UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Inocluation of ectomycorrhizal fungi in the IDFdk2 biogeoclimatic zone of British Columbia : new techniques,… Chapman, William Kenneth 1992

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

Item Metadata


831-ubc_1992_spring_chapman_william_ken.pdf [ 3.91MB ]
JSON: 831-1.0098903.json
JSON-LD: 831-1.0098903-ld.json
RDF/XML (Pretty): 831-1.0098903-rdf.xml
RDF/JSON: 831-1.0098903-rdf.json
Turtle: 831-1.0098903-turtle.txt
N-Triples: 831-1.0098903-rdf-ntriples.txt
Original Record: 831-1.0098903-source.json
Full Text

Full Text

INOCULATION OF ECTOMYCORRHIZAL FUNGI IN THE IDFdk2 BIOGEOCLIMATIC ZONE OF BRITISH COLUMBIA: FUNGI AND OUTPLANTING TRIALS  NEW TECHNIQUES,  By WILLIAM KENNETH CHAPMAN B.Sc.  Ag.,  The University of Alberta,  1978  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF SOIL SCIENCE  We accept this thesis as conforming t  1 L-’- d  n-ri  1 rerni r 4  /  1(1  -  THE UNIVERSITY OF BRITISH COLUMBIA December 1991 Øwiiiiam Kenneth Chapman,  1991  In presenting this thesis in  partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  /?  ABSTRACT  Ectomycorrhizal  fungi were used to alter seedling performance  in a normal reforestation situation. a  defined,  precisely  natural  This work was conducted in  ecological  situation,  so that  future work can be compared to this, and an understanding of the behaviour of ectomycorrhizal fungi, under the conditions defined in  this  study,  specific culture  can  problems of  developed  be  over  time.  In addition,  in applied mycorrhizal research, such as the  fungi,  inoculation in nurseries and examination of  ectomycorrhizae were addressed, and new techniques developed. Fungi  from  near-pure selected  a  variety  culture and  of sources were screened, using a new  synthesis  grown  apparatus.  volume,  to  using  Certain a  new  fungi  were  fungus culture  technique.  Seedlings in a commercial nursery were successfully  inoculated,  using  injection  two  procedures.  One procedure involved the  of mycelial slurry into container plugs and the other  involved application of the slurry to the surface of root plugs. The  performance  fungi  of  the  inoculated  seedlings and mycorrhizal  in  the  nursery  seedlings  was  increased or decreased, depending on the type of  fungus.  Even  tomentosus seedlings. that  was  very  (3.55%)  low  levels  increased  Seedlings  of  of  Shoot  infection  growth  the  lodgepole  growth  of  by  of the  Suillus  lodgepole  pine  pine and Enge].mann spruce  formed mycorrhizae with E—strain (sensu Mikola), Amphinema  byssoides (Fr.) J. Erikss. Snell  evaluated.  &  Dick  were  or Suillus tomentosus (Kauff.) Sing.,  transplanted  reforestation” situation.  to  the field into a “normal  In general, the differences in growth  from the nursery, persisted after one field season. 11  The  behaviour of the inoculated mycorrhizal fungi on egressed roots is described,  including the encroachment of wild fungi, the  growth of the inoculated fungus onto new roots and the behavior of Thelephora terrestris Ehrhart: Fr. low—toxicity dye  (FDA Blue No.  In the field.  A  1), not previously used for  examining ectomycorrhizae, was evaluated and a procedure for the relatively fast and detailed description of ectomycorrhizae is outlined. This  work  outplanting behaviour future sites  suggests  function  and  experiments. be  should  The major tenets of the protocol are that  described  system,  and  should long  outplanting and  of ectomycorrhizae can be collected in  using  a  comprehensive ecological  mycorrhizae on natural seedlings at the  site should be described, the mycorrhizae on seedlings at  outplantlng term  minimum basic protocol for conducting  trials to insure that meaningful information on the  classification study  a  be  described  and monitored over the short  term, a wide variety of fungi should be used in  trials in normal reforestation situations, cultural  inoculation  techniques  need to be improved and growth and  survival need to be monitored over the short and long terms. ectomycorrhizae information,  evaluation  trials  If  do not supply certain basic  it may be a very long time before it is understood  how ectomycorrhizal fungi respond to the type of site conditions found in normal reforestation situations.  