Perry 1985; Kropp et al. 1985). Perry (1985) stated that a higher proportion of western hemlock roots become colonized by C. geophilum while a high proportion of the Douglas-fir roots become colonized by R. vinicolor when both hosts were grown in the same s o i l . In the present study, the r e l a t i v e abundance of C. geophilum was t h i r d highest on most of the Douglas-fir s i t e s sampled and second highest on the western hemlock s i t e . The fungus was only present on one seedling sampled from the leveled gravel p i t (Site 6). On average, C. geophilum accounted for less than 1% of the mycorrhizal t i p s on Douglas-f i r and less than 2% on western hemlock. Vare (1989) also found C. geophilum colonization on pine seedlings at about 1%, much less than most of the other types encountered. There 143 have been attempts to inoculate seedlings with C. geophilum (Molina 1982; Molina and Chamard 1983; Hung and Molina 1986; Grossnickle and Reid 1982) , but there were benefits were n i l or not obvious. Inoculation with C. geophilum may not be appropriate because of the high mycorrhizal inoculum potential (MIP) of th i s fungus in almost a l l s o i l s , and because i t may only colonize a small percentage of young seedling roots (Wright 1964; Parke et al. 1983b; Danielson 1984; Vare 1989). Endogone-like ( Type 5) mycorrhizae Most ectomycorrhizae are formed by basidiomycetes, ascomycetes, or fungi imperfecti, but at least three species of Endogone (Phycomycetes) are also known to form ectomycorrhizae (Harley and Smith 1983). .Endog-one-like mycorrhizae were not detected in the nursery. However, Fassi et al. (1969) found E. lactiflua sporocarps near many conifer species including Douglas-fir in a bare root nursery in Italy . They noted that Douglas-fir seedlings with E. lactiflua mycorrhizae grew vigorously. Trappe (1962, in Fassi et al. 1969) also noted an abundance of E. lactiflua sporocarps around 3-year-old Douglas-fir in the f i e l d . Trappe and Gerdemann (1972, in Walker 1985) noted the fungus may be mistaken for E. flammicorona. Chu-Chou and Grace (1987) found E. flammicorona on very young to mature Douglas-fir. In the present study, the weight of seedlings with Endogone-lxke mycorrhizae was not different than those without. The species of Endogone could not be determined because no sporocarps were 144 found. It was probably an Endoggne species based on s i m i l a r i t i e s to descriptions given by Fassi et al. (1969) and Walker (1985). Warcup (1985) reported that Glomus tub iforme, another member of the Endogonaceae, formed ectomycorrhizae with a thin mantle and a Hartig net on several host species, but did not descibe the association in enough d e t a i l to make a comparison with the Endogone-like mycorrhizae encountered here. Tuber-like (Type 6) mycorrhizae Tuber species are t r u f f l e s and some are capable of forming mycorrhizae with Douglas-fir (Cho-Chou and Grace 1983a; 1987). Hunt and Trappe (1987) collected sporocarps of at least 6 species from a north-facing slope in a 35-50 year old second growth stand of Douglas-fir on a previous clearcut in western Oregon. Trappe (1977) noted that some Tuber species are r e s t r i c t e d to certain calcareous s o i l s of mediterranean climates and that inoculation of seedlings in bare root nurseries with certain species can improve seedling growth and y i e l d substantial p r o f i t s from the sale of t r u f f l e s . The Tuber-like mycorrhizae in the present study were i d e n t i f i e d based on descriptions by others (Cho-Chou and Grace 1983a, b; Danielson and Pruden 1989) . They were most common on the low elevation Brunisol s o i l s of Site 1 which were high in nitrogen. The weight of seedlings with Tuber-l i k e mycorrhizae were not different than those without. 145 Type 13 mycorrhizae For unknown reasons, Type 13 mycorrhizae only occurred on Site 2F. It occurred on only 4 of the 15 seedlings sampled and only on new roots. The c y s t i d i a produced were narrower but closely resembled those Taylor and Alexander (1989) described for Russula aeruginea on spruce. These researchers mentioned that many of the c y s t i d i a had a small spherical to e l l i p s o i d a l projection at the apex. Up to three such projections were formed on the c y s t i d i a of Type 13 mycorrhizae, and appeared spore-like because d i f f e r e n t sizes and shapes were seen and because a stub remained after the staining procedure. This stub may have been an a r t i f a c t or an actual s i t e of "spore" formation or detachment. R.M. Danielson (personal communication) has found a similar type (called Micky Mouse because the projections appeared as ears) but was not able to find attachment scars. Type 33 mycorrhizae Type 3 3 mycorrhizae were best developed on the leveled gravel p i t (Site 6) but also occurred on the logging roadway (Site 2R) . Sand grains were often incorporated into the mantle and the mycorrhizae frequently adhered t i g h t l y to small pebbles f a c i l i t a t e d by the production of red-brown exudates. The chemical composition and function of the exudates were not determined. 146 Other minor types of ectomycorrhizae Many of the minor mycorrhizal types only colonized one or a few t i p s of one seedling. Often a type occurred on seedlings from more than one s i t e but i t i s possible more than one species was involved in many of the types. I t i s also possible that some of the minor types were immature, distorted, or unusual forms of other types as influenced by microsite conditions. Clues to the identity of the mycobionts were not found in the l i t e r a t u r e reviewed. There may have been some host s p e c i f i c i t y of Types 23 and 34 to western hemlock because they were not found on any of the 215 Douglas-f i r sampled from the f i e l d . Species of Wilcoxina. and Complexipes (commonly refered to as E-strain) are common mycorrhiza formers in nurseries and clearcuts in Alberta and central B r i t i s h Columbia (Danielson 1985a). Ectomycorrhizal E-strain occurs on Douglas-fir on very dry s i t e s in the i n t e r i o r of B.C. (G. Hunt, personal communication). Egger and Fortin (1988) state that Wilcoxina species occur naturally in disturbed habitats, such as after f i r e s , and have adaptations, such as rapid growth rate, that allow them to thrive under these conditions. Danielson and Pruden (1989) give a brief description of Complexipes occurring on spruce. No mycorrhizal fungi f i t t i n g descriptions of E-strain were found in the nursery or on any of the field-sampled seedlings. This indicates d e f i n i t e host or s o i l - s i t e effects (R.M. Danielson, personal communication). 147 5.6 Further research into mycorrhiza management Douglas-fir and western hemlock Modifications for growing Douglas-fir and western hemlock should be t r i e d . This might include l i m i t i n g the use of fungicides and lowering the f e r t i l i z a t i o n l e v e l to allow a greater d i v e r s i t y of mycorrhizae to spontaneously form in the nursery. This may improve plug root quality by increasing root branching and the number of root t i p s which, along with fungal hyphae can create a more s o l i d plug that i s easier to plant (G. Hunt, personal communication). Rhizopogon vinicolor appears to be the obvious choice for the a r t i f i c i a l inoculation of Douglas-fir for several reasons. It greatly increases the number of root t i p s formed i n the nursery and has greatly improved growth and survival of Douglas-fir on clearcuts in Oregon where the isolates originated (Phil Hahn, personal communication). Inoculation methods have already been proven and are r e l a t i v e l y simple because spores can be used (Castellano et al. 1985). I t was very common on the seedlings sampled. It may convey drought tolerance (Parke et al. 1983a). Sporocarps of R. vinicolor should be collected, labeled as are seedlots on the basis of elevation and aspect, and used to inoculate seedlings. Preliminary conclusions on the value of such inoculations should only be made after methods for producing mycorrhizal seedlings are implemented in MacBean Nursery, and the plants are evaluated several years after being outplanted on hard to regenerate si t e s along with noninoculated seedlings. 148 The effect Thelephora terrestris has on growth and survival should be compared with R. vinicolor and completely nonmycorrhizal seedlings. Other mycorrhizal types or s o i l transfer studies may eventually be t r i e d , but e f f o r t s should probably focus on R. vinicolor inoculations and the lowering of f e r t i l i t y and fungicides to allow a d i v e r s i t y of mycorrhizae to form, and root morphology and possibly root growth capacity to improve. Western hemlock forms a large number of root t i p s in the nursery and i s planted on wetter s i t e s that are less l i k e l y to have low mycorrhizal colonization potential; therefore, a r t i f i c i a l inoculation of t h i s species may not be as important as i t i s for Douglas-fir. Western red cedar Mycorrhizal cedar should be grown using peat inoculated with VA mycorrhizal fungi (Premier Peat Moss, Quebec), or chopped roots of cedar (Morgan 1985), or plants more heavily dependent on VA mycorrhizae such as clover. If mycorrhizal cedar of acceptable size can be grown they should be outplanted along with noninoculated plants preferably of the same size also grown without fungicides or excessive f e r t i l i z a t i o n . 5.7 Characterization of ectomycorrhizae Choice of method Most minor types of mycorrhizae usually only had one, occasionally two or three d i s t i n c t i v e features and these were 149 often suspected of being quite variable. The main d i f f i c u l t y encountered was the low number of t i p s encountered of most of the minor types of mycorrhizae. Especially in cases where only one or a few t i p s were found, the range of morphological v a r i a b i l i t y could not be accurately determined. Many types may have been immature forms of other types, as was the case with Type 43 mycorrhizae probably being immature Tuber-like (Type 6) mycorrhizae in many cases and p a r t i c u l a r l y on Site 1. However, to have been more certain of the type divisions would have taken a great deal more time. Fortunately the major types of mycorrhizae were described elsewhere and could be recognized at least to genus with some certainty i f past a certain stage of development. More detailed characterizations were not warranted because the fungal identity could not be determined, the mycorrhizae were stained, and the morphological v a r i a b i l i t y within a type was uncertain. If the goal i s to characterize and identi f y p a r t i c u l a r mycorrhizae, several researchers have given excellent examples of how to do so (e.g., Trappe 1967b, 1969; Zak 1971; Zak and Larsen 1978; Agerer 1988; Agerer and Weiss 1989). Other researchers, dealing with several types of mycorrhizae on a pa r t i c u l a r host, only describe each mycorrhiza in enough d e t a i l to distinguish the types they encounter (e.g., Chilvers 1968; Danielson 1984, Chu-Chou and Grace 1983a, b) . Types that are less d i s t i n c t i v e , uncommon, or that cannot be i d e n t i f i e d are often ignored. In studies more concerned with treatment effects on the kinds of mycorrhizae, mycorrhizal 150 "types are either grouped using existing keys (Dominik 1959, 1969), or short descriptions and type categories usually based strongly on color are provided (e.g., Mejstrik 1971; Kropp 1982; Parke et al. 1983b, 1984; P i l z and Perry 1984). Even the simplest c l a s s i f i c a t i o n scheme usually requires well developed or mature mycorrhizae for accurate type designations. The resources to f u l l y characterize a l l the types encountered in the present study were not available or needed to address the objectives. The main objective of th i s study was to determine the mycorrhizal status before and after outplanting. This needed to involve some le v e l of mycorrhiza characterization to recognise nursery fungi amongst the other types that form after outplanting. However, time, experience, equipment, and copious amounts of fresh mycorrhizae of each type were not available for more detailed characterizations. Usefulness of the staining procedure Hyphal characteristics of mycorrhizae are considered less variable than are features such as color, form, and abundance of emanating hyphae (Zak 1973). Trappe (1967a) would base a key on stable hyphal characteristics and only use the less constant features as secondary characteristics. The color of Rhizopogon vinicolor, Mycelium radicis atrovirens, and Cenococcum geophilum, were quite d i s t i n c t i v e . However, color was only useful for i n i t i a l type designations i f i t was not affected by root color which meant that the mycorrhizae were beyond a certain stage of development and roots were not 151 heavily suberized. Of the mycorrhizae encountered that were brown, the staining procedure made M. radicis atrovirens and C. geophilum mycobionts much more obvious, but high magnification was usually s t i l l necessary to distinguish between the two types. The brown pigment in the hyphae of some mycobionts, such as Type 11, was a useful and d i s t i n c t i v e feature that may have been lost in the staining procedure. However, the type was not encountered i n the prestaining examination of seedlings or was mistaken for one of the other brown types. Like most of the types encountered, Thelephora terrestris mycorrhizae appeared brown due to the color of the root beneath. To make sections of fresh mycorrhizae and look only at hyphal color as a means of typing mycorrhizae as Dominik (1959, 1969) suggests was not possible because of the large number of t i p s on each of the hundreds of samples. In aggreement with Walker (1985), .Endogone-like mycorrhizae would have been overlooked i f the staining procedure had not been used. Rhizopogon vinicolor mycorrhizae would have been more easily seen without staining because the procedure removed a l l the pigment from the c y s t i d i a . The presence of t h i s fungus was noted before staining and then confirmed using microscopic features after staining. Often, many of the t i p s appeared to be d i f f e r e n t types at low magnification but were found to only have fewer c y s t i d i a but the same hyphal characteristics under high magnification. With practice i t became obvious they were just in di f f e r e n t stages of development and several type 152 designations were excluded. If bright white or yellow mycorrhizae were seen on the outplanted seedlings, a similar situation would probably have existed: stable and d i s t i n c t i v e c h a r a c t e r i s t i c s would need to be found to confirm groupings p a r t i c u l a r l y i f the mycorrhizae were immature. Although fresh samples could be used to estimate percentage colonization by yellow, white, black, or R. vinicolor mycorrhizae at low magnifications, many questionable types would have required high magnification before grouping. The staining procedure allowed for accurate and rapid estimates of t o t a l percent colonization. Chilvers (1968) found clearing and staining roots aided in observing mantle stucture. Alexander (1981) found mantle surface features were easier to see after clearing and staining roots which provided a more rapid means for assessment of mycorrhizal types. One of the main advantages of the clearing procedure was that i t helped to eliminate the effect of the root i n determining the morphology of the mycorrhiza: before staining root t i p s varied greatly depending on age and growing conditions and t h i s made grouping mycorrhizae into types more confusing. Once the most d i s t i n c t i v e characteristics of each type of mycorrhiza was recognized, and the key to the types was constructed, the typing of cleared and stained mycorrhizae based on stable hyphal characteristics became quite rapid. More research i s needed to construct a general key based on stable hyphal characteristics of related groups of mycobionts as seen on whole mounts of cleared and stained mycorrhizae. With 153 practice, t h i s key could be used by. forest researchers with limited experience to arrive at di f f e r e n t types which are a re s u l t of a particular mycobiont, not root color or stage of mycorrhiza maturation. The staining procedure i s recommended because many studies also involve estimates of percentage mycorrhizal colonization. Without staining, or a great deal of time making sections, estimates of percent colonization w i l l l i k e l y be low because a mantle i s not always present and the roots may appear nonmycorrhizal. 