iii  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  vii  List of Figures  ix  Acknowledgements  X  1.Introduction  1  2. Objectives  11  3. Literature Review 3.1 Experiment One  13  3.2 Experiment Two  15  3.3 A New Low Toxicity Stain Useful for the Examination of Ectomycorrhizal Fungi 3.3 Experiment Three  20  3.3.1 4. Experiment One:  17  34 In Vitro Growth of Ectomycorrhizal Fungi  On Dilute Agar  37  5. Experiment Two: Two Inoculation Techniques for Vegetative Mycorrhizal Inoculum In a Commercial Nursery. .41 5.lMaterialsandMethods  42  5.2 Results  46  5.3Discussion  50  6. A Low Toxicity Stain for ExaminingEctomycorrhizae  54  7. Experiment Three: The Selection and Evaluation of Ectomycorrhizal Fungi To Enhance Performance of Seedlings Planted tinder Normal Reforestation Conditions. .57  iv  7.1 Materials and Methods  .61  7.1.1  Site Selection and Description  61  7.1.2  Collection of Isolates  65  7.1.3  Isolation Techniques  66  7.1.4  Prescreening of Cultures  69  7.1.5  Near Pure Culture Synthesis  70  7.1.6  Trial Inoculations in the Nursery  74  7.1.7  Preparation of Inoculum for the Nursery  75  7.1.8  Inoculation of Seedlings in the Nursery  78  7.1.9  Evaluation of Nursery Trials  79  7.1.10 Outplanting Trials  83  7.1.11 Examination of Seedlings From the Outplanting Trials  84  7.1.12 Statistical Procedures  85  7.2 Results  87  7.2.1  Pure Culture Synthesis  87  7.2.2  Trial Inoculation in the Nursery  88  7.2.3  Observation of Nursery Seedlings  88  7.2.4  Observations of Seedlings After One Season in the Field  95 Observations on Pine Seedlings  96 Observations on Spruce Seedlings 7.3 Discussion  102 107  7.3.1 Pure Culture Synthesis Technique  107  7.3.2 Nursery Trials  110  7.3.3FieldTrials  114  8. Overall Conclusions  123  V  Literature Cited Appendix I:  132  Fungi Used in Pure Culture Synthesis and  Nursery Screening Trials  146  Appendix II: Descriptions of Mycorrhizae Appendix III: Nursery Fertilizer Regime, Appendix IV:  Summary of Statistics  vi  153 1989  163 164  LIST OF TABLES Table I. Mycelial Mass of Ectomycorrhizal Fungus After One Month of Growth in Liquid or 0.3% Agar  40  Table II. Comparison of Iniection Into the Plug Versus Top Application of Ectomycorrhizal Mycelial Slurry to Two Month Old Engelmann Spruce  47  Table III. Comparison of Injection Into the Plug Versus Top Application of, Ectomycorrhizal Mycelial Slurry to TwoMonthOidLodgepolePine Table IV.  Detailed Description of Outplanting Site  .49 63  Table V. Concentrations of Inoculum Used in the Nursery InoculationTrial  77  Table VI. Observations on Lodgepole Pine Seedlings Grown In the Heffley Reforestation Centre Nursery for 4 Months Following Inoculation With Ectomycorrhizal FungalMyceliumat2Months  89  Table VII. Observations on Engelmann Spruce Seedlings Grown In the Heffley Reforestation Centre Nursery for 4 Months Following Inoculation With Ectomycorrhizal Fungal Mycelium at 2 Months  92  Table VIII. Observations on Lodgepole Pine Seedlings Inoculated With Ectomycorrhlzae in the Nursery and Grown for 5 Months in the Field (F-ield 96  Measurements) Table IX. Observations on Lodgepole Pine Seedlings Inoculated With Ectomycorrhizae in the Nursery and Grown for 5 Months in the Field (Laboratory Measurements )  98  vii  Table X. Volunteer Fungi on Egressed Roots of Lodgepole Pine Grown in the Field For Five Months With 99  Different Mycorrhizal Treatments Table XI. Foliar Nutrient Analyses For Lodgepole Pine Grown in the Field For Five Months With Different  101  Mycorrhizal Treatments Table XII. Observations on Engelmann Spruce Seedlings Inoculated With Ectomycorrhlzae in the Nursery and Grown For 5 Months in the Field  (Field 103  Measurements) Table XIII. Observations on Engelmann Spruce Seedlings Inoculated With Ectomycorrhizae in the Nursery and Grown For 5 Months in the Field  (Laboratory 104  Measurements) Table XIV. Volunteer Fungi on Egressed Roots of Engeimann Spruce Grown in the Field For Five Months With Different Myeorrhizal Treatments  105  Table XV. Foliar Nutrient Analyses for Engelmann Spruce Grown in the Field For Five Months With Different Mycorrhizal Treatments  viii  106  LIST OF FIGURES FIG.  1.  Pure Culture Synthesis Apparatus  139  FIG.  2.  Inoculation in Pure Culture Synthesis  139  FIG.  3.  Dilute Agar Culture  140  FIG.  4.  Dilute Agar Liquid Cultures  140  FIG.  5.  E—strain Hyphae  141  FIG. 6.  E—strain growing From a Broken Root  141  FIG.  7.  E—strain Hartig net  141  FIG.  8.  Type 12 Suilloid Fungus  142  FIG.  9.  Type 10 Suilloid Rhizomorph  142  FIG.  10. Amphlnema byssoldes extramatrical hyphae  143  FIG.  11. Amphinema byssoldes in the field  143  FIG.  12. Type 3 mycorrhiza stained with FDA Blue No  FIG.  13. Type 3 mycorrhiza longitudinal section  144  FIG.  14. Type 8, Lactarlus showing lactifers  145  FIG.  15. Pine roots  145  ix  V  1....144  ACKNOWLEDGEMENTS  To my three dear children, Laticia, Oleh and Roman, who have had to live without so many of the amenities while their dad struggled from fellowship to grant. Thank you to Tim Ballard and Shannon Berch, two of the best teachers I have ever known. Tim Ballard loaned me money from his own pocket when times were bad. I will never forget his kindness and his love of learning. He is an inspiration to all teachers, and I hope he is not lost in the swollen maw of administration. Gary Hunt helped in many ways, from all manner of technical advice to arranging funding from Balco Canf or Reforestation Centre Ltd. (now the Heffley Thank you, Gary and Heffley Reforestation Centre Ltd.). Reforestation Centre Ltd. My friends in the department of Soil Science, especially Sharmin Gamiet and Guoping Xiao, helped to make the department one of the most lively and interesting places I have ever been. The Soil Science coffee room is a true centre of excellence. A special thanks to Ada and Jim Chapman, my mom and dad, who were also there to pull us through when times were so bad financially and emotionally. This thesis is dedicated to my collaborator in the experiment of life, my wife, my love Louisa, who has been raising three beautiful children under the most meagre of conditions. Everything I have done is nothing compared to what she has done.  x  INTRODUCTION  1. The in  existence of ectomycorrhizae has been formally recognized  our  culture  since the early 19th century.  ectomycorrhizae  was  Fogel,  Frank  1977).  studying also  truffles  suggested  proposed by Vittadini in 1842 named  in  it  this  abundantly  clear  association  (Trappe and  in  1885 while  the Kingdom of Prussia; at that time, he  was  mutualistic  a  Stribley and LeTacon,  (Mosse,  The function of  that  symbiotic relationship  Since then,  1981).  ectomycorrhizae  are  it has become  essential for the  normal growth of most temperate conifers and many angiosperms as well  (Harley  mycorrhizae seedlings where  play has  the  in  been  the  1983).  The  development  repeatedly symbiont  important survival  and  demonstrated is  role  missing,  that  of young  in circumstances such  as  in  the  exotic species (Mikola, 1970; Mikola, 1973) or  of  aftermath  However,  Smith,  fungal  introduction the  and  of  fungicide  use  (Trappe  and  Strand, 1969).  the application of mycorrhizal inoculum to more normal  reforestation situations has not been nearly as well studied and results are contradictory. Castellano and  10  (in  press)  (ecto  and VAM)  inoculation trials that involved outplanted seedlings.  Although  results fungal  unpublished  has thoroughly reviewed 103 published  are  mixed,  species.  Pisolithus  For  tinctorlus  studies  on  mycorrhizal  there are some useful observations on some example, among seedlings inoculated with Coker & Couch,  (Pers.)  only 46% of the  trials showed improved performance and 48% showed no  1  difference  from  Thelephora  growth  About  times  spp.  72  and  either  seedlings. growth  did  produced and  communication) consistently  not  what  fungus  has  circumstances.  It  trials,  Hebeloma and  affect growth or decreased it. (of these, only about 34 Danielson,  personal  is noticeably absent is a fungus that  growth.  the  17  three times, decreased  mycorrhizae,  improved  In  had no effect 11 times.  fungal species were examined  actually  every  improved  terrestris  three  Laccaria  uninoculated  It is also apparent that almost  potential to decrease growth under some  should be noted that the results summarized  by Castellano included studies in extreme circumstances, defined here  as situations likely devoid of the correct ectomycorrhizal  fungal  inoculum.  spoils,  reclamation  introduction we  Examples  might  of  of  borrow  of exotic species.  extreme pits,  situations  are  afforestation  and  the  These are the conditions where  expect the most dramatic response to inoculation, yet  no fungus increased growth in more than 5O of the cases. have  mine  very  been  few reports of results in normal reforestation  where  situations  There  one might expect less response to inoculation  than would be found in extreme circumstances. The and  work the  conditions symbiont in  of Marx (see Literature Review for a brief summary)  sometimes led  to  spectacular the  hope  results that  achieved  in  extreme  manipulation of the fungal  would be a direct means by which dramatic improvements  seedling  reforestation  performance situations.  