5.8 Conclusions Ectomycorrhizal status before outplanting Most Douglas-fir and western hemlock seedlings formed mycorrhizae in MacBean Nursery. Prior methyl bromide fumigation of potting mix did not deter subsequent mycorrhiza formation. Thelephora terrestris formed over 99% of the mycorrhizae. Percentage colonization was highly variable and not correlated to seedling growth. Ectomycorrhizal status after outplanting A l l the seedlings had formed mycorrhizae within the f i r s t f i e l d season. Thelephora terrestris colonized a large proportion of new roots and probably originated in the nursery. Rhizopogon vinicolor (on Douglas-fir only) and Cenococcum geophilum were present as inoculum on the s i t e s and occurred on most seedlings. Other major types included Mycelium radicis atrovirens, Endogone-like, and Tuber-like 154 mycorrhizae. The average mycorrhizal colonization l e v e l ranged from 72 to 93% per plot. Site 4 was probably the most harsh s i t e and had the lowest new root colonization l e v e l and the fewest mycorrhizal types. Inoculated seedlings from Oregon Douglas-fir inoculated with Rhizopogon vinicolor formed mycorrhizae in the nursery and were t a l l e r than nonmycorrhizal seedlings. After one season in the f i e l d , R. vinicolor inoculation did not result in greater seedling growth or survival. The effects of a r t i f i c i a l inoculation were absent or obscured for three reasons: both the control and the inoculated seedling had R. vinicolor mycorrhizae as well as many other types after one f i e l d season; most seedlings were deer browsed and many had root c o l l a r damage; Site 1 was not defi c i e n t in mycorrhizal inoculum or considered a harsh s i t e for seedling establishment. Western red cedar Western red cedar did not form mycorrhizae in the nursery. New root colonization averaged 15% with 36% of the seedlings remaining nonmycorrhizal after the f i r s t growing season. Most seedlings were chlorotic; possibly due to a deficiency of vesicular-arbuscular mycorrhizae. 155 Mycorrhizal inoculum potential Sampling seedlings once in the f a l l a fter one season of being outplanted did not indicate a deficency in ectomycorrhizal inoculum. The new roots of most seedlings had high percentage mycorrhizal colonization on each of the 7 s i t e s including the leveled gravel p i t , the logging roadway, and the steep, high elevation, southwest facing slope that had been burned. However, because of the study's design, i t could not determined whether there was an actual deficency i n the f i r s t few months following outplanting or a lack of be n e f i c i a l mycobionts. If T. terrestris did not colonize the new roots the colonization levels may have been lower. There are no studies in the l i t e r a t u r e of the a b i l i t y of t h i s fungus to improve seedling growth on clearcuts of the P a c i f i c Northwest. The l i t e r a t u r e reviewed suggests T. terrestris could be reduced by a r t i f i c i a l inoculation with R. vinicolor and altered growing conditions in the nursery. 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Bot. 5 6 : 1416-1424 . 175 APPENDIX 1 Percent cover and mycorrhizal status of non-crop species and naturally regenerated conifers Percent cover was estimated at the time of seedling sampling as the percentage of the plot area containing at least one individual per square meter. If a l l the individuals in the plot were considered to occupy less than one square meter in t o t a l then cover was recorded as less than 1 percent. Mycorrhizal status was determined using methods outlined in section 3.5 and 3.6: NM = nonmycorrhizal, ETM = ectomycorrhizae, ERM = e r i c o i d mycorrhizae, VA = vesicular-arbuscular (VA) mycorrhizae, VAF = VA mycorrhizae with fine endophyte, VAC = VA mycorrhizae with coarse endophyte, VAB = VA mycorrhizae both fine and coarse endophtye present. Abundances are indicated as +, ++, and +++ for low (1 - 15 % ) , medium (16 - 60 % ) , and high (61 - 100 %) levels of colonization respectively. A question marked indicates mycorrhizal status was unknown because the sample went mouldy in cold storage. Site 2F Berberis nervosa Pursh Cirsium arvense (L.) Scop. Cirsium vulgare (Savi.) Tenore Epilobium munitum L i n d l . Gaultheria shallon Pursh PERCENT COVER 2 < 1 < 1 < 1 100 MYCORRHIZAL STATUS NM VAC++ ERM+ 176 Hypochaeris radicata L. Lactuca muralis (L.) Fresen. Linnaea borealis L. Madia exigua (J.E. Smith) Gray Rosa sp. Rubus ursinus Cham. & Schlecht. Senecio sylvaticus L. Vaccinium parvifolium Smith < 1 < 1 < 1 < 1 < 1 2 2 < 1 VAC+ NM VAC++ NM VAC+ VAC+ VAF+ NM Site 2H PERCENT COVER Berberis nervosa Pursh 2 Bromus sp. 2 Cirsium arvense (L.) Scop. 5 Cirsium vulgare (Savi.) Tenore 3 0 Deschampsia elongata (Hook) Munro < 1 Epilobium munitum L i n d l . 5 Festuca microstacys Nutt. 1 Gaultheria shallon Pursh 100 Hypochaeris radicata L. 15 Lactuca muralis (L.) Fresen. 5 Polystichum munitum (Kaulf.) Presl. < 1 Psuedotsuga menziesii (Mirbel) Franco. < 1 Pteridium aquilinum (L.) Kuhn. < 1 Rosa sp. 2 Rubus leucodermis Dougl. 15 Rubus ursinus Cham. & Schlecht. < 1 MYCORRHIZAL STATUS NM VA++ VAC++ VAB++ VAF++ NM VAB++ NM VAC & ER+ NM VAB++ ETM+++ VAC+ VAC+ 177 Senecio sylvaticus L . Trientalis latifolia Hook. 60 < 1 VAB+ VAF+++ Site 2R Contained only the planted Douglas-f ir . Site 3A Berberis nervosa Pursh Cirsium vulgare (Savi.) Tenore Deschampsia elongata (Hook) Munro Epilobium munitum L i n d l . Gaultheria shallon Pursh Hypochaeris radicata L . Lactuca muralis (L. ) Fresen. Rubus leucodermis Dougl. Senecio sylvaticus L . Tsuga heterophylla (Raf.) Sarg. Vaccinium sp. PERCENT COVER < 1 < 1 < 1 < 1 100 2 < 1 < 1 30 < 1 10 MYCORRHIZAL STATUS NM ? NM NM ERM+ NM NM NM ETM+++ ERM+ Site 3B Berberis nervosa Pursh Chamaecyparis nootkatensis (D. Don) Spach Cirsium arvense (L.) Scop. Cirsium vulgare (Savi.) Tenore Deschampsia elongata (Hook) Munro PERCENT COVER < 1 < 1 < 1 < 1 < 1 MYCORRHIZAL STATUS VA++ VA++ NM NM 178 Epilobium munitum L i n d l . Epilobium sp. Gaultheria shallon Pursh Hypochaeris radicata L. Lactuca muralis (L.) Fresen. Pinus sp. Psuedotsuga menziesii (Mirbel) Franco. Rubus leucodermis Dougl. Senecio sylvaticus L. Tsuga heterophylla (Raf.) Sarg. Vaccinium parvifolium Smith Vaccinium sp. 2 < 1 100 3 3 < 1 < 1 2 20 < 1 2 5 NM ERM++ VAB++ NM ETM+++ 7 VAB++ NM ERM+ ERM++ Site 4 PERCENT COVER Cirsium arvense (L.) Scop. 2 Cirsium vulgare (Savi.) Tenore 5 Epilobium munitum L i n d l . 75 Festuca bromoides L. < 1 Festuca subulata Trin. < 1 Gaultheria shallon Pursh 60 Hypochaeris radicata L. 4 0 Lactuca muralis (L.) Fresen. 95 Linnaea borealis L. < 1 Polystichum munitum (Kaulf.) Presl. < 1 Psuedotsuga menziesii (Mirbel) Franco. < 1 Rubus leucodermis Dougl. 40 MYCORRHIZAL STATUS NM NM VAC++ VAB++ VAB++ NM VAB++ VAB++ NM VAB+++ 7 VAB++ 179 Senecio sylvaticus L. 80 VAC+ Thuja plicata Donn ex D. Don 4 VAB+++ Site 5 PERCENT COVER MYCORRHIZA] STATUS Berberis nervosa Pursh 2 Cirsium arvense (L.) Scop. 2 VAC+++ Cirsium vulgare (Savi.) Tenore 1 Epilobium munitum L i n d l . 1 VAC+ Gaultheria shallon Pursh 60 ERM+ Gymnocarpium dryopteris (L.) Newm. 5 VAB+ Hypochaeris radicata L. 3 VAB+++ Lactuca muralis (L.) Fresen. 2 VAB++ Rosa sp. 1 VAC++ Rubus leucodermis Dougl. 10 VAB+++ Rujbus ursinus Cham. & Schlecht. 20 VAB++ Rubus sp. 10 VAF+++ Senecio sylvaticus L. 5 VAB++ Sonchus asper (L.) H i l l < 1 7 Tsuga heterophylla (Raf.) Sarg. < 1 ETM+++ Vaccinium parvifolium Smith 2 7 Site 6 Trifolium sp. PERCENT COVER 20 MYCORRHIZAL STATUS VAM+++ 180 Site 7H PERCENT MYCORRHIZAL COVER STATUS Abies sp. 20 ETM+ Achlys triphilla (Smith) DC. 3 Bromus sp. < 1 NM Cirsium sp. < 1 7 Epilobium angustifolium L. 30 NM Epilobium munitum L i n d l . < 1 7 Hypochaeris radicata L. 10 VAC++ Lactuca muralis (L.) Fresen. 1 VA++ Psuedotsuga menziesii (Mirbel) Franco. < 1 ETM+++ Senecio sylvaticus L. 10 VAC+ Smilacina stellata (L.) Desf. < 1 7 Tsuga heterophylla (Raf.) Sarg. 5 ETM+++ Vaccinium sp. 15 7 Site 7C PERCENT COVER MYCORRHIZA] STATUS Abies sp. 2 7 Achlys triphilla (Smith) DC. 2 7 Berjberis nervosa Pursh < 1 7 Cirsium vulgare (Savi.) Tenore < 1 7 Epilobium angustifolium L. 70 7 Gaultheria shallon Pursh < 1 7 Gymnocarpium dryopteris (L.) Newm. < 1 7 Hypochaeris radicata L. 20 7 Lactuca muralis (L.) Fresen. 2 7 Linnaea borealis L. 3 VAC+ 181 Senecio sylvaticus L. 10 ? Smilacina stellata (L.) Desf. < 1 ? Tsuga heterophylla (Raf.) Sarg. 10 ETM+++ Vaccinium sp. 30 ? 182 APPENDIX 2 Means of estimating dry root weight from fresh root weight Douglas-fir: PLUG ROOTS— (X) NEW ROOTS-(X) 4PLE FRESH DRY ESTIMATE FRESH DRY ESTIMATE WEIGHT WEIGHT WEIGHT WEIGHT 327 3551 1090 1110 1780 490 477 328 6372 1950 1753 2334 730 616 333 4252 1440 1270 480 120 149 335 3652 1160 1133 2370 790 625 339 4485 1400 1323 1255 406 344 341 2233 700 810 419 110 134 343 5564 1640 1569 1049 340 293 344 3726 1210 1150 815 260 234 345 4039 1300 1221 896 290 254 348 2570 930 887 919 290 260 352 5235 1247 1494 1690 404 454 354 3581 985 1117 1020 269 285 355 3130 904 1014 924 232 261 361 5464 1634 1546 3506 769 911 363 3963 1349 1204 3539 985 919 368 7980 2133 2119 2088 490 554 371 5048 1650 1451 2311 598 610 372 4098 1040 1235 55 15 43 373 6554 1686 1794 2171 444 575 375 6917 1630 1877 765 187 221 Regression Output: Constant 301.81 Std Err of Y Est 144.02 R Squared 0.856 No. of Observations 20 Degrees of Freedom 18 X Co e f f i c i e n t 0.228 Std Err of Coef. 0.022 Y = 0.228X + 301.8 +Note: Fd9766 from Site 2 was used. Regression Output: Constant 28.72 Std Err of Y Est 75.34 R Squared 0.918 No. of Observations 20 Degrees of Freedom 18 X Coe f f i c i e n t 0.252 Std Err of Coef. 0.0177 Y = 0.252X + 28.7 183 Western hemlock: (X) (Y) DRY wt./ SAMPLE FRESH DRY Y FRESH NUMBER WEIGHT WEIGHT ESTIMATE Wt. 526 2587 411 403.24 0. 16 527 2685 383 425.81 0. 16 528 3993 640 726.97 0. 18 529 2898 515 474.85 0. 16 530 4321 771 802.49 0. 19 531 3381 662 586.06 0. 17 532 5277 856 1022.61 0. 19 533 5437 1226 1059.45 0. 19 534 3337 583 575.93 0. 17 535 4734 917 897.59 0. 19 536 5752 1143 1131.98 0. 20 AVG: 0.179 Regression Output: Constant -192.413 Std Err of Y Est 90.599 R Squared 0.904 No. of Observations 11 Degrees of Freedom 9 X C o e f f i c i e n t 0.23 Std Err of Coef. 0.025 Y = 0.23X - 192 = equation used to obtain estimates of dry-plug root weights from fresh root weights. Y = 0.179X = factor multiplied to fresh root weights to obtain an estimate of dry new weight. +Note: Hw7321 from MacBean Nursery was used. 184 Western red cedar: Corrected dry plug root weights were calculated as dry plug weight minus 1.0 gm subsample that was stained plus the average weight of ten other such subsamples that were dried instead of stained. Corrected dry root weights for new roots were calculated by multiplying the f r a c t i o n derived above by the fresh root weights. FRESH CORR. DRY PERCENT CONSTANT SAMPLE ROOT ROOT Wt. Y WATER TO GET DRY NUMBER WEIGHT (PLUG mg) ESTIMATE CONTENT NEW ROOT WT. 226 4300 953 883.88 77. 8 0. 22 230 5290 1226 1102.274 76. 8 0. 23 232 9150 2146 1953.79 76. 5 0. 23 233 6970 1458 1472.882 79. 1 0. 21 234 6100 12*96 1280.96 78. 8 0. 21 235 10700 2452 2295.72 77. 1 0. 23 238 8020 1612 1704.512 79. 9 0. 20 240 8420 1650 1792.752 80. 4 0. 20 241 4700 934 972.12 80. 1 0. 20 242 6790 1228 1433.174 81. 9 0. 18 243 4030 824 824.318 79. 6 0. 20 244 8900 1782 1898.64 80. 0 0. 20 247 3280 711 658.868 78. 3 0. 22 0.21 AVG. Regression Output: Constant -64.703 Std Err of Y Est 124.576 R Squared 0.947 No. of Observations 13 Degrees of Freedom 11 X C o e f f i c i e n t 0.221 Std Err of Coef. 0.0158 Y = 0.221X - 64.702 185 APPENDIX 3 Analysis of variance summary tables Part A: Fd9766 (Table VI) Plug roots: percentage mycorrhizae Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 8021.379 4 2005.3447 3.801 Within groups 48013.428 91 527.6201 Total 56034.807 95 P = 0 .0067 ** Plug roots: mycorrhizal t i p s Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 535786.7 4 133946.67 1.347 Within groups 9051843.8 91 99470.81 Total 9587630.5 95 P = 0.2588 Plug roots: t o t a l t i p s Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 248641.8 4 62160.443 .686 Within groups 8249421.2 91 90652.980 Total 8498063.0 95 P = 0.6037 Plug roots: weight Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 4527048.6 4 1131762.2 11.833 Within groups 8703895.5 91 95647.2 Total 13230944 95 P < 0. oooi *** 186 New roots: percentage mycorrhizae Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 1117.713 Within groups 14033.367 3 67 372.57085 209.45324 1.779 Total 15151.079 70 P = 0.1596 New roots: mycorrhizal t ip s Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 25725.0 Within groups 3700040.8 3 67 8575.015 55224.490 .155 Total 3725765.9 70 P = 0.9259 New roots: t o t a l t ip s Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 132121.0 Within groups 4726934.9 3 67 44040.325 70551.267 .624 Total 4859055.9 70 P = 0.6018 New roots: weight Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 157403.7 Within groups 2917232.6 3 67 52467.898 43540.785 1.205 Total 3074636.3 70 P = 0.3147 1 8 7 Shoot height Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 232015.38 4 58003.844 35.391 Within groups 149145.03 91 1638.956 Total 381160.41 95 P < .0001 *** Cal iper Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 29.290554 4 7.3226385 17.231 Within groups 38.671842 91 .4249653 Total 67.962396 95 P < .0001 *** Shoot weight Source of variation Sum of Squares d.f. Mean square F-ratio 28.039 Between groups Within groups 98769027 80138521 4 91 24692257 880643 Total 1.7891E0008 95 P < .0001 *** Root over shoot mass r a t i o Source of variation Sum of Squares d.f. Mean square F-ratio Between groups 4 .2156644 4 1.0539161 40.545 Within groups 2 .3654292 91 .0259937 Total 6 .5810936 95 P < .0001 *** 188 Part B: Fd4503 (Table VII) Plug roots: percentage mycorrhizae Source of variation Sum of Squares d.f. Mean square F-ratio Between groups Within groups 98081.511 45189.852 4 113 24520.378 399.910 61.315 Total 143271.36 117 P < .0001 *** Plug roots: mycorrhizal t ip s Source of variation Sum of Squares d.f. Between groups Within groups 7853722.3 9688057.0 4 113 Total 17541779 117 Plug roots: t o t a l t ip s Source of variation Sum of Squares d.f. Mean square F-ratio Between groups Within groups 3007958.0 9138445.0 4 113 751989.49 80871.19 9.299 