not been fulfilled.  could  be  achieved  in  normal  To this date, the early promise has  This has resulted in a backlash, where many  now believe that ectomycorrhizal fungi cannot be used to  2  manipulate However,  seedling there  growth,  have  except  been  some  in extreme circumstances. studies,  Involving  normal  reforestation situations, where inoculation with ectomycorrhizae did  improve  performance  undeniably  establishes  potential  to  reforestation which to  situations.  ectomycorrhizal seedling  fungi  growth  This  review).  In  have  some  the  normal  What is not understood is the way in  ectomycorrhizal fungi, trees and the environment interact  influence  will  growth  affect  growth  possibility failure  and  survival.  Without this fundamental  it is very difficult to predict how any given fungus  knowledge,  as  that  influence  literature  (see  that  conditions  as  vary.  There  is a strong  mixed results do not reflect the general  the  of ectomycorrhizal fungi to improve performance so much  our  lack  of  understanding  the  of  ectomycorrhizal  relationship. Investigation  into the effects of ectomycorrhizae on seedling  growth  has  expect  that the relationship should be well understood by now.  A  primary  tremendous  been  obstacle  range  virtually alter by  the  within  in any  on for at least 60 years and one might  to  diversity  characteristics the  going  this understanding has been and is the  of conditions, species and within-species  found in many temperate forests. microsite  characteristics  plantation  seedling growth. diversity  is  usually  For example,  that can be found in  enough to significantly  The microsite variability is compounded  resulting  from  normal genetic variability  a tree species and by the almost incredible diversity in  ectomycorrhizal preferences  fungi.  The  variability  in  the  habitat  of higher plants is so well documented that it does  3  not  further discussion. As Trofymow and van den Driessche  need  have  (1991)  optimally  at  nutrient  levels.  different  ectomycorrhizal response  to  nitrogen and  decomposition, other  also grow  within—species  the  differences  of  temperature and moisture, phosphate solubilization  metabolites  are  species  moisture levels, temperatures and  pH,  Even  fungal  fungi are very large in such characteristics as  uptake,  and  different  summarized,  utilization,  antibiotics  and  production resistance  of  as summarized by Trappe and Fogel  examples  of  enzymes, hyphae  to  There  (1977).  of the diversity of ectomycorrhizal fungi,  and this has no doubt contributed to the difficulty in selecting a  to  fungus  the  task  achieve a particular result.  becomes  particular  fungal  particular  more  isolate  provenance  environmental  apparent  when  might  of  be  tree  circumstances.  one considers that any optimally  and  Even  But the enormity of  if  a one  adapted  particular selects  set  to a of  the best  fungus for a particular provenance of tree at a particular site, there  is  resulting creates  a  still  considerable  from  uncontrollable  room for environmental variation factors  such as weather.  This  high level of uncertainty in any experiment designed  to monitor the effects of mycorrhizal fungi. The  effort  to  develop  guidelines  describing  the  circumstances where a particular fungus might give a predictable result make result that  is it  confounded by the ecotypic variations in fungi which possible  that  the same species may not give the same  in similar circumstances. changes  There is even the possibility  occurring within an isolate during storage (e.g.,  sectoring) might result in different responses with the same  4  isolate. is  This In  holding  species  makes it very difficult to even define what one  or  one’s  set  of  hand,  let alone prescribe it for a tree  environmental  conditions that may also be  undefineable, except at very crude levels. The  diversity  implausibly in  one  the  in  fungal  characteristics  begins  to  sound  complex unless one remembers that trees may persist  place for centuries and while the genetic complement of  tree  is essentially fixed, the fungal component can change  with  the inevitable climatic, soil and other changes that occur  over  time.  likely over  that the  Fungi  are  every  tree would be exposed to a myriad of spores  life  profuse  of a forest.  