Total 12146403 117 P < .0001 *** Plug roots: weight Source of variation Sum of Squares d.f. Mean square F-ratio Between groups Within groups 14179710 13483132 4 113 3544927.5 119319.8 29.709 Total 27662842 117 P < .0001 *** 189 New roots: percentage mycorrhizae Source of variation Sum of Squares d. f. Mean square F-ratio Between groups Within groups 6075.727 37382.392 3 89 2025.2423 420.0269 4.822 Total 43458.119 92 P = .0037 ** New roots: mycorrhizal t ip s Source of variation Sum of Squares d, .f. Mean square F-ratio Between groups Within groups 12667636 23741600 3 89 4222545.4 266759.5 15.829 Total 36409236 92 P < .0001 *** New roots: t o t a l t ip s Source of variation Sum of Squares d, .f. Mean square F-ratio Between groups Within groups 15225859 23667772 3 89 5075286.3 265930.0 19.085 Total 38893631 92 P < .0001 *** New roots: weight Source of variation Sum of Squares d .f. Mean square F-ratio Between groups Within groups 17312031 16270341 3 89 5770676.9 182812.8 31.566 Total 33582371 92 P < .0001 *** 190 Shoot height Source of variation Sum of Squares d.f. Mean square F-ratio Between groups Within groups 65887.38 315049.41 4 113 16471.845 2788.048 5.908 Total 380936.79 117 P = .0002 ** Cal iper Source of variation Sum of Squares d.f. Mean square F-ratio Between groups Within groups 44.347908 49.489719 4 113 11.086977 .437962 25.315 Total 93.837627 117 P < .0001 *** Shoot weight Source of variation Sum of Squares d.f. Mean square F-ratio Between groups Within groups 1.0866E0008 1.5521E0008 4 113 27164659 1373529 19.777 Total 2.6387E0008 117 P < .0001 *** Root over shoot mass r a t i o Source of variation Sum of Squares d.f. Mean square F-ratio Between groups Within groups 1.6902784 5.0481023 4 113 .4225696 .0446735 9.459 Total 6.7383807 117 P < .0001 *** 191 APPENDIX 4 Seedling Data Collected F u l l variable names used i n the following tables: SWT shoot weight (mg) SHT shoot height (mm) CAL caliper (mm) FRWP fresh root weight plug (mg) RWP root weight plug (mg) FRWN fresh root weight new (mg) RWN root weight new (mg) ROS root over shoot mass r a t i o LDR leader length or longest new branch growth i f browsed (mm) MTP mycorrhizal t i p s plug roots TTP t o t a l t i p s plug roots PMP percentage mycorrhizae plug roots MYCP mycorrhizal types plug roots MTN mycorrhizal t i p s new roots TTN t o t a l t i p s new roots PMN percentage mycorrhizae new roots MYCN mycorrhizal types new roots 192 Fd9509 - NURSERY, NOVEMBER 19, 1987.+ CONTROL FUMIGATED 1PLE SWT SHT CAL PMP SAMPLE SWT SHT CAL PMP 2 1050 235 2.2 41 26 1610 297 2.9 43 3 1186 232 2.3 95 27 1887 287 3.0 52 4 1326 226 2.8 33 28 2145 273 3.2 16 5 1813 268 3 66 30 1114 329 2.8 6 6 1785 295 2.8 72 31 1717 297 3.0 86 8 1187 257 2.6 50 32 1911 305 2.8 82 9 1650 254 3 13 33 1412 277 2.9 24 10 2146 284 3 5 34 1051 276 2.3 14 11 1604 271 2.7 49 35 2142 305 3.3 88 12 1257 252 2.3 98 36 1688 281 2.9 75 13 1496 251 2.8 67 37 996 321 2.2 42 14 1346 267 2.6 84 38 1730 304 2.8 7 15 1700 273 2.8 51 39 1159 287 2.8 1 16 1324 246 3 19 40 1851 303 3.0 0 17 2036 410 3.3 23 41 1447 273 2.8 5 18 1331 274 2.8 1 42 2393 317 3.7 0 19 757 235 2.1 40 43 1852 291 3.3 33 20 1484 283 2.7 26 44 1745 283 3.1 26 21 1760 288 3.1 92 45 1705 275 3.1 0 22 679 177 2.3 0 46 1603 287 2.8 33 23 605 197 2.3 49 47 1966 288 3.1 19 24 1102 224 2.5 25 48 1441 298 3.0 69 25 1663 241 2.6 51 50 2330 340 3.7 0 n= 23 23 SUM: 32287 5940 61.6 1050 38895 6794 68.5 719 AVG: 1404 258 2.7 46 1691 295 3.0 31 VAR: 156339 1836 0.097 852 141421 320 o . i i 880 STD: 395 43 0.3 29 376 18 0.34 30 SE: 82 9 0.1 6 78 4 0.07 6 +Note: Type 1 mycorrhizae accounted for over 99% of the mycorrhizal t i p s . !<T3 Fd3290C (CONTROL) - NURSERY, MAY 19, 1988. SAMPLE SWT 76 1096 77 845 79 870 80 829 83 1343 88 1628 90 1313 91 1312 92 1656 93 1926 95 1099 96 1440 98 1234 99 1459 103 927 105 1490 n= 16 SUM: 20467 AVG: 12 79 VAR: 96355 STD: 310 SE: 78 SHT CAL 300 2.3 266 2.1 174 1.9 192 2.7 321 2.9 322 2.6 287 2.5 281 2.6 328 2.8 313 2.9 271 2.2 314 2.6 247 3.0 326 2.5 285 2.2 309 2.6 4536 40.4 284 2.5 1974 0.09 44 0.30 11 0.08 RWP ROS 637 0.58 480 0.57 499 0.57 554 0.67 765 0.57 1006 0.62 687 0.52 689 0.53 1106 0.67 1213 0.63 446 0.41 680 0.47 710 0.58 930 0.64 505 0.54 924 0.62 11831 9.18 739 0.57 51342 0.01 227 0.07 57 0.02 MTP TTP 0 491 26 427 1 276 0 576 322 483 0 1019 9 877 204 849 0 877 775 815 135 455 131 786 0 879 0 1059 0 296 25 1081 1628 11246 102 703 38565 68555 196 262 49 65 PMP MYCP 0 1 6 1 0 1 0 -67 1 0 -1 18 24 1 0 -95 1 30 1 17 1 0 -0 -0 -2 1 242 15 726 27 7 Fd3290I (INOCULATED) - NURSERY, MAY 19, 1988. SAMPLE SWT SHT CAL RWP 78 1601 323 2.5 1076 81 2377 377 3.0 941 82 1081 295 2.2 491 84 1596 292 2.3 1113 85 1220 296 2.2 549 86 1688 349 3.0 1283 87 1860 369 3.0 955 89 1081 285 2.2 550 94 1648 303 3.0 852 97 1443 306 3.0 723 100 1021 306 2.2 399 101 1888 339 3.2 913 102 1433 317 2.3 818 104 1226 294 2.3 623 n= 14 SUM: 21163 4451 36.4 11286 AVG: 1512 318 2.6 806 VAR: 133273 813 0.15 63261 STD: 365 29 0.39 252 SE: 98 8 0.10 67 ROS MTP TTP PMP MYCI 0.67 17 879 2 2 0.40 678 1284 53 2 0.45 228 493 46 2 0.70 762 897 85 2 0.45 324 476 68 2 0.76 76 1134 7 2 0.51 770 1039 74 2 0.51 161 361 45 2 0.52 18 864 2 2,1 0.50 586 797 74 2 0.39 304 435 70 2 0.48 300 710 42 2 0.57 116 832 14 2 0.51 557 767 73 2 7.42 4897 10968 654 0.53 350 783 47 0.01 68508 68159 809 0.11 262 261 28 0.03 70 70 8 /9S Fd3290C (CONTROL) - SITE 1, JUNE 21, 1988.+ >AMPLE SWT SHT CAL RWP PMP RWN PMN ROS 161 1090 213 2.5 560 0 8 0 0.52 162 3153 310 4.4 2709 80 54 80 0.88 163 2390 266 4.0 1439 20 143 20 0.66 164 4761 391 4.4 1203 20 140 60 0.28 165 2866 C 307 4.6 1356 75 38 90 0.49 n = SUM: 14260 1487 19.9 7267 195 383 250 2.83 AVG: 2852 297 4.0 1453 39 77 50 0.57 VAR: 1410633 3424 0.59 489418 1044 3027 1200 0.04 STD: 1188 59 0.77 700 32 55 35 0.20 SE: 531 26 0.34 313 14 25 15 0.09 Fd3290I (INOCULATED) - SITE 1, JUNE 21, 1988.+ >AMPLE SWT SHT CAL RWP PMP RWN PMN ROS 156 1986 245 4.0 1190 100 41 90 0. 62 157 3070 361 5.0 1504 40 169 80 0. 54 158 2760 314 4.6 1244 98 92 50 0.48 159 2221 292 4.1 1584 20 202 40 0.80 160 1820 C 132 3.1 802 80 9 0 0.45 n— SUM: D 11857 1344 20.8 6324 338 513 260 2.90 AVG: 2371 269 4.2 1265 68 103 52 0.58 VAR: 222849 6073 0.41 75863 1031 5391 1016 0.02 STD: 472 78 0.64 275 -^32 73 32 0.13 SE: 211 35 0.29 123 14 33 14 0.06 +Note: Samples 156 to 165 were collected as a preliminary investigation to determine i f the inoculated fungus was beginning to colonize new roots and to obtain practice handling f i e l d samples. 116 Fd3290C (CONTROL) - SITE 1, NOVEMBER 23, 1988. SAMPLE SWT SHT CAL FRWP RWP FRWN RWN ROS LDR 392 9080 392 5.5 10152 2613 3411 887 0.39 82 393 5682 325 6.3 8255 2181 1000 280 0.43 81 394 4182 313 3.9 4469 1319 815 234 0.37 71 395 7271 339 6.0 88 322 1341 366 0.09 88 396 7859 335 6.9 7567 2025 1246 342 0.30 56 397 8797 316 6.9 10119 2606 5745 1474 0.46 74 398 8042 431 6.3 8122 2151 2073 550 0.34 93 399 6325 329 6.2 4842 1404 4842 1247 0.42 51 400 2510 281 5.0 4585 1346 1196 330 0.67 0 401 8980 357 6.9 9241 2406 3052 797 0.36 66 402 10690 367 6.6 6823 1855 1000 280 0.20 92 403 6403 336 6.1 10326 2653 2855 747 0.53 101 404 8224 279 8.0 11693 2964 1948 519 0.42 41 405 7359 231 6.6 9308 2421 3369 876 0.45 68 406 4153 341 3.8 5343 1518 2850 746 0.55 44 407 4877 344 5.0 4551 1338 1825 488 0.37 61 408 3546 324 5.5 6855 1863 1542 417 0.64 29 409 4013 318 4.5 5558 1567 930 263 0.46 31 410 12959 415 3.9 14465 3595 5727 1469 0.39 54 411 2436 266 3.9 2370 841 1288 353 0.49 59 412 5560 364 6.0 4339 1290 1468 398 0.30 23 413 3339 207 4.1 3339 1062 495 153 0.36 50 414 4180 401 4.3 4815 1398 1355 370 0.42 120 415 7015 315 6.5 8288 2189 2849 745 0.42 56 416 7900 306 7.3 9316 2423 2168 574 0.38 57 n= 25 SUM: 161382 8232 142.0 174829 47352 56390 14904 10.22 1548 AVG: 6455 329 - 5.7 6993 1894 2256 596 0.41 62 VAR: 6635695 2621 1.44 10049713 521005 2058054 130259 0.01 683 STD: 2576 51 1.20 3170 722 1435 361 0.12 26 SE: 515 10 0.24 634 144 287 72 0.02 5 /<?7 Fd3290C (CONTROL) - SITE 1, NOVEMBER 23, 1988. iMPLE MTP TTP PMP MYCP MTN TTN PMN MYCN 392 866 949 91 45,46,2,P 849 856 99 5,2 393 687 752 91 32,2,14,43 204 221 92 1,45,2 394 551 566 97 2,6,43 257 265 97 2,6 395 682 750 91 2,6,32,4 409 411 100 2,6,4 396 463 819 57 6,35,2,3 429 431 100 6,2,32,3 397 362 371 98 6 947 947 100 6,43 398 312 332 94 6,33,43 150 159 94 6,33,43,4 399 360 383 94 43,1,4 779 780 100 43,2,4 400 633 824 77 1,6,43 201 270 74 1,2,4,6 401 1119 1131 99 2,6 686 695 99 2,6 402 464 502 92 5,6,32,3 250 255 98 6,32,1,4 403 324 623 52 6 524 547 96 6,2 404 877 941 93 5,1,2,4 369 371 99 1,2 405 397 566 70 6,2,5 752 768 98 6,2 406 123 155 79 43,2 346 389 89 2,43 407 470 495 95 17,6,3 398 417 95 17,6 408 425 584 73 5,1,3 258 303 85 5,1,43 409 178 447 40 6,17,2 140 152 92 6,2,17,4 410 420 537 78 5,2,17 441 524 84 17,5,2,47 411 159 170 94 5,2,32 275 278 99 2,27,32 412 97 286 34 3,5 249 400 62 6,5,3 413 138 150 92 6 13 89 15 6,2 414 226 579 39 6,5 418 442 95 6,5,10 415 149 263 57 1,2,6 235 239 98 6,2,5,1 416 308 631 49 43,2,5 235 250 94 43,2,4 SUM: 10790 13806 1926 9814 10459 2255 AVG: 432 552 77 393 418 90 VAR: 66101 65128 433 55393 51354 313 STD: 257 255 21 235 227 18 SE: 51 51 4 47 45 4 Fd3290I (INOCULATED) - SITE 1, NOVEMBER 23, 1988. SAMPLE SWT SHT CAL FRWP RWP FRWN RWN ROS LDR 417 10214 412 7.0 10541 2702 5736 1472 0.41 51 418 5958 284 3.9 3732 1152 1236 340 0.25 24 419 6041 378 6.9 9065 2366 2065 548 0.48 58 420 4839 335 4.6 4260 1272 1478 401 0.35 31 421 6062 315 6.5 8872 2322 3727 966 0.54 57 422 2354 318 4.2 5096 1462 2376 626 0.89 47 423 11308 417 8.5 9947 2567 2689 705 0.29 88 424 5867 313 6.2 6887 1870 3534 918 0.48 19 425 7663 396 6.6 7927 2107 4460 1151 0.43 49 426 5111 305 5.4 9069 2367 2528 665 0.59 42 427 8934 377 6.8 7668 2048 2980 778 0.32 41 428 6180 313 5.8 5787 1619 2122 563 0.35 57 429 7661 374 7.3 10608 2717 4823 1242 0.52 80 430 2480 317 4.2 3190 1028 608 182 0.49 58 431 5565 415 4.7 4860 1408 950 268 0.30 82 432 5825 272 6.8 9924 2561 3402 885 0.59 51 433 6200 330 6.4 6399 1759 2969 776 0.41 59 434 5434 315 6.3 8289 2189 2747 720 0.54 66 435 5912 384 5.8 7557 2022 1561 421 0.41 62 436 3911 285 5.4 4897 1417 3556 923 0.60 28 437 4671 302 5.6 6580 1800 2539 667 0.53 46 438 3118 260 4.7 4086 1232 1576 425 0.53 50 439 2783 292 4.2 4156 1248 2313 611 0.67 39 440 2439 303 4.4 4058 1226 561 170 0.57 41 441 4754 364 5.4 5527 1560 1709 459 0.42 52 n— SUM: 141284 8376 143.6 168982 46021 64245 16880 11.95 1278 AVG: 5651 335 5.7 6759 1841 2570 675 0.48 51 VAR: 4980498 2125 1.35 5257059 272540 1590734 100682 0.02 276 STD: 2232 46 1.16 2293 522 1261 317 0.14 17 SE: 446 9 0.23 459 104 252 63 0.03 3 Fd3290I (INOCULATED) - SITE 1, NOVEMBER 23, 1988. VMPLE MTP TTP PMP MYCP MTN TTN PMN MYCN 417 903 1007 90 2 1031 1055 98 2 418 467 473 99 2 325 377 86 2,20,5,4 419 786 835 94 2,5,4 209 315 66 1,2,4 420 351 386 91 1,21,43,2,P 115 138 83 1,2 421 498 590 84 2,P,3 242 302 80 43,P,2 422 387 401 97 5,43,3 301 311 97 2,5,43 423 494 617 80 2,43 194 210 92 35,2,4 424 144 173 83 6,2,3 364 365 100 6,2 425 950 1041 91 2,6,5 602 639 94 2,6,16,4 426 617 669 92 2,1,5,P 526 551 95 2,5 427 445 521 85 43,6,5,3 314 319 98 43,6,46,5 428 502 515 97 2 558 572 98 2,5 429 855 866 99 2,6 729 764 95 43,6,2 430 235 242 97 43,2 42 52 81 43,2 431 358 401 89 20,43,40,5,2,3 162 168 96 43,20,2 432 552 552 100 5,2 1051 1051 100 2,5 433 809 830 97 2,6,43 760 762 100 6,43,2 434 760 850 89 2,5 383 405 95 42,2,5 435 975 996 98 2,43,29,6 339 342 99 43,2 436 352 387 91 2,5 601 614 98 5,2 437 654 694 94 2 712 740 96 2,5,42 438 515 534 96 2,5 334 368 91 2,5,42 439 468 481 97 2,5 452 465 97 2,42 440 168 232 72 2,5 13 36 36 2,42 441 521 606 86 2,42 428 457 94 2,20,43 SUM: 13766 14899 2291 10787 11378 2266 AVG: 551 596 92 431 455 91 VAR: 53100 57546 45 72403 70737 184 STD: 230 240 7 269 266 14 SE: 46 48 1 54 53 3 boo Fd9766 - NURSERY, MAY 19, 1988. SAMPLE SWT SHT CAL RWP ROS MTP TTP PMP MYC] 106 959 161 2.6 920 0.96 600 638 94 1 107 1010 183 3.1 1263 1.25 1096 1175 93 1 108 863 164 2.6 768 0.89 730 779 94 1 109 1088 173 3.0 1092 1.00 213 540 39 1 110 809 165 2.8 836 1.03 557 618 90 1 111 837 172 2.8 970 1.16 404 1031 39 1 112 669 176 2.2 738 1.10 784 794 99 1 113 774 166 2.3 772 1.00 748 757 99 1 114 975 194 3.6 947 0.97 422 475 89 1,3 115 919 179 2.9 1051 1.14 606 685 88 1 116 733 157 2.5 728 0.99 699 835 84 1,3 117 704 170 2.3 800 1.14 846 957 88 1 118 744 179 3.4 844 1.13 771 815 95 1,3 119 490 161 2.1 555 1.13 488 586 83 1 120 1119 186 2.9 1206 1.08 738 746 99 1 121 903 179 2.9 861 0.95 932 967 96 1 122 830 163 3.0 836 1.01 505 681 74 1,3 123 1105 191 3.1 1295 1.17 255 544 47 1 124 668 162 2.6 684 1.02 612 709 86 1 125 916 181 2.8 868 0.95 639 678 94 1 126 646 151 2.4 730 1.13 910 933 98 1 127 861 181 2.8 1011 1.17 596 611 98 1 128 953 168 3.0 873 0.92 531 667 80 1 129 647 160 3.0 781 1.21 860 882 98 1 130 1484 208 3.5 1360 0.92 958 971 99 1 n= 25 SUM: 21706 4330 70.2 22789 26.43 16500 19074 2142 AVG: 868 173 2.8 912 1.06 660 763 86 VAR: 39847 166 0.14 39687 0.01 44726 28818 304 STD: 200 13 0.38 199 0.10 211 170 17 SE: 40 3 0.08 40 0.02 42 34 3 £0 1 Fd9766 - SITE 2F, SEPTEMBER, 1988. SAMPLE SWT 351 2256 353 2133 356 2122 357 3993 358 2168 359 3951 360 2806 362 2879 364 3257 365 3323 366 2384 367 2204 369 2100 370 2324 374 1259 n= 15 SUM: 39159 AVG: 2611 VAR: 527872 STD: 727 SE: 188 SHT CAL 268 4. 1 252 3. 7 306 3. 7 388 5. 9 292 4. 0 315 5. 6 295 3. 3 305 4. 5 310 4. 2 304 4. 5 275 4. 1 279 4. 0 269 3. 3 264 3. 4 243 3. 1 4365 61.4 291 4.1 1128 0.60 34 0.77 9 0.20 FRWP RWP 8260 2183 4650 1361 4060 1226 5750 1611 2600 894 4464 1318 3950 1201 5520 1559 4254 1270 4210 1260 7190 1939 3590 1119 3560 1112 4104 1236 2220 807 68382 20097 4559 1340 2351078 121886 1533 349 396 90 FRWN RWN 679 200 1500 406 700 205 590 177 790 227 493 153 2200 582 1710 459 1400 381 1810 484 2070 549 1580 426 780 225 446 141 265 95 17013 4711 1134 314 385279 24385 621 156 160 40 ROS LDR 1.06 76 0.83 58 0.67 106 0.45 156 0.52 66 0.37 145 0.64 295 0.70 106 0.51 96 0.52 79 1.04 81 0.70 81 0.64 56 0.59 51 0.72 44 9.96 1496 0.66 100 0.04 3694 0.19 61 0.05 16 Fd9766 - SITE 2F, SEPTEMBER, 1988. SAMPLE MTP TTP PMP MYCP MTN TTN PMN MYCN 351 1172 1513 77 1,2,4 290 333 87 1,13,2 353 623 659 95 1,2 448 467 96 1,2,4 356 928 1027 90 1,2,4 262 286 92 1,2 357 658 689 96 1,4 114 123 93 1 358 313 511 61 2,3 122 246 50 2,1,3 359 621 629 99 1 117 120 98 1,4 360 322 327 98 1,4,3,2 572 580 99 1,2,4 362 178 1066 17 5,2,4,3 404 432 94 2,4 364 461 494 93 1 393 395 99 1,4,2 365 209 315 66 1,2,4,3 429 492 87 13,1,2 366 1506 1566 96 1,2,4,3 776 782 99 1,2,4 367 433 546 79 1,2,4 448 499 90 2,1,4 369 304 559 54 1,2,4 233 264 88 1,13,2,4 370 49 599 8 20,2 60 110 55 1,20,13 374 86 122 70 1 11 18 61 1 SUM: 7863 10622 1101 4679 5147 1286 AVG: 524 708 73 312 343 86 VAR: 157800 160737 766 41684 39898 256 STD: 397 401 28 204 200 16 SE: 103 104 7 53 52 4 $03 Fd9766 - SITE 2H, SEPTEMBER 28, 1988. >AMPLE SWT SHT CAL FRWP RWP FRWN RWN ROS LDR 326 3955 334 4.8 6030 1675 680 200 0.47 85 329 3012 273 4.1 4237 1267 3823 990 0.75 61 330 4188 305 4.3 4446 1314 3112 812 0.51 71 331 3991 325 3.8 4950 1429 1160 321 0.44 77 332 5091 343 4.6 4518 1331 1656 445 0.35 74 334 3755 339 4.0 4724 1377 3136 818 0.58 86 336 2614 292 3.9 3786 1164 595 178 0.51 65 337 3180 262 4.4 4398 1303 2296 606 0.60 66 338 2749 325 4.5 5179 1481 399 129 0.59 44 340 2281 255 3.9 3604 1122 1356 370 0.65 43 342 4369 349 3.9 4720 1376 1398 380 0.40 81 346 2329 229 4.0 3688 1142 395 128 0.55 46 347 4166 389 5.3 5874 1639 989 278 0.46 116 349 1317 230 2.3 1615 670 94 52 0.55 49 350 2937 i c; 312 3.8 3816 1171 524 161 0.45 95 n = SUM: 49934 4562 61.6 65585 19460 21613 5868 7.87 1059 AVG: 3329 304 4.1 4372 1297 1441 391 0.52 71 VAR: 927114 2014 0.40 1039484 53890 1235920 78224 0.01 397 STD: 963 45 0. 63 1020 232 1112 280 0.10 20 SE: 249 12 0.16 263 60 287 72 0.03 5 soy Fd9766 - SITE 2H, SEPTEMBER 28, 1988. SAMPLE MTP TTP PMP MY CP MTN TTN PMN MYCN 326 1420 1458 97 1 134 135 99 1 329 339 428 79 1,2,25 854 901 95 25,1,2, 330 216 465 46 1,2,P 677 1021 66 2,25 331 176 748 24 2,40 495 530 93 2,40 332 149 412 36 29,3,4 356 422 84 17,29,2 334 495 565 88 1 798 820 97 1,25,15 336 537 891 60 1,2,3 185 207 89 2/5,3/ 337 688 939 73 1,2,5,3,4 879 1019 86 2,1 338 687 741 93 1 31 38 82 31,4,2 340 139 495 28 2,5,4 276 492 56 1,2,4 342 425 625 68 1,2 250 430 58 1,2 346 434 604 72 1,2 80 110 73 1,4 347 694 755 92 1,4 132 153 86 1 349 58 223 26 1,4 8 19 42 2,1,4 350 245 696 35 1,2,4,3 251 264 95 1,2,4 SUM: 6702 10045 918 5406 6561 1203 AVG: 447 670 61 360 437 80 VAR: 109916 78411 657 86616 116617 281 STD: 332 280 26 294 341 17 SE: 86 72 7 76 88 4 2,0.5 Fd9766 - SITE 2R, SEPTEMBER 28, 1988. SAMPLE SWT SHT CAL FRWP RWP FRWN RWN ROS LDR 376 4825 291 4.7 5743 1609 1211 333 0.40 71 377 2449 226 4.4 4890 1415 233 87 0.61 46 378 4038 297 3.7 4866 1410 2833 741 0.53 73 379 3319 274 3.9 3711 1147 2914 762 0.58 81 380 2541 283 3.0 4892 1416 919 260 0.66 57 381 4117 336 3.9 6475 1776 1073 299 0.50 95 382 3346 309 4.1 3568 1114 1536 415 0.46 66 383 2533 269 3.7 2827 945 599 179 0.44 49 384 2184 243 2.8 4820 1399 522 160 0.71 50 385 3207 245 4.1 5255 1498 2691 706 0.69 37 386 4952 283 4.5 5614 1580 1769 474 0.41 77 387 2523 252 3.5 3472 1092 460 144 0.49 37 388 3930 310 5.0 5565 1569 1001 281 0.47 57 389 4933 298 4.6 6331 1743 1933 515 0.46 73 390 2099 275 3.3 1972 751 468 146 0.43 38 391 4817 i fi 298 4.5 4693 1370 2248 594 0.41 58 n = SUM: J. Q 55813 4489 63.7 74694 21836 22410 6097 8.26 965 AVG: 3488 281 4.0 4668 1365 1401 381 0.52 60 VAR: 1015977 776 0.37 1475431 76490 764376 48379 0.01 277 STD: 1008 28 0.61 1215 277 874 220 0.10 17 SE: 252 7 0.15 304 69 219 55 0.02 4 Fd9766 - SITE 2R, SEPTEMBER 28, 1988. SAMPLE MTP TTP PMP MYCP 376 390 629 62 1,2 377 763 783 97 1,43, 378 410 538 76 2,1,4 379 128 369 35 1 380 552 l l l l 50 2,1,4 381 291 932 31 2,3 382 519 579 90 2,1,4 383 158 457 35 33 384 606 1139 53 2,1 385 266 528 50 1,2,3 386 1564 1634 96 1,2,4 387 373 709 53 2,33, 388 765 906 84 1 389 834 1320 63 2,1 390 194 269 72 1 391 367 651 56 1,2 5136 8180 12554 1003 384 511 785 63 21 118548 127458 430 5 344 357 21 1 86 89 5 MTN TTN PMN MYCN 206 222 93 2,1 89 108 82 1,43 679 870 78 2,1,4 909 1182 77 1,2,4 379 402 94 2,1 126 216 58 2 430 521 83 2,1,4 132 177 75 33,6,4 232 296 78 2,1,43 664 745 89 1,2,4 600 655 92 1,2,4 153 184 83 33,2,1 398 426 93 1 352 572 62 2,1 108 131 82 1 365 684 53 2,1 5822 7391 1273 364 462 80 55398 88189 147 235 297 12 59 74 3 Fd9766 - SITE 3A, SEPTEMBER 30, 1988. SAMPLE SWT SHT CAL FRWP RWP FRWN RWN ROS LDR 301 2531 272 3.5 3047 996 783 226 0.48 102 302 4783 342 4.8 6610 1807 783 226 0.42 123 303 1496 182 3.4 5089 1461 696 204 1.11 90 304 966 171 2.8 2177 797 135 63 0.89 51 305 2091 272 3.7 6800 1850 332 112 0.94 122 306 1745 226 3.1 4186 1255 1613 434 0.97 111 307 5457 335 5.1 7830 2085 981 275 0.43 134 308 3064 259 4.0 4633 1357 1063 296 0.54 83 309 2434 175 4.3 4777 1389 1568 423 0.74 54 310 2862 194 4.6 6526 1788 1030 288 0.73 84 311 3758 250 5.4 7660 2046 1639 441 0.66 177 312 3840 334 4.1 4348 1292 1013 284 0.41 133 313 2954 274 4.1 4746 1382 1093 304 0. 57 90 314 3109 190 4.8 5992 1666 1344 367 0. 65 41 315 3515 279 3.6 4857 1408 1274 349 0.50 103 316 2953 234 4.4 7170 1934 506 156 0.71 25 317 4420 339 4.3 2803 940 1005 282 0.28 99 318 2138 181 3.7 4079 1231 542 165 0.65 74 319 1509 212 3.4 4093 1234 299 104 0.89 58 320 1939 230 3.4 4217 1262 484 150 0.73 69 321 3827 274 4.2 3637 1130 431 137 0.33 75 322 2119 262 3.3 4331 1288 1924 513 0.85 83 323 2081 226 3.3 3299 1053 1025 287 0.64 36 324 6527 394 6.0 8624 2265 567 171 0.37 132 325 2738 264 4.5 7736 2063 3067 800 1.05 80 n = SUM: 74856 6371 101.8 129267 36978 25197 7057 16.55 2229 AVG: 2994 255 4.1 5171 1479 1008 282 0.66 89 VAR: 1642476 3417 0.57 2971483 154050 381731 24161 0.05 1223 STD: 1282 58 0.75 1724 392 618 155 0.23 35 SE: 256 12 0.15 345 78 124 31 0.05 7 305 Fd9766 - SITE 3A, SEPTEMBER 30, 1988. IMPLE MTP TTP PMP MYCP MTN TTN PMN MYCN 301 339 497 68 1,2,4 377 430 88 1,2,4 302 293 689 43 2,3 248 254 98 43,2,4 303 398 863 46 1,2,4 53 63 84 1,2 304 554 578 96 1,22,3 186 199 . 93 43,1 305 254 433 59 1,2 201 304 66 1,2,4 306 259 275 94 1,2 388 390 99 1,2 307 305 685 45 2,44,38,43,4 208 224 93 38,2,4 308 783 794 99 1,43,2,4 544 556 98 1,2 309 467 477 98 1,2 425 452 94 1,2,4 310 1154 1191 97 1,3 631 637 99 43,1,2 311 835 848 98 20,43,1,36 514 514 100 1 312 483 647 75 2,43,4 349 414 84 2 313 682 689 99 1 661 667 99 1,2,4 314 239 488 49 2,3 232 442 52 2,4 315 695 710 98 1,2,4 282 286 99 1,2,4 316 420 746 56 1,2,3 372 440 85 2 317 323 324 100 43,1,4 199 205 97 1,2,4 318 291 309 94 1,4 169 170 99 1,2,3 319 166 468 35 43,3,2 90 112 80 1,2 320 384 589 65 1,2 143 186 77 1,2,4 321 429 460 93 1,2,3 591 632 94 1,2 322 670 875 77 43,1,3 414 429 97 25,26,1,2 323 157 290 54 5,32,P,2 132 194 68 32,2,4 324 1246 1264 99 43,1,3 634 691 92 1,2,4 325 829 1253 66 1,2 663 812 82 1,25,2 SUM: 12655 16442 1902 8706 9703 2217 AVG: 506 658 76 348 388 89 VAR: 80848 76097 487 35567 38728 145 STD: 284 276 22 189 197 12 SE: 57 55 4 38 39 2 Fd4503 - NURSERY, MAY 19, 1988. SAMPLE SWT SHT CAL RWP ROS MTP TTP PMP MYC1 51 1823 290 3.8 933 0.51 117 657 18 1 52 1716 253 3.5 875 0.51 0 599 0 -53 1543 248 3.1 946 0.61 0 844 0 -54 1882 286 3.7 1372 0.73 2 736 0 1 55 1391 297 2.7 653 0.47 1 354 0 1 56 1285 288 2.9 746 0.58 0 650 0 -57 1669 252 3.4 990 0.59 215 934 23 1 58 1342 266 3.2 626 0.47 0 207 0 -59 1201 245 3.0 673 0.56 54 492 11 1,3 60 1618 284 3.0 985 0.61 83 1020 8 1 61 1871 255 3.0 1032 0.55 32 493 6 1,3 62 1314 253 3.0 989 0.75 68 874 8 1 63 994 256 2.6 557 0. 56 80 485 16 1 64 1609 226 3.1 1178 0.73 270 827 33 1 65 1665 268 3.3 1105 0.66 94 637 15 1 66 1723 235 3.3 1091 0.63 38 813 5 1,3 67 1949 281 3.1 1030 0.53 1 750 0 1 68 1416 234 3.0 772 0.55 0 744 0 -69 2372 304 3.7 1250 0.53 0 872 0 -70 1598 296 3.1 888 0.56 187 643 29 1 71 1683 287 2.6 808 0.48 327 1116 29 1 72 1840 267 3.1 1129 0. 61 59 747 8 1 73 1303 230 3.1 996 0. 76 596 1001 60 1 74 1145 257 2.5 855 0.75 137 647 21 1,3 75 1260 231 2.6 849 0.67 0 831 0 -n= 25 SUM: 39212 6589 77.4 23328 14.97 2361 17973 290 AVG: 1568 264 3.1 933 0.60 94 719 12 VAR: 90550 533 0.12 38212 0.01 18454 42646 204 STD: 301 23 0.34 195 0.09 136 207 14 SE: 60 5 0.07 39 0.02 27 41 3 2\0 Fd4503 - SITE 3B, SEPTEMBER 30, 1988. SAMPLE SWT 201 2477 202 4860 203 2678 204 3263 205 1560 206 2010 207 2584 208 6331 209 1735 210 875 211 3950 212 2541 213 2976 214 2429 215 2267 216 3048 217 1708 218 3324 219 2296 220 4494 221 1094 222 2234 223 3118 224 2113 225 1707 n= 25 SUM: 67672 AVG: 2707 VAR: 1414783 STD: 1189 SE: 238 SHT CAL 324 4. 5 326 5. 3 306 4. 5 323 3. 9 172 3. 1 161 3. 4 273 4. 0 352 5. 8 198 3. 9 162 2. 7 344 4. 1 227 4. 5 313 4. 1 206 5. 0 297 4. 1 303 4. 2 223 4. 3 308 3. 8 322 3. 9 352 5. 7 206 3. 1 264 3. 8 275 4. 0 227 4. 1 232 3. 2 6696 103.0 268 4.1 3629 0.55 60 0.74 12 0.15 FRWP RWP 5395 1530 9385 2439 7779 2073 6387 1756 4109 1237 9842 2543 4933 1425 10767 2753 5384 1528 3056 998 5522 1559 6827 1856 6222 1718 7846 2088 6856 1863 6857 1863 6014 1671 5441 1541 9250 2408 9438 2451 4421 1308 7661 2046 7428 1993 5293 1507 4061 1226 166174 45381 6647 1815 3840147 199084 1960 446 392 89 FRWN RWN 1614 435 826 237 2465 649 299 104 1654 445 886 252 559 169 830 238 266 96 92 52 1614 435 604 181 1680 451 373 123 1127 312 622 185 526 161 530 162 170 71 893 253 190 77 1093 304 1453 394 953 268 275 98 21594 6150 864 246 348010 22026 590 148 118 30 ROS LDR 0.79 125 0.55 128 1.02 64 0.57 90 1.08 42 1.39 49 0.62 82 0.47 127 0.94 68 1.20 33 0.50 83 0.80 56 0.73 27 0.91 115 0.96 86 0.67 72 1.07 74 0.51 62 1.08 64 0.60 117 1.27 40 1.05 31 0.77 99 0.84 48 0.78 41 21.17 1823 0.85 73 0.06 961 0.25 31 0.05 6 51! Fd4503 - SITE 3B, SEPTEMBER 30, 1988. SAMPLE MTP TTP PMP MY CP MTN TTN PMN MYCN 201 509 746 68 1,4 220 241 91 1 202 0 1170 0 - 12 59 20 32 203 912 994 92 1,2 1201 1214 99 1,2,4 204 614 741 83 1,2,3 150 152 99 1 205 878 913 96 1,2,3 678 681 100 1,2 206 1298 1445 90 1,2,36,3 372 385 97 1,2,4 207 1145 1273 90 1,2 161 180 89 1,2 208 1785 1918 93 1,2,4 183 224 82 1,2 209 910 1039 88 1,3 158 173 91 1,2 210 500 508 98 1,28,2,25 49 51 96 1,2,25 211 925 1000 93 1,2,3 1026 1061 97 1,2 212 1026 1058 97 1,2 134 143 94 1,2 213 310 525 59 2,36,3 471 488 97 36,2 214 162 1055 15 2,4 48 119 40 2,4,P 215 953 986 97 1,3,35,2 480 512 94 1,35,2,4 216 1446 1542 94 1,2,3 593 608 98 35,32,2,3 217 1042 1256 83 1 183 183 100 1 218 1388 1394 100 1,2,4 184 186 99 1,2 219 988 1045 95 1,37,2,3 37 40 93 1,33 220 687 796 86 2,1,3 412 419 98 2,3,4 221 428 461 93 37,28,1,3 32 35 91 28,1 222 1171 1322 89 1,39,3 231 307 75 1,2,39,3 223 625 672 93 1,2 163 197 83 1,2,4 224 873 908 96 1,P 273 290 94 1,2 225 713 975 73 1,2 42 59 71 1,2,28 SUM: 21288 25742 2059 7493 8007 2187 AVG: 852 1030 82 300 320 87 VAR: 163098 112248 578 89001 88739 345 STD: 404 335 24 298 298 19 SE: 81 67 5 60 60 4 Fd4503 - SITE 4, SEPTEMBER 27, 1988. SAMPLE SWT SHT CAL FRWP RWP FRWN RWN ROS LDR 276 2760 310 3.5 2894 961 399 129 0.39 90 277 1404 211 3.4 3135 1016 1308 358 0.98 19 278 1143 195 3.4 2200 803 239 89 0.78 31 279 2323 281 3.6 3618 1126 307 106 0.53 67 280 1582 213 3.1 2458 861 600 180 0.66 28 281 1999 237 3.1 2708 918 808 232 0.58 43 282 2614 350 4.8 4162 1249 259 94 0.51 76 283 1966 294 3.3 3402 1076 312 107 0.60 78 284 2641 335 4.4 4765 1387 352 117 0. 57 62 285 2732 347 3.8 3671 1138 471 147 0.47 63 286 1457 201 4.0 2479 866 493 153 0.70 16 287 2265 296 3.9 3622 1126 719 210 0.59 66 288 3974 413 4.4 4876 1412 328 111. 0.38 127 289 3308 255 5.2 6931 1880 1367 373 0.68 82 290 1470 246 2.9 1770 705 449 142 0.58 73 291 2359 307 4.1 4886 1414 783 226 0.70 45 292 2901 321 4.4 4581 1345 1422 386 0.60 62 293 2033 223 3.7 3390 1074 512 158 0.61 52 294 2697 372 3.6 4709 1374 370 122 0. 55 104 295 1542 286 3.2 2943 972 138 63 0.67 69 296 2311 340 3.7 3961 1204 307 106 0.57 64 297 2157 308 3.3 2978 980 188 76 0.49 68 298 3471 376 4.1 3986 1209 453 143 0.39 130 299 4437 321 4.3 4416 1307 415 133 0.32 77 300 1762 225 4.8 5228 1492 619 184 0.95 86 n= 25 SUM: 59308 7263 96.0 93769 28895 13618 4144 14.85 1678 AVG: 2372 291 3.8 3751 1156 545 166 0.59 67 VAR: 639092 3500 0.35 1262316 65442 120312 7615 0.02 779 STD: 799 59 0.59 1124 256 347 87 0.15 28 SE: 160 12 0.12 225 51 69 17 0.03 6 S/3 Fd4503 - SITE 4, SEPTEMBER 27, 1988. VMPLE MTP TTP PMP MYCP MTN TTN PMN MYCN 276 375 408 92 1,2 128 142 90 1,2 277 305 378 81 1,2 285 386 74 1,2,4 278 497 532 93 1,4 43 60 72 1,4 279 382 505 76 1,2 99 136 73 1,2 280 562 570 99 1,2 229 245 93 1 281 316 824 38 2,1,3 276 341 81 2,1,4 282 731 757 97 1 112 119 94 1 283 512 648 79 1 57 183 31 1 284 600 609 99 1 78 82 95 1 285 976 1033 94 1 69 77 90 1 286 429 444 97 1,2 156 171 91 1,4 287 519 798 65 1,3,2,36 138 248 56 1,3,2 288 412 436 94 1,3 22 36 61 1 289 383 739 52 1,2 295 328 90 2,1,3 290 222 250 89 1 201 204 99 1,4 291 4 660 1 1,2 16 75 21 2,1 292 790 796 99 1,4 419 431 97 1,2,4 293 212 366 58 1,4 42 144 29 1 294 509 542 94 1,P 37 39 95 1 295 708 761 93 1,2,4 29 44 66 1 296 743 851 87 1,3,4,2 364 387 94 1 297 386 402 96 2,1,4,3 3 48 6 2 298 568 591 96 1 76 126 60 1,2 299 500 681 73 1,2,3 158 177 89 1,2,4 300 223 514 43 1,3 109 208 52 1,4,36 SUM: 11864 15095 1985 3441 4437 1800 AVG: 475 604 79 138 177 72 VAR: 43724 33584 572 12684 13590 675 STD: 209 183 24 113 117 26 SE: 42 37 5 23 23 5 Fd4503 - SITE 5, NOVEMBER 22, 1988. SAMPLE SWT 442 2801 443 6385 444 4702 445 2996 446 6539 447 2207 448 2740 449 3841 450 4452 451 3978 452 1994 453 7180 454 5087 455 4853 456 4556 457 2404 458 3611 459 4203 460 3156 461 4942 462 2425 463 4566 464 2196 465 3110 466 3609 467 3440 468 5869 469 2571 n= 28 SUM: 110413 AVG: 3943 VAR: 1912825 STD: 1383 SE: 261 SHT CAL 294 4. 3 436 6. 1 291 4. 9 320 3. 7 390 5. 4 285 4. 1 317 4. 1 315 5. 8 433 5. 9 233 4. 9 304 3. 8 411 6. 3 392 5. 1 384 4. 2 307 5. 3 178 3. 6 310 5. 4 330 5. 0 246 5. 0 399 5. 1 255 4. 5 332 5. 4 259 3. 1 337 4. 3 370 5. 0 321 4. 7 322 5. 2 294 3. 9 9065 134.1 324 4.8 3668 0.62 61 0.78 11 0.15 FRWP RWP 4169 1251 8669 2276 8078 2141 3221 1035 7704 2056 4418 1308 5000 1440 7343 1974 4354 1293 5835 1630 3867 1182 6486 1779 5469 1547 5216 1489 7593 2031 3511 1101 4887 1415 3900 1190 6197 1713 7282 1960 3302 1054 6720 1832 4783 1391 5085 1460 4436 1312 4684 1368 5932 1652 4112 1238 152253 43117 5438 1540 2293464 118899 1514 345 286 65 FRWN RWN 1424 387 4759 1226 2818 738 1869 499 3673 953 1208 333 906 257 1998 531 448 141 7570 1933 1027 287 4416 1140 3569 927 1965 523 2233 590 1163 321 2551 670 1530 414 2339 617 2468 650 1693 455 2306 609 2002 532 1831 489 1295 354 3756 974 1935 516 1487 403 66239 17468 2366 624 2066681 130805 1438 362 272 68 ROS LDR 0.58 78 0.55 156 0.61 96 0.51 94 0.46 156 0.74 51 0.62 906 0.65 148 0.32 181 0.90 81 0.74 68 0.41 167 0.49 115 0.41 109 0.58 77 0.59 78 0.58 91 0.38 107 0.74 74 0.53 117 0.62 71 0.53 65 0.88 74 0.63 88 0.46 120 0.68 80 0.37 63 0.64 75 16.20 3586 0.58 128 0.02 23558 0.14 153 0.03 29 3U5 Fd4503 - SITE 5, NOVEMBER 22, 1988. \MPLE MTP TTP PMP MY CP MTN TTN PMN MYCN 442 939 1015 93 1,2,3,4 780 807 97 1,3 443 1562 1641 95 1,4 785 789 99 1,4 444 1471 1612 91 1,36,2,3 737 750 98 1,36,2,4 445 239 391 61 P,3,2 730 731 100 P,41,2,4, 446 574 579 99 36,1,4 829 851 97 1,36,4 447 722 740 98 1,36,3,4 441 447 99 36,1,3 448 976 1161 84 1,40,2,3 491 505 97 40,1,3 449 979 991 99 1,16,19,3 539 548 98 1,19,4 450 45 183 25 40,16,4 14 70 20 16,40 451 780 785 99 1,40,29,14 2098 2103 100 1,29,40,2 452 435 577 75 2,30,14,29, 265 281 94 40,2,3 6,3,4 453 503 523 96 1 612 633 97 1,4 454 221 288 77 40,29,3,4 947 1056 90 45,40,4, 455 576 588 98 1 704 721 98 1,3 456 474 767 62 1,2,3,4 902 1016 89 1,2,5,4 457 242 336 72 2 234 359 65 2,4 458 562 615 91 1,36,3 867 893 97 1,36,3 459 370 374 99 1 311 312 100 1 460 598 636 94 5,3 792 796 99 5,2,3 461 1008 1037 97 1,3,4 489 491 100 1 462 175 201 87 1,4 188 194 97 1,5 463 769 778 99 1,2,3 979 1105 89 1,4 464 699 935 75 1,2,17,3 987 1098 90 1,2,17 465 674 752 90 1,2,3 715 758 94 1,2,3,4 466 249 323 77 1 420 441 95 1,29,4 467 823 874 94 1,2,4 1584 1665 95 1,2,4 468 359 369 97 1,36 441 451 98 1,4 469 890 902 99 1,36,2,5,4 530 536 99 1,3 SUM: 17914 19973 2423 19411 20407 2591 AVG: 640 713 87 693 729 93 VAR: 128974 133129 269 168554 176640 239 STD: 359 365 16 411 420 15 SE: 68 69 3 78 79 3 2 / £ Fd4503 - SITE 6, NOVEMBER 24, 1988. SAMPLE SWT SHT CAL FRWP 470 3336 300 4.2 6821 471 3659 347 5.4 5332 472 9129 343 5.6 11031 473 5104 275 5.3 6250 474 2540 264 3.8 3914 475 3470 346 4.1 5029 476 4036 261 5.1 8155 477 4768 301 3.9 5249 478 6237 355 5.5 7620 479 5186 348 5.0 8346 480 2621 318 4.3 7961 481 2110 250 4.0 7320 482 5529 315 5.2 6274 483 3198 330 4.6 3938 484 2329 249 3.5 5661 n= 15 SUM: 63252 4602 69.5 98901 AVG: 4217 307 4.6 6593 VAR: 3202601 1387 0.46 3324801 STD: 1790 37 0. 68 1823 SE: 462 10 0.18 471 RWP FRWN RWN ROS LDR 1855 4326 1117 0. 89 83 1516 5099 1312 0. 77 89 2813 12426 3155 0. 65 158 1725 4429 1143 0. 56 95 1193 3043 794 0. 78 66 1447 2559 672 0. 61 84 2159 10166 2586 1. 18 83 1497 5262 1353 0. 60 66 2037 6814 1743 0. 61 99 2202 2941 769 0. 57 99 2114 12795 3248 2. 05 83 1968 1742 467 1. 15 64 1730 6801 1740 0. 63 100 1198 2200 582 0. 56 92 1591 2018 536 0. 91 66 >7046 82621 21216 12. 52 1327 1803 5508 1414 0. 83 88 72367 12499383 791117 0. 14 498 415 3535 889 0. 38 22 107 913 230 0. 10 6 Xii-Fd4503 - SITE 6, NOVEMBER 24, 1988. IMPLE MTP TTP PMP MYCP MTN TTN PMN MYCN 470 365 507 72 2,33,1,3,P 759 790 96 33,1,2,3 471 331 362 91 33,1 970 983 99 33,2,1 472 930 1106 84 1,33 3599 3599 100 1,33 473 388 478 81 1 678 717 95 1 474 271 305 89 33,1,P 403 888 45 33,5,1,3 475 621 784 79 2,1 473 495 96 2,1 476 279 321 87 33,1 3458 3462 100 33,1 477 501 613 82 2,1,12 1025 1071 96 2,1,4,12 478 411 682 60 43,1,2 2365 2456 96 43,2,3 479 428 615 70 2,1 298 401 74 2,1 480 163 482 34 1,2 865 2109 41 1,2 481 417 793 53 33,1 652 660 99 33 482 394 438 90 33,1,2,36 1573 1605 98 33,1,2,36 483 316 351 90 1,2,12,33 272 286 95 1,12,2,33 484 498 767 65 12 627 717 87 12 SUM: 6313 8604 1127 18017 20239 1317 AVG: 421 574 75 1201 1349 88 VAR: 29659 46594 253 1098665 1077577 345 STD: 172 216 16 1048 1038 19 SE: 44 56 4 271 268 5 2/0 Hw7321 - NURSERY, MAY 19, 1988. SAMPLE SWT 131 1438 132 1880 133 2765 134 1189 135 1398 136 1804 137 1229 138 1532 139 1765 140 1502 141 2273 142 1463 143 904 144 1994 145 2357 146 1356 147 1425 148 2141 149 1343 150 1997 151 518 152 1842 153 1212 154 1299 155 1544 n= 25 SUM: 40170 AVG: 1607 VAR: 226225 STD: 476 SE: 95 SHT CAL RWP ROS 195 4.0 773 0.54 212 3.3 1393 0.74 232 3.6 2020 0.73 177 3.1 576 0.48 199 3.1 539 0.39 242 3.5 1431 0.79 227 2.5 665 0.54 168 3.5 833 0.54 217 3.0 1165 0. 66 279 3.9 764 0.51 231 3.9 1555 0.68 234 2.9 883 0.60 150 2.8 426 0.47 234 3.6 1252 0.63 219 3.9 1483 0.63 201 2.8 722 0.53 193 4.0 765 0.54 250 3.2 1495 0.70 273 2.9 478 0.36 172 3.1 974 0.49 186 3.2 1064 2.05 217 3.8 988 0.54 204 3.7 784 0.65 181 3.3 555 0.43 218 2.8 666 0.43 5311 83.4 24249 15.65 212 3.3 970 0.63 955 0.19 159363 0.10 31 0.43 399 0.31 6 0.09 80 0.06 MTP TTP PMP MYCP 35 500 7 1 197 489 40 1 103 1836 6 1 525 607 86 1 641 768 83 1 201 500 40 1,3 0 160 0 -4 355 1 1 0 500 0 -0 500 0 -3 1620 0 -69 500 14 1,3 194 298 65 1 6 500 1 1,3 0 500 0 -405 1003 40 1 1103 1218 91 1 1015 1247 81 1 0 500 0 -0 500 0 -56 329 17 1 413 563 73 1 65 496 13 1 0 500 0 -370 419 88 1 5405 16408 749 216 656 30 )5085 162222 1195 308 403 35 62 81 7 Hw7321 - SITE 7H, SEPTEMBER 29, 1988. 3AMPLE SWT SHT CAL FRWP RWP FRWN RWN ROS LDR 251 6036 335 6.0 6978 1421 3988 714 0.35 105 252 2810 268 4.1 4639 880 2506 449 0.47 58 253 12156 504 6.5 10287 2186 3391 607 0.23 202 254 4007 327 5.5 5502 1080 2108 377 0.36 87 255 5633 314 5.9 7997 1657 2803 502 0.38 71 256 2685 302 4.0 2931 485 720 129 0.23 104 257 3594 226 5.5 4969 957 2944 527 0.41 74 258 6163 324 6.4 8163 1695 3456 619 0.38 122 259 2431 248 3.8 3315 574 403 72 0.27 31 260 5068 316 5.1 5731 1133 2299 412 0.30 134 261 3625 264 4.4 5517 1083 962 172 0.35 51 262 5160 286 5.8 5401 1057 1801 322 0.27 93 263 5266 377 5.6 11307 2422 2438 436 0.54 83 264 2978 233 4.5 5680 1121 1394 250 0.46 61 265 7333 359 6.5 9234 1943 4951 886 0.39 173 266 7717 329 5.4 9065 1904 7170 1283 0.41 84 267 5248 335 5.1 7800 1611 1352 242 0.35 97 268 9622 413 7.0 9957 2110 7332 1312 0.36 199 269 10001 507 5.7 9913 2100 7626 1365 0.35 230 270 7213 412 6.5 6757 1370 3055 547 0.27 178 271 9003 552 6.1 9126 1918 6809 1219 0.35 298 272 8847 518 5.2 7413 1522 2797 501 0.23 166 273 6702 446 5.8 10283 2186 3994 715 0.43 158 274 4994 0 A 283 5.2 5735 1134 3222 577 0.34 95 n— SUM: Z H 144292 8478 131.6 173700 35551 79521 14234 8.48 2954 AVG: 6012 353 5.5 7238 1481 3313 593 0.35 123 VAR: 6380100 8500 0.70 5199511 278052 4193724 134371 0.01 4046 STD: 2526 92 0.84 2280 527 2048 367 0.08 64 SE: 516 19 0.17 465 108 418 75 0.02 13 Hw7321 - SITE 7H, SEPTEMBER 29, 1988. SAMPLE MTP TTP PMP MY CP MTN TTN PMN MYCN 251 1614 1622 100 1,4 2911 2918 100 1,4 252 1146 1268 90 1 666 689 97 1,4 253 2764 2768 100 1,4 1603 1639 98 1,3 254 348 501 69 5,4,3 896 899 100 5,4 255 1360 1430 95 1,3 202 307 66 1,4 256 264 397 66 43,1,4 74 90 82 43,4 257 531 615 86 14,43,5,3 1098 1121 98 14,43,3 258 505 510 99 1 282 322 88 1,5 259 450 500 90 1,4 117 131 89 1,43 260 485 500 97 1 679 701 97 1 261 1365 1705 80 1 267 330 81 1,4 262 487 500 97 1 2493 2513 99 1,28 263 2053 3057 67 1,5,4,3 622 622 100 1 264 1909 2149 89 34,1,4 443 491 90 34,4 265 913 944 97 1 1622 1642 99 1,23,3,4 266 432 500 86 1 2342 2350 100 1,4 267 316 500 63 1,3 287 339 85 1,3 268 708 1812 39 43,3 1138 1515 75 43,3 269 500 500 100 1,4 1698 1705 100 1,4 270 442 600 74 1,4 733 808 91 1,4 271 294 500 59 43,4 1669 1746 96 43,14,4 272 496 500 99 1 698 736 95 1 273 475 500 95 1 1657 1683 98 1,4 274 441 527 84 28,43,4 905 984 92 28,43,5,4 SUM: 20298 24405 2022 25102 26281 2213 AVG: 846 1017 84 1046 1095 92 VAR: 424367 587842 253 602588 596171 77 STD: 651 767 16 776 772 9 SE: 133 157 3 158 158 2 Cw4511 - NURSERY, MAY 19, 1988. SAMPLE SWT SHT CAL RWP ROS PMP 501 1237 280 2.4 744 0. 60 0 502 1203 277 2.6 485 0.40 0 503 1518 289 2.5 787 0.52 0 504 838 205 2.9 603 0.72 0 505 645 198 1.7 580 0.90 0 506 1923 304 3.0 1000 0.52 0 507 1467 308 2.7 600 0.41 0 508 949 225 2.0 460 0.48 0 509 1449 305 2.9 822 0.57 0 510 1453 282 2.9 610 0.42 0 511 1146 304 2.8 469 0.41 0 512 1436 261 2.6 918 0.64 0 513 1425 359 3.0 488 0.34 0 514 1180 258 2.2 748 0.63 0 515 1279 284 2.4 582 0.46 0 516 1785 316 2.8 535 0.30 0 517 1346 336 2.8 298 0.22 0 518 1375 267 2.2 628 0.46 0 519 1314 288 2.3 472 0.36 0 520 1923 303 3.2 823 0.43 0 521 1177 243 2.9 647 0.55 0 522 1151 291 2.8 500 0.43 0 523 993 219 2.3 842 0.85 0 524 1421 276 2.9 722 0.51 0 525 1133 284 2.5 473 0.42 0 n= 25 SUM: 32766 6962 65.3 15836 12.55 0 AVG: 1311 278 2.6 633 0. 50 0 VAR: 86538 1420 0.12 27501 0.02 0 STD: 294 38 0.35 166 0.15 0 SE: 59 8 0.07 33 0.03 0 222 Cw4511 - SITE 7C, SEPTEMBER 29, 1988. SAMPLE SWT SHT CAL 226 2977 336 4.1 227 2643 312 4.2 228 2398 326 4.1 229 4385 394 4.1 230 2473 327 3.5 231 2228 309 2.9 232 5239 372 4.8 233 2410 306 3.6 234 2608 327 3.9 235 5252 437 5.4 236 3321 380 4.7 237 3956 381 4.5 238 3076 295 4.2 239 2950 227 3.9 240 2650 276 3.9 241 3358 369 3.9 242 1279 225 2.7 243 1922 259 3.0 244 3216 335 4.3 245 2673 282 3.2 246 3766 366 4.5 247 2441 315 3.3 248 1873 323 2.8 249 2624 353 4.0 250 1837 287 3.2 n= 25 SUM: 73555 8119 96.7 AVG: 2942 325 3.9 VAR: 920077 2470 0.44 STD: 959 50 0.66 SE: 192 10 0.13 RWP FRWN RWN ROS 953 2830 594 0. 52 1302 510 107 0. 53 848 610 128 0. 41 1551 620 130 0. 38 1226 1210 254 0. 60 981 220 46 0. 46 2146 2110 443 0. 49 1458 3310 695 0. 89 1296 960 202 0. 57 2452 2510 527 0. 57 1617 1543 324 0. 58 2017 2270 477 0. 63 1612 990 208 0. 59 1099 1610 338 0. 49 1650 1310 275 0. 73 934 5010 1052 0. 59 1228 1740 365 1. 25 824 180 38 0. 45 1782 4200 882 0. 83 416 2540 533 0. 36 2176 4240 890 0. 81 711 1840 386 0. 45 841 1540 323 0. 62 1167 2480 521 0. 64 804 1250 263 0. 58 33091 47633 10003 15. 03 1324 1905 400 0. 60 55671 1557330 68678 0. 03 506 1248 262 0. 19 101 250 52 0. 04 3&3 Cw4511 - SITE 7C, SEPTEMBER 29, 1988.+ SAMPLE PMP MYCP PMN MYCN 226 2 C 7 C 227 0 - 3 C 228 53 c 56 C 229 1 F 10 F,C 230 34 F,C 64 F,C 231 0 0 -232 0 - 0 C 233 33 F,C 64 F,C 234 0 - 0 -235 0 - 0 -236 0 - 2 C,F 237 0 - 0 -238 1 C 30 C,F 239 0 - 1 F,C 240 0 - 0 -241 27 F,C 45 F,C 242 0 - 2 C 243 0 - 0 -244 11 C,F 41 C, F 245 28 F,C 37 F,C 246 1 F 1 F 247 0 - 0 -248 0 - 5 F,C 249 0 - 0 -250 0 - 0 -SUM 191 368 AVG 8 15 VAR 209 483 STD 14 22 SE 3 4 +Note: F = f i n e endophyte, C = coarse endophyte. 32 APPENDIX 5 Characterization of ectomycorrhizal types Major types Type 1 FUNGUS: Thelephora terrestris Ehrenb.: Fr. SOURCE: Various Douglas-fir and western hemlock. MOST DISTINCTIVE CHARACTERISTICS: Hyphoid c y s t i d i a about 100 um long with clamp at base ar i s i n g from mantle and rhizomorphs. HABIT: Tips unbranched and appearing nonmycorrhizal (color of the root; root hairs sometimes present) in the nursery. Black sporocarps on undersurface of styroblocks and around a i r -pruned zone of some seedlings occurred in MacBean Nursery. Mycorrhizae from f i e l d once to twice pinnate to irregular, sometimes with a grey or s i l v e r y color. Mantle texture smooth to velvety. Rhizomorphs common, off white to buff in nursery and buff to dark brown in f i e l d (Figs. 26-28, 39 and 40). CYSTIDIA: Very common to rare but usually a few present on each t i p i f closely examined. Hyaline, hyphoid, and generally straight, (25-)75-150(-340) um x 2-3 um wide near the clamped septum at the base of the c y s t i d i a attaching i t to the mantle, gradual taper to 1.5-2 um at 10 um from rounded t i p , walls thicker than emanating hyphae (Figs. 29-33). Each cystidium with 2-4 (-8) retraction septa (Schramm 1966). EMANATING HYPHAE: Hyphae of indefinite length uncommon or easily l o s t in procedures, hyaline, 2.5-5(-7) um wide with 225 clamps on most septa, forming mycelial strands or rhizomorphs (Fig. 34). MYCELIAL STRANDS: Rhizomorphs common when colonization l e v e l greater than about 20%. Usually not d i r e c t l y connected to mantle, 15-2 65 um wide, off white to l i g h t brown with age in nursery to dark brown in the f i e l d . Core hyphae (2-)3.5-5.5(-8.5) um wide and only seen in sections or sometimes in squash mounts of larger strands. Outer hyphae l i k e mantle c y s t i d i a (Figs. 26, 28, and 31). In nursery, emanating hyphae sometimes forming simple strands of several hyphae 2-4 um wide twisted together or running p a r a l l e l , c y s t i d i a absent (Fig. 34) . MANTLE: Usually absent or < 5 um thick in nursery to 27 um in the f i e l d . Net prosenchymous with irregular pattern tending towards textura epidermoidia or "jigsaw". Hyphae smooth and hyaline. Outer hyphae 3-5 um wide with clamps on most septa. Clamps hard to discern at deeper mantle depths; hyphal swellings 6-10(-14) um wide may occur mostly at 3- or 4-way intersections (Figs. 35-37). HARTIG NET: Hyphal invaginations 1-3 um wide; regular beading sometimes seen; some epidermal c e l l s may have coiled hyphae on them, no clamps seen (Figs. 37 and 38). OTHER DESCRIPTIONS: Agerer and Weiss (1989) give a much more detailed decription of t h i s fungus on spruce and a summary of other descriptions found in the l i t e r a t u r e . 226 FIGS. 26 and 27. Type 1 (Thelephora terrestris) mycorrhizae on Douglas-fir from MacBean Nursery. FIG. 26. Mycorrhizal (m) t i p , rhizomorph (r) , and hyphae (arrows). x2l5. FIG. 27. Part of Thelephora terrestris sporocarp (*) at base of root plug. x86. FIGS. 28-30. Habit of Type 1 (Thelephora terrestris) mycorrhizae from f i e l d . FIG. 28. Mycorrhizal root of Douglas-f i r from Site 3B with rhizomorph (r) attached. x86. FIGS. 29 and 30. Mycorrhizal root tips of western hemlock from Site 7H showing c y s t i d i a (arrows). xl38. FIG. 29. Mature t i p . FIG. 30. Senescent t i p . 228 FIGS. 31 and 32. Type 1 (Thelephora terrestris) mycorrhizae af t e r staining procedure. FIG. 31. Rhizomorph a x i l showing c y s t i d i a l outer hyphae. X1348. FIG. 32. Cy s t i d i a (arrows) emanating from mantle surface. X2150. 34 FIGS. 33 and 34. Type 1 (Thelephora t e r r e s t r i s ) mycorrhizae. X5375. FIG. 33. Mycorrhiza from Site 5 with c y s t i d i a emanating from mantle showing t i p (t) , clamp (c) at base near mantle, and r e t r a c t i o n septum (arrow). FIG. 34. Hyphal morphology of mycelial strand formed i n MacBean Nursery. 2 3 0 J 3 36 F I G S . 35 a n d 3 6 . P l a n v i e w o f m a n t l e o f T y p e 1 (Thelephora terrestris) m y c o r r h i z a o n D o u g l a s - f i r f r o m S i t e 5 . x 5 3 7 5 . F I G . 3 5 . M i d m a n t l e w i t h t e x t u r a e p i d e r m o i d i a p a t t e r n . F I G . 3 6 . I n n e r m a n t l e h y p h a e a f t e r s t a i n i n g p r o c e d u r e ( p h a s e c o n t r a s t i l l u m i n a t i o n ) . 33i FIGS. 37 and 38. Type 1 (Thelephora terrestris) mycorrhizae. x5375. FIG. 37. Cross-sect ion of Douglas - f i r mycorrhiza from S i t e 1C showing c o r t i c a l c e l l s (c) , H a r t i g net (n) , t ann in -f i l l e d epidermal c e l l s (e) , and mantle (m) . FIG. 38. H a r t i g net of Doug las - f i r mycorrhiza from S i t e 5 seen i n plan view with hyphae forming c o i l s (arrow) on epidermal c e l l s . 0.32 Type 2 FUNGUS: Rhizopogon vinicolor A.H.Smith. SOURCE: Fd3290I and various Douglas-f ir from the f i e l d . MOST DISTINCTIVE CHARACTERISTICS: Form and co lor . Narrow hyphoid c y s t i d i a , often kinked, with brown contents removed in s ta in ing procedure. HABIT: Spec i f i c to Douglas - f ir . Mycorrhizae black with white areas where a i r i s trapped under hyphae. Rhizomorphs concolorous, very common, and attached d i r e c t l y to mantle. Branching i s in two dimensions forming t r i a n g u l a r l y once-pinnate short roots with t ips c lose ly packed together; however unbranched short roots do occur (Figs. 3 9-4 2). Sometimes a r i n d encloses the ent ire short root to form a tubercle (Fig . 43) . CYSTIDIA: Usually very abundant and comprising a l l the emanating hyphae of the mantle, rhizomorphs and tubercle r i n d when present (Figs. 41-43). Cys t id ia a r i s i n g from mantle are 100-230 x 1.5-2 (-2.5) um near the center with gradual taper to a rather pointed t i p . Cys t id ia a r i s i n g from rhizomorphs are 100-650 x 1-1.5(-2) um. The brown contents of numerous c y s t i d i a gave the mycorrhizae i t s black appearance at low magnif ication. The contents of the c y s t i d i a were removed during the root c lear ing procedure and sometimes accumulated around the mantle hyphae. The c y s t i d i a were hyaline once the contents were removed because the walls usual ly d id not s ta in with Trypan blue. , C y s t i d i a often with a kink (usually one but rare ly two per cystidium). Kinked c y s t i d i a a dichotomy with 233 one branch at least 2 0% shorter than the other and often the ti p s of both branches were seen. Some kinks with part of a very thin walled, colorless hypha attached, but direc t connection to mantle surface hyphae was not observed (Figs. 44 and 45). Brownish exudates were sometimes present on f i e l d samples. EMANATING HYPHAE: Hyphae from mantle usually d i r e c t l y involved in rhizomorph formation beginning at the mantle surface. Rarely, hyphae emanating from the mantle were amongst c y s t i d i a and not part of a rhizomorph. These hyphae were smooth, not clamped, thin walled, 1.5-4 um wide, staining with Trypan Blue and often ribbon-like. MYCELIAL STRANDS: Most rhizomorphs were 8 0-3 00 um wide. Core hyphae arose deep within the mantle and were only seen after removal of c y s t i d i a . Core hyphae smooth, not clamped, blue-staining, and occasionally branched, 4-11 um wide, narrowing at septa, and 4 to 10 in number usually with one being much larger than the others (Figs. 39, 41, 46-48). Brownish exudates sometimes present on f i e l d samples. MANTLE: Fel t prosenchymous in plan view and textura globosa in cross-section, compact, 5-30(-375) um thick. Hyphae (1.5-)2-3(-5) um wide in outer mantle to 7 (-9) um wide near epidermis, smooth, staining poorly with Trypan blue. Clamp connections were seen only on some ti p s (Figs. 49-51). HARTIG NET: Invagination 1-3 um wide, presence of clamps suspected; not beaded, c e l l separation one hypha wide (Figs. 51 and 52). 234 OTHER DESCRIPTIONS: Zak (1971) gives a more detailed characterization of th i s symbiosis and includes references to descriptions by other authors. NOTE: When stained, immature types with exudates may be mistaken for Type 3 3 unless presence of c y s t i d i a confirmed. 235 FIGS. 39 and 40. Habit of Type 1 (Thelephora terrestris) mycorrhizae (*) contrasted with Type 2 mycorrhizae. x34. FIG. 39. Mycorrhizae from Site 3B showing rhizomorphs ( r ) . FIG. 40. Mycorrhizae from Site 3A aft e r staining procedure. 336 FIGS. 41 and 42. Habit of Type 2 {Rhizopogon vinicolor) mycorrhizae from Site 3B. xl38. FIG. 41. Rhizomorph (r) and c y s t i d i a (c) . FIG. 42. Removal of c y s t i d i a (c) to reveal whitish mantle (m). a3? FIGS. 43-45. Type 2 (Rhizopogon vinicolor) mycorrhizae from S i t e 6. FIG. 43. Cross-section of a mycorrhizal t i p showing mantle (m) , c y s t i d i a (c) , and r i n d of t u b e r c l e (t) . x538. FIGS. 44 and 45. Mantle c y s t i d i a . X 5 3 7 5 . FIG. 44. Midway kink (k) with part of a t h i n walled hyaline hyphae (arrow) a t -tached. FIG. 45. Kink (k) and one of the two rounded ends (e). 238 48 FIGS. 46-48. Rhizomorphs of Type 2 (Rhizopogon v i n i c o l o r ) mycorrhizae. FIG. 46. Plan view of Type 2 rhizomorph contrasted with a rhizomorph of Type 33 (*) mycorrhizae from Site 6. x538. FIG. 47. Cross-section snowing water pipe anatomy. x538. FIG. 48. Outer c y s t i d i a l hyphae of rhizomorph from Site 1C as seen in plan view. x2150. 239 FIGS. 49 and 50. Mantle of Type 2 (Rhizopogon vinicolor) mycorrhizae as seen i n plan view. x5375. FIG. 49. Mycorrhiza from S i t e 5 with f e l t prosenchymous pattern of outer mantle hyphae some with clamp connections (arrows) . FIG. 50. Inner mantle of mycorrhiza from Site 1C. FIGS. 51 and 52. Type 2 (Rhizopogon vinicolor) mycorrhizae. x5375. FIG. 51. Cross-section of mycorrhiza from S i t e 1C showing c o r t i c a l c e l l s (c) , Hartig net (n) , and mantle (m) . FIG. 52. Hartig net of mycorrhiza from S i t e 5 seen i n plan view showing clamped hyphae around epidermal c e l l (arrow). Type 3 FUNGUS: Mycelium radicis atrovirens Melin. SOURCE: Various Douglas-fir and western hemlock. MOST DISTINCTIVE CHARACTERISTIC: Narrow, brown, usually f i n e l y ornamented, emanating hyphae. HABIT: In MacBean Nursery these mycorrhizae usually appeared l i k e nonmycorrhizal roots because root hairs were sometimes present, mantles were poorly developed, and emanating hyphae were few so that the color of the root predominated. Other ti p s had a thin mantle and abundant emanating hyphae giving a cottony or woolly appearance (Fig. 53). Tips were easily spotted after staining procedure, but higher magnification was needed to distinguish t i p s from Cenococcum in f i e l d samples. Unbranched in nursery to once pinnate in f i e l d . CYSTIDIA: Absent. EMANATING HYPHAE: Dark brown-green pigment becoming l i g h t brown after staining procedure. Hyphae 1.5-2.5 um wide, not clamped, straight, infrequently branched, growing ends not seen; walls f i n e l y ornamented (verruculose) becoming smooth and losing a l l or some pigment close to mantle or Hartig net (Figs. 54 and 58). MYCELIAL STRANDS: Rare; only one seen that was 60 um wide. Hyphae sometimes involved with T. terrestris strands in MacBean Nursery. MANTLE: Absent or thin and patchy in the nursery to 6 um thick in f i e l d samples, f e l t prosenchymous to synenchymous with jigsaw pattern or sometimes with a s t e l l a t e pattern, loose to 242 compact; hyphae 1.5-2 um wide, l i g h t to dark brown (Figs. 55-57) . HARTIG NET: Invaginations 1-2 um wide; not beaded; hyphae hyaline on nursery samples but usually brown (at least around epidermal c e l l s ) on f i e l d samples. Pigment remaining after staining procedure (Fig. 58). OTHER DESCRIPTIONS: Wilcox and Wang (1985) give Melin's o r i g i n a l description. Several different species have similar characteristics and different strains of the same species can vary i n the degree of i n t r a c e l l u l a r penetration in monoxenic cultures (Wilcox and Wang 1987). NOTE: A l l mycorrhizae with emanating hyphae of these characteristics were put into this type. Hyphae seen in Douglas-fir root hairs in the nursery. Cortex was not investigated for i n t r a c e l l u l a r penetration. 243 FIGS. 53 and 54. Type 3 (Mycelium radicis atrovirens) mycorrhizae from MacBean Nursery. FIG. 53. Mycorrhizal t i p showing emanating hyphae (arrows). x538. FIG. 54. Emanating hyphae. x5375. 2HH FIGS. 55 and 56. Variation i n mantle pattern of Type 3 (Mycelium radicis atrovirens) mycorrhizae from S i t e 3B as seen i n plan view. X2150. FIG. 55. Net prosenchymous mantle. FIG. 56. Regular synenchymous mantle with repeating s t e l l a t e pattern. 245 FIGS. 57 and 58. Type 3 (Mycelium radicis atrovirens) mycorrhizae from Site 5 as seen i n plan view. x5375. FIG. 57. Mantle with textura epidermoidia pattern. FIG. 58. Mycorrhiza without mantle showing emanating hyphae (h) becoming smooth and hyaline to form Hartig net (n). 2HC Type 4 FUNGUS: Cenococcum geophilum Fr. SOURCE: Various Douglas-fir and western hemlock. MOST DISTINCTIVE CHARACTERISTICS: Jet black mycorrhizae with s t i f f , wide, emanating hyphae. Mantle hyphae synenchymous forming s t e l l a t e pattern. HABIT: Unbranched, stubby, with black mantle that i s smooth to woolly depending on the amount of emanating hyphae (Fig. 59). CYSTIDIA: Absent. EMANATING HYPHAE: Few to abundant, 4-5 um wide, smooth or sometimes ornamented, not clamped, growing ends rarely seen, infrequently branched, dark brown wall pigment becoming lighter brown after staining procedure (Figs. 59-61). MYCELIAL STRANDS: Absent. Mantle: Regular synenchymous with hyphae forming a repeating s t e l l a t e pattern soon after the fungus colonises the root, hyphae 3-5 um wide. Some of the emanating hyphae (when present) a r i s i n g from center of s t e l l a t e pattern (Fig. 61). HARTIG NET: Invaginations 1-3 um wide; not beaded; hyphae (at least around epidermis) remaining brown after staining procedure (Figs. 62 and 63). OTHER DESCRIPTIONS: Chilvers (1968). 247 FIGS. 59-61. Type 4 (Cenococcum geophilum) mycorrhizae. FIG. 59. Habit of western hemlock mycorrhizae. x86. FIG. 60. Emanating hypha. x5375. FIG. 61. Plan view of mantle with regular synenchymous repeating s t e l l a t e pattern showing emanating hyphae (arrows) on Douglas-fir from S i t e 1C. X 2 1 5 0 . MS FIGS. 62 and 63. Hartig net of Type 4 (Cenococcum geophilum) mycorrhizae from Site 5 as seen i n plan view. x5375. FIG. 62. Mantle (m) removed to reveal Hartig net (n) around epidermal c e l l s . FIG. 63. Hartig net (n) hyphae becoming hyaline around c o r t i c a l c e l l s . 3^1 Type 5 . FUNGUS: Endogone-like. SOURCE: Douglas-fir on Sites IC and 2F. MOST DISTINCTIVE CHARACTERISTIC: Hyphae without septa at regular intervals that were usually dark staining. HABIT: Colonized roots appear nonmycorrhizal unless f i r s t cleared and stained; emanating hyphae and mantle absent except for a low abundance of root surface hyphae (Figs. 64 and 65). CYSTIDIA: Absent. EMANATING HYPHAE: Lost in staining procedure. MYCELIAL STRANDS: Absent. MANTLE: Absent except for a few surface hyphae that were d i r e c t l y involved in Hartig net formation; 3-7 um wide, width variable along length, i r r e g u l a r l y branching, without septa at regular intervals, and usually staining dark blue (Figs. 66 and 67). Some ti p s from Site 2F had narrower hyphae 1-3 um wide (Fig. 65). HARTIG NET: Patchy occurrence to well developed. Pattern di f f e r e n t from other mycorrhizal types in the combination of aseptate hyphae and invaginated hyphae. Usually staining dark blue (Figs. 64-67). OTHER DESCRIPTIONS: Closely resembles descriptions of Endogone lactiflua on members of the Pinaceae (Fassi et al. 1969, Chu-Chou and Grace 1987). If unstained samples were available an ani l i n e blue stain test turning hyphae v i o l e t blue would probably confirm genus (Walker 1985), but sporocarps are required to identify species. 250 FIGS. 64 and 65. Colonization pattern of Type 5 (Endogone-like) mycorrhizae on Douglas-fir as seen i n plan view a f t e r staining procedure. FIG. 64. Type 5 fungus on root t i p from Site IC showing lack of mantle and patchy Hartig net (n) development. x538. FIG. 65. Type 5 mycorrhiza from S i t e 2F with narrow hyphae. X1348. as. FIGS. 66 and 67. Hyphae (h) on root surface becoming invaginated (i) and forming Hartig net of Type 5 (Endogone-like) mycorrhizae on Douglas-fir from Site IC as seen i n plan view. x2150. 2S2. Type 6 FUNGUS: Tuber-like. SOURCE: Douglas-fir on Site 1C. MOST DISTINCTIVE CHARACTERISTICS: Mantle with jigsaw pattern and b r i s t l e - l i k e c y s t i d i a without clamp connection at base. HABIT: Once to i r r e g u l a r l y pinnate branching pattern, brown-orange to brown-yellow and staining dark blue (Figs. 68 and 69) . CYSTIDIA: Absent to common, usually at least a few present. Straight, b r i s t l e - l i k e , a r i s i n g mostly at right angles from outer mantle hyphae, 70-120 (-140) x (2-)4-5(-6) um near the mantle with gradual taper to 1.5 um near the rounded t i p , yellow, staining darker blue than mantle (FIGS. 70-72). EMANATING HYPHAE: None seen. MYCELIAL STRANDS: None seen in Site 1. However, a strand with similar c y s t i d i a was found with a sample from Site 2H but none of these seedlings formed th i s mycorrhizae type. One seedling from Site 2R had t h i s type (FIG. 72). MANTLE: In plan view outer mantle net prosenchymous becoming more tetura epidermoidia and then "jigsaw" pattern with depth. Textura prismatica to angularis in cross section, 15-30 um thick; hyphae 2-10 um wide across septa, smooth, not clamped, yellow, staining dark blue (FIGS. 70, 71, 73, and 74). HARTIG NET: Invaginations 1-3 um wide; not beaded; hyphae yellow, staining l i g h t blue (Fig. 74). OTHER DESCRIPTIONS: Chu-Chou and Grace (1983a,b), and Danielson and Pruden (1989). 253 FIGS. 68-70. Type 6 (Tuber-1ike) mycorrhizae on Douglas-fir from S i t e 1C. FIG. 68. Habit. x34. FIG. 69. Habit compared to nonmycorrhizal (*) t i p (after staining procedure). x52. FIG. 70. Mantle as seen i n plan view following s t a i n i n g procedure showing c y s t i d i a (arrows) and net prosenchymous outer mantle (m) pattern. x2150. 3LSH FIGS. 71 and 72. Type 6 (Tuber-like) mycorrhiza. FIG. 71. Douglas-fir mycorrhiza from Site 1C with cystidium emanating from mantle (m) surface showing two t h i n septa (arrows) near base. X 5 3 7 5 . FIG. 72. Rhizomorph found amoungst Douglas-fir roots from S i t e 2H as seen aft e r staining procedure showing c y s t i d i a (arrows) that are very s i m i l a r to those of Type 6 mycorrhizae. X1348. 3 55 74 FIGS. 73 and 74. Type 6 (Tuber-like) mycorrhizae on Douglas-f i r from S i t e IC. x5375. FIG. 73. Mid to outer mantle as seen i n plan view with textura epidermoidia or "jigsaw" pattern. FIG. 74. Cross-section showing c o r t i c a l c e l l s (c) , Hartig net (n), and mantle (m). a s6 Minor types Note: Descriptions were based on whole mounts of t i p s i n Hoyer's medium after staining procedure unless otherwise indicated. Number in parentheses refer to sample number (found i n Appendix 4) in which the type was f i r s t encountered and described (p =• plug roots and n = new roots). See Fig. 25 for the occurrence of each type on other s i t e s . Type 10 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 1C (# 414n). MOST DISTINCTIVE CHARACTERISTIC: Mantle texture and brown pigment. HABIT: Branching once pinnate, smooth mantle texture, l i g h t blue-staining but with brown areas where mantle i s thick. CYSTIDIA: Absent. EMANATING HYPHAE: Absent. MYCELIAL STRANDS: Absent. MANTLE: Textura angularis to prismatica or epidermoidia with depth; outer hyphae 10-25(-30) x 4-17 um wide (at septa); outer hyphae brown after staining procedure while inner hyphae smaller and staining l i g h t blue (FIGS. 7 5 and 76). HARTIG NET: Not invaginated; not beaded; hyphae 2-6 um wide, walls not staining but dark blue-staining deposits within some c e l l s (FIG. 75). NOTE: Type 10 was mistaken for Type 4 at low magnification and would be Type 43 i f not for pigmented hyphae. 257 t 75 76 FIGS. 75 and 76. Type 10 mycorrhizae on Douglas-fir from Site 1C as seen i n plan view a f t e r staining procedure. FIG. 75. Pigmented outer mantle (m) hyphae and hyaline inner mantle hyphae and Hartig net (n) showing dark blue s t a i n i n g deposits (arrows). X 1 3 4 8 . FIG. 76. Outer mantle hyphae. x5375. 2 SB Type l l FUNGUS: Unknown. SOURCE: Douglas-fir on Site 1C (# 164). MOST DISTINCTIVE CHARACTERISTIC: Dark brown emanating hyphae with clamps. HABIT: Mature mycorrhizae dark brown to black; 1.5 mm x 1 mm, unbranched (Fig. 77). CYSTIDIA: Absent. EMANATING HYPHAE: Common, 4-5 um wide, clamps on most septa, smooth, frequently branched, l i g h t to dark brown. (Fig. 78). MYCELIAL STRANDS: Absent. MANTLE: Irregular synenchymous to textura angularis; hyphae (5-)7-9 um wide with swellings 9-14 um wide, dark brown (Fig. 78) . HARTIG NET: Invaginations 2-5 um wide; not beaded; around c o r t i c a l c e l l s only (Fig. 79). NOTE: Type 11 found only on June sampled seedlings from Site 1U which were not stained. Therefore, i t i s not certain i f pigment would remain. Abundant, nonpigmented hyphae 2-3 um wide that were similar to Type 17 appear to be associated with t h i s type and Type 17 ti p s were also found on the same sample. 259 FIGS. 77-79. Type 11 mycorrhizae found on Douglas-fir from Site II. FIG. 77. Habit. x86. FIG. 78. Plan view of outer mantle and emanating hyphae showing clamp connections (arrows). X 5 3 7 5 . FIG. 79. Plan view of noninvaginated hyphae around epidermal c e l l s . x5375. 260 Type 12 FUNGUS: Unknown. SOURCE: Douglas- f ir on Site 6. MOST DISTINCTIVE CHARACTERISTIC: Emanating hyphae. HABIT: Grey with brown-black emanating hyphae. Staining blue but emanating hyphae remaining pigmented. CYSTIDIA: Absent. EMANATING HYPHAE: Few to abundant but not causing t ips to appear a l l black, smooth, not clamped, growing ends sometimes seen; 1.5-2 um wide and sometimes swell ing up to 6 um wide near mantle, black, becoming l i g h t brown after s ta in ing procedure (Figs. 80 and 82). MYCELIAL STRANDS: Rhizomorphs black-brown, < 2 0 hyphae. Core hyphae 2-5 um wide, not clamped, smooth. Outer hyphae l i k e emanating hyphae (Fig . 81). MANTLE: Net prosenchymous, compact, 2 5-50 um th ick; hyphae smooth and not clamped. Outer hyphae 2-3 um wide, turning l i g h t brown to blue after s ta ining procedure. Inner hyphae hyal ine , up to 7.5 um wide with swellings to 15 um wide, s ta in ing with Trypan blue (Figs. 80 and 82). HARTIG NET: Invaginations 2-4 um wide; c e l l separation 1-2 hyphae wide; nonpigmented (Fig . 82). 261 FIGS. 80-82. Type 12 mycorrhizae on Douglas-fir from S i t e 6. x5375. FIG. 80. Plan view of mantle (m) surface showing emanating hyphae (arrows). FIG. 81. Rhizomorph showing outer hyphae (arrows) l i k e emanating hyphae. FIG. 82. Cross-section showing c o r t i c a l c e l l s (c) , Hartig net (n) , mantle (m) , and emanating hyphae (arrows). 1 £ \ Type 13 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 2F (# 351n). MOST DISTINCTIVE CHARACTERISTIC: Cystidia producing spore-like structures from t i p s . HABIT: Branching two-dimensionally, once pinnate and closely packed. Mantle rough textured and staining dark blue. CYSTIDIA: Common to very abundant, 25-45 x 1-4.5 um with some forming 1 to 3 round spore-like structures at t i p that were mostly 2 um round and appear to be produced from within the cystidium (Figs. 83 and 84). EMANATING HYPHAE: Common, mostly < 50 um long due to breakage, (1.5-)2-3 um wide, smooth, not clamped, frequently branched, blue-staining and ribbon-like. MYCELIAL STRANDS: Strand 3 0 um wide with hyphae as above. MANTLE: Felt prosenchymous becoming textura epidermoidia near epidermis in plan view; textura i n t r i c a t a and globosa i n edge view; moderately compact, 30-80 um thick; hyphae without clamps, smooth, forming 3-way intersections, staining dark blue (Fig. 85). HARTIG NET: Invaginations 0.5-4 um wide; beaded; c e l l separation one hypha wide; blue-staining (Fig. 86). NOTE: Cystidia had a close resemblence to Russula aerugina Lindblad: Fr. on spruce described by Taylor and Alexander (1989). 263 FIGS. 83 and 84. Cystidia of Type 13 mycorrhizae on Douglas-f i r from Site 2F with most showing spore-like (arrows) structures (as seen i n edge view a f t e r s t a i n i n g procedure using phase contrast illumination). X 5 3 7 5 . FIG. 83. Cystidia with spore and stub (s) where another may form. FIG. 84. Cystidium without spore (*) . FIGS. 85 and 86. Type 13 mycorrhizae on Douglas-fir from Site 2F as seen i n plan view after staining procedure. FIG. 85. Inner mantle hyphae over epidermis. x5375. FIG. 86. Hartig net showing regular beading (arrows) of hyphae viewed i n cross-section between c e l l s and invaginations (i) of hyphae seen i n plan view. x2150. 265 Type ;L4 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 4 (# 287p), western hemlock on Site 7H (# 257). MOST DISTINCTIVE CHARACTERISTIC: Cystidia. HABIT: After staining, mantle texture rough to cottony. CYSTIDIA: Few to common 35-50(-70) um x 2-3 um wide, some swollen to 5 um wide near center, staining blue (Fig. 87). EMANATING HYPHAE: Common to highly abundant, 2-3 um wide, smooth, not clamped, frequent branching, blue-staining. MYCELIAL STRANDS: Absent. MANTLE: Fel t prosenchymous, < 10 um thick, loose to compact; hyphae l i k e emanating hyphae but more frequently branched and up to 4 um wide, forming 3- or 4-way intersections (Figs. 87-88) . HARTIG NET: Invaginations 1-4 um wide; l i g h t blue-staining; irregular beading. 266 FIGS. 87 and 88. Type 14 mycorrhizae after staining procedure on Douglas-fir from Site 4. FIG. 87. Mantle (m) as seen in edge view showing cystidia (arrows). X 2 1 5 0 . FIG. 88. Felt prosenchymous mantle pattern as seen in plan view. X 5 3 7 5 . 2 6 7 Type 15 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 2H (# 334n). MOST DISTINCTIVE CHARACTERISTIC: Long, thin c y s t i d i a without clamp connection or kink. HABIT: Smooth mantle texture. CYSTIDIA: Common, 41-105 x 0.5-1 um along at least half the length, base 1-3 um wide, with gentle curves, kinks absent, clamps absent, single septum at base, smooth, blue-staining. EMANATING HYPHAE: Absent. MYCELIAL STRANDS: Absent. MANTLE: Textura epidermoidia in plan view and globosa to angularis in plan view, 3 0 um thick, very compact; hyphae 5-9 um wide, not clamped, blue-staining, 3-way intersections v i s i b l e in some areas. HARTIG NET: Invaginations 1-3 um wide; beaded; staining l i g h t blue. NOTE: Mantle may be mistaken for Type 45. Type 16 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 449p). MOST DISTINCTIVE CHARACTERISTIC: Very narrow emanating hyphae with clamps. HABIT: Mantle texture cottony. CYSTIDIA: Absent. 268 EMANATING HYPHAE: Usually abundant, smooth, clamps on most septa, 1-1.5 um wide, frequent branching, blue-staining. MYCELIAL STRANDS: Absent. MANTLE: Usually absent or < 5 um thick, net prosenchymous, hyphae as above. HARTIG NET: Invaginations 1-2 um wide; irregular beading; blue-staining. Type 17 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 464n). MOST DISTINCTIVE CHARACTERISTICS: Abundant emanating hyphae with clamps and lack of true mantle. HABIT: Mantle texture cottony. CYSTIDIA: Absent. EMANATING HYPHAE: Abundant, smooth, blue-staining, clamps on every septum, 2-3 um wide, branching frequent. MYCELIAL STRANDS: Usually lost in staining procedure, of about 25 hyphae, 50 um wide, hyphae as above. MANTLE: Of abundant emanating hyphae, 2-4 um wide. HARTIG NET: Invaginations 1-2 um wide; beaded; clamps on noninvaginated hyphae; l i g h t blue-staining. Type 18 FUNGUS: Unknown. SOURCE: Douglas-fir3290C nursery (# 90). 269 MOST DISTINCTIVE CHARACTERISTIC: Keyhole clamps on most septa of emanating hyphae. HABIT: Wiry mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Abundant, smooth, keyhole clamps on most septa, swelling near septa, 2-4 um wide, blue-staining. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, loose, 3-6 um thick; hyphae 2-6 um wide, wider near septa, swellings to 10 um wide, clamps common, forming 3-way intersections, walls staining l i g h t blue, septa staining darker. HARTIG NET: Invaginations 1-3 um wide; not beaded; hyphae staining blue. Type 19 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 449n). MOST DISTINCTIVE CHARACTERISTIC: Grey-blue wavy synenchymous mantle after staining. HABIT: Smooth mantle texture, may be glued to s o i l p a r t i c l e s . CYSTIDIA: Absent. EMANATING HYPHAE: Few to abundant, smooth, poorly stained, ribbon-like, clamps on a l l septa, short celled, < 100 (due to breakage) x (l-)2-3(-4) um. MYCELIAL STRANDS: Absent. MANTLE: Synenchymous, appearing wavy, very compact, < 10 um thick; individual hyphae not seen, grey-blue-staining. 270 HARTIG NET: Not v i s i b l e in whole mounts. Type 2 0 FUNGUS: Unknown. SOURCE: Douglas-fir on Site II (# 418n). MOST DISTINCTIVE CHARACTERISTIC: Large textura angularis outer mantle hyphae. HABIT: Rough mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Low abundance, < 100 (due to breakage) x 3-5(-7) um, smooth, clamps rare, frequent branching, staining l i g h t blue. MYCELIAL STRANDS: Strands of < 20 hyphae with clamps and septation more frequent than in above hyphae. MANTLE: Outer mantle textura angularis, very compact, 10-2 0 um thick; hyphae without clamps or intersections, 10-25 um wide, blue-staining. Inner mantle net prosenchymous, very compact, < 10 um thick; hyphae not clamped, 3-5 um wide, forming 3-way intersections, hyphal fusions common, blue-staining. HARTIG NET: Invaginations 1-3 um wide; beaded; staining l i g h t blue. Type 21 FUNGUS: Unknown. SOURCE: Douglas-fir on Site II (# 420p). MOST DISTINCTIVE CHARACTERISTIC: Mantle with clamped hyphae forming abundant 3-way intersections. 271 HABIT: Smooth mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Low abundance 2-4 (-5) um wide, smooth, clamps on some septa, blue-staining. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, very compact, about 10 um thick; hyphae 2-5 um wide with swellings to 9 um wide, septa 10-20(-40) um apart and mostly clamped, forming abundant 3-way intersections, staining l i g h t blue. HARTIG NET: Invaginations 1-2 um wide; not beaded; blue-staining. Type 22 FUNGUS: Unknown. SOURCE: Douglas-fir on Site II (# 417n). MOST DISTINCTIVE CHARACTERISTICS: Mycorrhizal t i p s staining dark blue with clamps present on emanating hyphae only. HABIT: Smooth mantle texture, dark blue-staining. CYSTIDIA: Absent. EMANATING HYPHAE: Common, (1.5-)2-3 um wide smooth, frequent branching, clamps on most septa, staining dark blue. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous; very compact, 5-2 0 um thick, hyphae staining dark blue, 2-3(-4) um wide, not clamped, septa and branching frequent, forming abundant 3-way intersections. HARTIG NET: Invaginations 1-2 um wide; beaded; blue-staining. 272 Type 2 3 FUNGUS: Unknown. SOURCE: Western hemlock on Site 7H (# 265n). MOST DISTINCTIVE CHARACTERISTIC: Width of emanating hyphae variable along short lengths. HABIT: Found only on western hemlock. Wiry to cottony mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Abundant, clamps on some septa, < 100 um (due to breakage) x 1.5-8 um wide, width varying along short distances, branching frequent and angular, staining blue and not ribbon-like (Fig. 89). MYCELIAL STRANDS: Absent. MANTLE:. Net prosenchymous in plan view and textura globosa in plan view, loose to compact, 5-40 um thick; hyphae 2-5 um wide, clamped, branching to form 3- to multi-way intersections (Figs. 89 and 90). HARTIG NET: Invaginations 1-3 um wide; several hyphae between c e l l s forming a multi-beaded appearance. 273 FIGS. 89 and 90. Type 23 mycorrhizae on western hemlock from Site 7H after staining procedure. FIG. 89. Edge view showing mantle (m) and emanating hyphae (h). x2150. FIG. 90. Plan view of net prosenchymous mantle showing clamp connections (arrows). X 5 3 7 5 . 274 Type 2 4 FUNGUS: Laccaria laccata (Scop.: Fr.) Berk. & Br. SOURCE: Douglas-fir in MacBean Nursery. MOST DISTINCTIVE CHARACTERISTICS: Hyaline hyphae and brown mushrooms in nursery (Fig. 91). HABIT: Unbranched with smooth white mantle and white rhizomorphs becoming transparent i f bruised or moistened. CYSTIDIA: Absent. EMANATING HYPHAE: Uncommon, clamps on a l l septa, hyaline, thin walled, smooth 2-4(-5) um wide. MYCELIAL STRANDS: Strands of loosely arranged smooth hyphae, white, becoming transparent when bruised MANTLE: Net prosenchymous, compact; hyphae 2-4(-6) um wide, thin walled, hyaline, staining poorly with Trypan Blue. HARTIG NET: Invaginations 1-4 um wide; not beaded; not staining with Trypan Blue. OTHER DESCRIPTIONS: Chu-Chou and Grace (1983a, b). NOTES: Not found on sampled seedlings. Identified using sporocarp characteristics and tracing the rhizomorphs ar i s i n g from a mushroom to a mycorrhizae. 275 FIG. 91. Douglas-fir seedling from MacBean Nursery with mushroom and Type 24 mycorrhizae formed by Laccaria laccata. Type 2 5 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 3A (# 322n). MOST DISTINCTIVE CHARACTERISTICS: Emanating hyphae clamped, highly abundant, and very frequently branched. HABIT: Cottony mantle texture. CYSTIDIA: Absent. 276 EMANATING HYPHAE: Very abundant, (1.5-)2-3 um wide, smooth, very frequent branching, ribbon-like, blue-staining; septa < 50 um apart a l l with clamps mostly closed. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous to textura epidermoidia with depth, loose to moderately compact, < 10 um thick (not including the loose outer hyphae); hyphae 2-5 um wide (at septa), clamps very common, blue-staining. HARTIG NET: Invaginations 2-3 um wide; staining l i g h t blue; beading absent to irregular. Type 2 6 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 3A (# 322n). MOST DISTINCTIVE CHARACTERISTICS: Clamps on larger emanating hyphae and in mantle. HABIT: Cottony mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Abundance low to high, 1-4 um wide, smooth, staining l i g h t blue, clamps on some septa of larger hyphae. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, very compact, < 10 um thick; hyphae 2- 4(-5) um wide, staining l i g h t blue, clamps common, forming 3- way intersections. HARTIG NET: Invaginations 1-2 um wide; not beaded; poorly stained. 277 Type 27 FUNGUS: Unknown. SOURCE: Douglas-fir on Site IC (# 411n). MOST DISTINCTIVE CHARACTERISTIC: Medium width hyphae with clamps, emanating from a compact, irregular net prosenchymous mantle. HABIT: Rough mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Common, 3.5-4 um wide, smooth, blue-staining, closed clamps on most septa, frequent branching. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, very compact, irregular (pattern variable along tip) about 10 um thick; hyphae 3-6 um wide, clamps present but d i f f i c u l t to see, forming 3-way intersections, blue-staining. HARTIG NET: Invaginations 1-3 um wide; not beaded; staining dark blue. Type 2 8 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 3B (# 210p), western hemlock on Site 7H (# 262n). MOST DISTINCTIVE CHARACTERISTIC: Abundant emanating hyphae 1.5-4 um wide with elongated clamps on some septa. HABIT: Cottony mantle texture. CYSTIDIA: Absent. 278 EMANATING HYPHAE: Very abundant and long, 1.5-4 um wide, smooth, clamps on some septa, some clamps elongated (not hemispherical), branching frequent, blue-staining, some hyphae ribbon-like. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, moderately compact, 10-2 0 um thick; hyphae 2-5 um wide, septa 5-2 0 um apart, some septa clamped, forming 3-way intersections, blue-staining. HARTIG NET: Invaginations 0.5-3 um wide; not beaded; blue-staining. Type 2 9 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 451p). MOST DISTINCTIVE CHARACTERISTICS: Very narrow emanating hyphae lacking clamps, with thin mantle of similar hyphae. HABIT: Cottony mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Common to very abundant, long, smooth, not clamped, 1-1.5 (-2) um wide, infrequently branched, staining l i g h t blue. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, loose, < 5 um thick, hyphae as above. HARTIG NET: Invaginations < 1 um wide; narrow beading; blue-staining. 279 Type 3 0 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 452p). MOST DISTINCTIVE CHARACTERISTIC: Mantle textura epidermoidia. HABIT: Mantle smooth. CYSTIDIA: Absent. EMANATING HYPHAE: Few, smooth, blue-staining, some ribbon-l i k e , not clamped, 1-2 um wide, branching infrequent. MYCELIAL STRANDS: Absent. MANTLE: Textura epidermoidia in plan view, 25 um thick; hyphae blue-staining, not clamped, 3-6 um wide, septa hard to see. HARTIG NET: Invaginations 1-3 um wide; beaded; hyphae l i g h t blue. Type 31 FUNGUS: Unknown SOURCE: Douglas-fir on Site 4 (# 276p) MOST DISTINCTIVE CHARACTERISTICS: Abundant, narrow, emanating hyphae and mantle with narrow 3-way intersections. HABIT: Cottony mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Very abundant, smooth, not clamped, c e l l s short, 1.5-2 um wide, blue-staining. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, moderately compact, 5-2 5 um thick; hyphae 1-1.5 um thick some widening to 3 um near the 280 epidermis, blue-staining, septation and branching frequent, forming abundant 3-way intersections, clamps absent. HARTIG NET: Invaginations 1-2 um wide; beaded; not staining with Trypan blue. Type 32 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 3A (# 323p). MOST DISTINCTIVE CHARACTERISTICS: Lack of mantle, root hair and i n t r a c e l l u l a r penetration often seen. HABIT: Appearing nonmycorrhizal and root hairs were usually present. CYSTIDIA: Absent. EMANATING HYPHAE: Low abundance, < 100 (due to breakage) x 2-3 um, smooth, staining blue, not clamped, infrequent branching. MYCELIAL STRANDS: Absent. MANTLE: Absent. HARTIG NET: Invaginations 1-2 um wide; not beaded; blue-staining; hyphae occasionally i n t r a c e l l u l a r including root hairs. Type 33 FUNGUS: Unknown SOURCE: Douglas-fir on Site 6 (# 471). MOST DISTINCTIVE CHARACTERISTICS: Red-brown exudates from emanating hyphae and the lack of cy s t i d i a . HABIT: White to grey becoming reddish-brown with age due to 281 exudates from emanating hyphae and rhizomorphs. Usually with sand grains incorporated into mantle or t i p s firmly attached to small rocks. Crowded, three-dimensional once or twice pinnate branching (Fig. 92). CYSTIDIA: Absent. EMANATING HYPHAE: Abundant, 1.5-3 um wide, hyaline. Exudates reddish-brown, globular, up to 11 um wide and c l e a r l y v i s i b l e at 32x magnification (Figs. 94 and 95). MYCELIAL STRANDS: Rhizomorphs 70-100 um wide. Outer hyphae as above, abundance low so that core hyphae were ea s i l y seen in whole mounts. Central core hypha 6-21 um wide and aseptate. Surrounding hyphae 2-4 um wide, smooth, and with regular septation (Figs. 46, 92, and 93) MANTLE: Net prosenchymous in plan view. In cross section, inner mantle textura porrecta and outer mantle textura globosa; compact, 10-85 um thick. Hyphae not clamped, 2-4 um wide with swellings to 8 um wide, hyaline, thin walled, and poorly staining with Trypan blue (Figs. 96 and 97). HARTIG NET: Invaginations 2-3 um wide; not beaded; c e l l separation one hyphae wide (Fig. 97). COLOR REACTIONS: KOH (15%) caused whitish hyphae to turn pink then dark red. Concentrated NH4OH turned these whitish hyphae dark purple. 282 FIGS. 92 and 93. Type 33 mycorrhizae on Douglas-fir from Site 6. FIG. 92. Habit showing red-brown emanating hyphae (h) , rhizomorphs (arrows), and the incorporation of sand grains (s) . x l38. FIG. 93. Rhizomorph showing large core hypha (h) . X5375. 283 FIGS. 94 and 9 5 . Emanating hyphae of Type 3 3 mycorrhizae on Douglas-fir from Site 6 . x 5 3 7 5 . FIG. 9 4 . Hyphae with red-brown globular exudates (arrows). FIG. 9 5 . Hyphae after staining procedure showing agglutination of exudates (e). 2 8 4 FIGS. 96 and 97. Type 33 mycorrhizae on Douglas-fir from Site 6. FIG. 96. F e l t prosenchymous mantle as seen i n plan view. FIG. 97. Cross-section showing c o r t i c a l c e l l s (c) , Hartig net (n), tannin f i l l e d epidermal (e) c e l l s , and mantle (m). x5375. 28S Type 3 4 FUNGUS: Unknown. SOURCE: Western hemlock on Site 7H (# 264p). MOST DISTINCTIVE CHARACTERISTICS: Fel t prosenchymous mantle and emanating hyphae combination. HABIT: Found only on western hemlock. Mantle texture smooth to velvety. CYSTIDIA: Absent. EMANATING HYPHAE: Few to very abundant, smooth, not clamped, (1.5-)2-3 um wide, mostly not ribbon-like, blue-staining. MYCELIAL STRANDS: Strands of 50-200 hyphae as above. MANTLE: Felt prosenchymous, moderately to very compact, 15-2 0 um thick; hyphae (1.5-)2-3(-4.5) um wide, not clamped, blue-staining. HARTIG NET: Invaginations 0.5-2 um wide; not beaded; staining l i g h t blue and d i f f i c u l t to see in plan view. Type 35 FUNGUS: Unknown. SOURCE: Douglas-fir on Site II (# 423n). MOST DISTINCTIVE CHARACTERISTICS: Mycorrhizal t i p s darkly stained with undamped, closely-septate hyphae forming a mantle of 3-way intersections. HABIT: Tips staining dark blue in Trypan Blue and folding i n Hoyer's medium. Mantle texture smooth. CYSTIDIA: Absent. 286 EMANATING HYPHAE: Low abundance, < 200 (due to breakage) x 2-3 um, smooth, staining l i g h t blue, septa often > 50 um apart and not clamped, branching common. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, moderately to very compact, about 15 um thick; hyphae 2-3(-5) um wide, staining dark blue, septa 3-20 um apart and not clamped, forming abundant 3-way intersections. HARTIG NET: Invaginations uncommon; mostly l i k e mantle hyphae; beaded; blue-staining. Type 3 6 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 444). MOST DISTINCTIVE CHARACTERISTIC: Mantle very thick. HABIT: Yellow, staining blue, mantle texture rough. CYSTIDIA: Absent. EMANATING HYPHAE: Common, 1.5-3 um wide, not clamped, smooth, very frequently branched, blue-staining. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous in plan view, textura i n t r i c a t a in edge view, and textura globosa in cross section, loose to compact, 20-80(-150) um thick; hyphae 1-3 um wide, not clamped, smooth, septa mostly > 20 um apart, forming 3-way intersections, blue-staining. HARTIG NET: Invaginations 1-3 um wide; not beaded; c e l l separation 1-3 hyphae wide; blue-staining. 287 Type 3 7 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 3B (# 219p). MOST DISTINCTIVE CHARACTERISTICS: Mantle and emanating hyphae combination. HABIT: Smooth mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Low abundance, 1.5-3 um wide, not clamped, smooth, frequently branched, staining l i g h t blue. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous to textura epidermoidia in plan view and textura i n t r i c a t a in edge view, highly compact, 10-20 um thick; hyphae 2-4 um wide, not clamped, septa often > 20 um apart, 3- or 4-way intersections, staining l i g h t blue. HARTIG NET: Invaginations 1-3 um wide; not beaded; blue-staining . Type 38 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 3A (# 307n). MOST DISTINCTIVE CHARACTERISTICS: Lack of mantle and abundant, ribbon-like, emanating hyphae. HABIT: Cottony mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: High abundance, 2-4 um wide, smooth, not clamped, staining l i g h t blue, ribbon-like, frequent branching. MYCELIAL STRANDS: Absent. 288 MANTLE: Absent. HARTIG NET: Invaginations (l-)2-3 um wide; not beaded; stain intensity variable. Type 39 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 3B (# 222) . MOST DISTINCTIVE CHARACTERISTICS: Mantle and emanating hyphae combination. HABIT: Mantle texure rough. CYSTIDIA: Absent. EMANATING HYPHAE: Abundance high, most < 100 (due to breakage) x 2-4 um, not clamped, smooth, highly branched, staining l i g h t blue. MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous, moderately compact, about 10 um thick; hyphae 2-5 um wide, not clamped, forming 3-way intersections, blue-staining. HARTIG NET: Invaginations 1-3 um wide; not beaded; blue-staining . Type 4 0 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 448) . MOST DISTINCTIVE CHARACTERISTICS: Mantle net prosenchymous with multi-way intersections. HABIT: Cottony mantle texture. 289 CYSTIDIA: Absent. EMANATING HYPHAE: Abundant, smooth, not clamped, 2-4 um wide, frequently branched, blue-staining. .MYCELIAL STRANDS: Absent. MANTLE: Net prosenchymous; hyphae as above with frequent branching forming 3- or 4-way swollen intersections. Some large c e l l s with several hyphae attached forming multi-way intersections. HARTIG NET: Invaginations 1-3 um wide; beading in some areas; l i g h t blue-staining. Type 4 1 FUNGUS: Unknown. SOURCE: Douglas-fir on Site 5 (# 445n). MOST DISTINCTIVE CHARACTERISTICS: Gelatinized mantle hyphae and wide emanating hyphae with blue-staining encrustations. HABIT: Smooth mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Uncommon, blue-staining, not clamped, 5-7 um, walls encrusted. MYCELIAL STRANDS: Absent. MANTLE: Synenchymous; hyphae appear glued together with blue-staining substance. HARTIG NET: Invaginations 3-5 um wide; blue staining; possible exudates. 290 Type 42 FUNGUS: Unknown. SOURCE: Douglas-fir on Site II (# 434n). MOST DISTINCTIVE CHARACTERISTICS: Wide Hartig net hyphae without invaginations and compact mantle in which intersections were s t i l l apparent. HABIT: Rough mantle texture, staining l i g h t blue. CYSTIDIA: Absent. EMANATING HYPHAE: Low abundance, 2-6 um wide, smooth, not clamped, staining l i g h t blue. MYCELIAL STRANDS: Absent. MANTLE: Textura epidermoidia to "jigsaw", moderately compact, < 20 um thick; hyphae staining l i g h t blue, 3-9 um wide (across septa), septa < 20 um apart and not clamped, forming mostly 3-way intersections. HARTIG NET: Hyphae up to 8 um wide (across septa) and resulting in c e l l separations of up to 6 um. Type 43 FUNGUS: Unknown. SOURCE: Various Douglas-fir and western hemlock. MOST DISTINCTIVE CHARACTERISTIC: Closely septate wide blue-staining hyphae. HABIT: May appear nonmycorrhizal without staining;.unbranched, mantle thin, color of root. CYSTIDIA: Absent. 291 EMANATING HYPHAE: Low to medium abundance, (3-)4-7(-11) um wide, most compartments < 20 um long, not clamped, simple sometimes wavy septa that usually s ta in better than wal ls . MYCELIAL STRANDS: Absent. MANTLE: F e l t to net prosenchymous, < 10 um th ick; hyphae (4-) 5-10(-13) um wide and l i k e emanating hyphae. HARTIG NET: Invaginations in parts of some t i p s . Other t ips with noninvaginated hyphae in cortex which were s imi lar in s ize as those i n mantle. Not beaded; s ta in ing l i g h t blue. NOTE: In some cases, p a r t i c u l a r l y on Si te 1, Type 43 mycorrhizae were suspected of being immature Type 6. Type 44 FUNGUS: Unknown. SOURCE: Douglas-f ir on Site 3A (# 307p). MOST DISTINCTIVE CHARACTERISTIC: Darkly stained Hart ig net with beading v i s i b l e at lOOx magnification. HABIT: Smooth mantle texture. CYSTIDIA: Absent. EMANATING HYPHAE: Low abundance, < 50 (due to breakage) x 4-9 um, smooth, b lue-s ta in ing , not clamped. MYCELIAL STRANDS: Absent. MANTLE: Present or absent. Net prosenchymous to textura epidermoidia with depth, 0-30 um th ick; hyphae 3-6(-9) um wide, some septa appearing complex, not clamped, blue-s ta in ing , branching very i r r e g u l a r , hyphal fusions common. 292 HARTIG NET: Invaginations (2-)3-7 um wide causing c e l l separation of 2-4 um; beaded; b l u e - s t a i n i n g . Type 4 5 FUNGUS: Unknown. SOURCE: Dou g l a s - f i r on S i t e 5 (# 454n). MOST DISTINCTIVE CHARACTERISTIC: Mantle and emanating hyphae combination. HABIT: Mantle texture rough, t i p s long and unbranched. CYSTIDIA: Absent. EMANATING HYPHAE: Low abundance, short due to breakage, 4-5 um wide, smooth, not clamped, no branching seen, s t a i n i n g l i g h t blue. MYCELIAL STRANDS: Absent. MANTLE: F e l t prosenchymous, compact, 2 0-3 0 um t h i c k ; hyphae (2-)3-4 um wide, not clamped, s t a i n i n g l i g h t blue with septa s l i g h t l y darker, no i n t e r s e c t i o n s . HARTIG NET: Invaginations 1-3 um; not beaded; s t a i n i n g l i g h t blue. Type 4 6 FUNGUS: Unknown. SOURCE: Dou g l a s - f i r on S i t e I I (# 427n). MOST DISTINCTIVE CHARACTERISTIC: H a r t i g net, wide mantle hyphae, and emanating hyphae combination. HABIT: Rough mantle texture, s t a i n i n g blue. CYSTIDIA: Absent. 293 EMANATING HYPHAE: A b u n d a n c e l o w , 2-5 um w i d e , s m o o t h , n o t c l a m p e d , i n f r e q u e n t l y b r a n c h e d , s t a i n i n g l i g h t b l u e . M Y C E L I A L STRANDS: S t r a n d s o f a b o u t 2 0 h y p h a e ; h y p h a e 3 -5 um w i d e . M A N T L E : F e l t t o n e t p r o s e n c h y m o u s , m o d e r a t e l y c o m p a c t , 2 0-4 0 um t h i c k ; h y p h a e 3-9 um w i d e , s e p t a 7-40 um a p a r t , n o t c l a m p e d , b l u e - s t a i n i n g , b r a n c h i n g i n f r e q u e n t l y t o f o r m 3 - o r 4-way i n t e r s e c t i o n s . HARTIG N E T : I n v a g i n a t i o n s 1-2 um w i d e ; n o t b e a d e d ; b l u e -s t a i n i n g . Type 47 FUNGUS: Unknown. SOURCE: D o u g l a s - f i r on S i t e 1C (# 4 1 0 n ) . MOST D I S T I N C T I V E C H A R A C T E R I S T I C : H a r t i g n e t , w i d e m a n t l e h y p h a e , a n d e m a n a t i n g h y p h a e c o m b i n a t i o n . H A B I T : Smooth t o c o t t o n y m a n t l e t e x t u r e . C Y S T I D I A : A b s e n t . EMANATING HYPHAE: A b u n d a n c e low t o common, 2-6 um w i d e , s m o o t h , n o t c l a m p e d , b r a n c h i n g i n f r e q u e n t , l i g h t b l u e -s t a i n i n g , s o m e t i m e s r i b b o n - l i k e . M Y C E L I A L STRANDS: A b s e n t . M A N T L E : N e t p r o s e n c h y m o u s , v e r y c o m p a c t , a b o u t 2 0 um t h i c k ; h y p h a e 4 . 5 - 9 um w i d e ( a t s e p t a ) , b l u e - s t a i n i n g , n o t c l a m p e d , b r a n c h i n g f r e q u e n t l y t o f o r m 3-way i n t e r s e c t i o n s . HARTIG N E T : N o t v i s i b l e t h r o u g h m a n t l e . I n v a g i n a t i o n s a b s e n t , h y p h a e 2-4 um w i d e , s t a i n i n g l i g h t b l u e ; n o t b e a d e d . 294 APPENDIX 6 Glossary of technical terms Beading: Refers to Hartig net seen i n whole mount when focusing on cross-sections of hyphae between c e l l s . If round in cross-section the hyphae appear as a beaded st r i n g (Fig. 86). Irregular beading i s when the hyphae are more rectangular i n cros's-section. Not beaded means the hyphae are collapsed or flattened between the c e l l s . Clamp: A semispherical outgrowth from one compartment to an adjacent compartment on the same hypha (Fig. 78). Compartment: One " c e l l " of a hypha deliniated by two septa. Cross-section: A thin s l i c e (made by making two transverse cuts) of a mycorrhizal t i p seen under high magnification. Also refers to the appearance of Hartig net when focused on hyphae as they would appear i f a cross-section i s made. Cystidia: Emanating hyphae or outer rhizomorph hyphae of d i s t i n c t i v e shape and length. B r i s t l e - l i k e c y s t i d i a are generally straight and gradually tapper to a point (Fig. 70). Edge view: The appearance of the end or side of a whole mount of a mycorrhizal t i p (Fig. 87). Emanating hyphae: Hyphae ar i s i n g from the mantle surface. Endophyte: The mycobiont involved in forming vesicular-arbuscular mycorrhizae (Fig. 13). Exudates: Deposits of irregualar shape apparently passed through the hyphal wall and i s not part of i t (Fig. 94). 295 Habit: Appearance of a mycorrhiza under low magnification including color, branching pattern, and mantle texture. Hart ig net: Hyphae that grow in the root cortex and epidermis without entering the c e l l s (Fig. 37). Hyaline: Hyphae that lack dark pigment making i t transparent or nearly so. Hypha: (pl. hyphae) A filament comprised of one or more compartments that are the basic unit of the vegetative fungal growth (Fig 60). Inoculum: Any viable propagule of a mycobiont including hyphae, mycelial strands, s c l e r o t i a , and sexual and asexual spores. Intersect ions: Branching of hyphae in prosenchymous mantles to form a dichotomy or a 3-way intersection (Fig. 90). Branching may also be a trichotomy to form a 4-way intersection or less commonly a polytomy to form a multi-way intersection. Invaginations: Branching of Hartig net hyphae (Fig. 86). Keyhole clamp: A clamp comnnection with a conspicuous a i r space enclosed by the clamp and the main part of the hypha. Mantle: A sheath of compacted hyphae over fine root t i p s . Mycel ia l strands: Bundles of hyphae running together to form tiny cords. Ornamentation: (1) Wall outgrowths that appear as tiny bumps (Fig. 54). (2) Exudates. Plan view: Microscopic examination, usually of whole mounts, perpendicular to the surface and at various depths. 296 Prosenchymous: A moderately compact mantle i n which spaces exist between the hyphal elements making them c l e a r l y distinguishable. Felt prosenchyma consists of infrequently branched hyphae (Fig. 88). Net prosenchyma consists of hyphae that frequently branched at wide angles (Chilvers 1968) to form intersections (Fig. 90). Rhizomorph: A mycelial strand with one or more larger central core hypha(e) (Fig. 93). Ribbon-like hyphae: Thin-walled hyphae that are collapsed and twisted after the staining procedure. Septa: A cross-wall in a hypha usually between two compartments. Synenchymous: A thoroughly compacted mantle with l i t t l e obvious interhyphal space, in which the hyphal basis i s d i f f i c u l t or impossivle to discern. Irregular synenchyma consists of highly branched hyphae with no regular pattern (Fig. 57) or appear as textura "jigsaw" (Fig. 73). Regular synenchyma has hyphal compartments that are about the same size and have f a i r l y straight walls (Fig. 61; Chilvers 1968). Textura: The pattern made by the mantle hyphae as seen in plan view under high magnification. Examples include textura epidermoidia Fig. 35, textura angularis Fig. 76, s t e l l a t e pattern Fig. 61, and "jigsaw" pattern Fig. 73. 297 Texture: The appearence of the mantle under disecting microscore as influenced by the abundance of emanting hyphae. Varied from woolly (Fig. 59) to cottony (Fig. 41) to velvety (Fig. 29) to smooth (Fig. 68) as emanating hyphae decreased in abundance and diameter. Noncompact mantles (comprised of loose hyphae) with few or no emanating hyphae may appear rough instead of smooth. Verr u c u l o s e : Having small rounded processes or xwarts' Fig 54. Whole mount: The process of putting an unsectioned mycorrhizal t i p on a s l i d e under a cover s l i p for viewing under high magnification. 298 

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