spore  formers and it is very  The heterokaryotic nature of many  ectomycorrhizal fungi further increases the possibility that the fungal  thalli  at  any  site might each have a large and unique  pool of genetic material to draw on to assure optimum adaptation to  that  site.  Given  the  length  site,  the  opportunity for exposure to a wide  of time that fungi have to  persist  on  one  variety  of  genetic material, and the malleability of fungi,  is  unrealistic  not  adaptation. fungi  The  at  all  to  possibility  it  expect site and tree specific  of  extreme  site specificity by  is borne out by the fact that there are many thousands of  species of ectomycorrhizal fungi and subgroups of those species. It  is  hard  to imagine why this diversity would exist if there  were not sufficient niches to support it. It  is  symbiosis supplies some  becoming cannot  more  simply  carbohydrates  apparent be  defined  that  the  ectomycorrhizal  as  one  where  the host  and any ectomycorrhizal fungus provides  minerals and a few incidental benefits.  5  If Pirozynski and  (1975) are correct in their hypothesis that terrestrial  Malloch plants  a result of the combination of aquatic plants and a  are  fungus,  the  then  mycorrhizal  relationship  is,  as  old  as  terrestrial plant life itself and almost certainly very complex. If  one  why  it  is  so  reforestation manipulation effect  suggest  that  it  difficult  situations.  to  manipulate  However,  this  fungi is  in  normal  not to say that  of ectomycorrhizal fungi could be expected to have  little  the  this complexity, then it is easy to understand  accepts  on  tree  altering  growth.  It makes little more sense to  the mycorrhizal fungus would not affect  growth of seedlings in normal reforestation situations than does to suggest that altering the tree species or provenance  would have no effect on performance.  Manipulation of the fungus  to achieve specific results is much more difficult because it is so  hard  one  is  very  to precisely specify the characteristics of the fungus  set  specific  complement  of  the  that  circumstances,  seedling  ectomycorrhizal  Nevertheless, growth  a  of  may  change,  the  desired  fungal  in the nursery or the  with exposure to any stray inoculum and the exact nature  field, of  with, the fungus may confer benefits only in a  working  cannot will  the be  relationship  fungal written  contribution  growth  major  components  to  tree nutrition and  off as the component of tree growth  be looked after by nature.  their  not well understood.  is  Trees and how to improve  will never be adequately understood until the two that  constitute  a  tree, the fungal and the  plant, are both understood. The  complexity  of  the mycorrhizal relationship does hint at  the improbability of selecting a single fungus that will  6  consistently need  improve  seedling performance.  The questions that  to be answered in order to select the best fungus are such  things as, how important are the fungi on the roots of seedlings at a  outplanting, how long does it take for a seedling to develop full  complement  adapted  to  normally site, in  its  of  fungal  genetic material to be properly  environment,  found  on  a  how  particular  many  species  of fungi are  tree species at a particular  can introduction of exotic fungal genetic material result changes  large  population  in  result  conditions  performance better  in  vary?  One  and  does a diverse fungal  performance  possibility  is  as  environmental  that there are so many  fungal spores and the inoculum potential of normal reforestation sites  may  be at  seedlings  be  high,  outplanting  that  the  mycorrhizal populations of  are  not  important.  While this is  it may not be probable for several reasons.  possible, found  so  The fungi  a mature forest, that was present before logging, may  in  able to form mycorrhizae with seedlings and even replace the  fungi  on  the  possibility young  that  seedlings  the  outplanting,  but  there  is  a  they might not be the most suitable fungi for  seedlings.  between  at  There  is  almost  outplanting  of  seedlings  certainly and  some  lag time  infection  by the  optimum complement of fungi, even though infection by some fungi may occur quickly. roots  of  infection mycorrhizae stress  seedlings by  There is a possibility that the fungi on the from  the  nursery  more beneficial fungi.  could  interfere  with  Seedlings with inadequate  may be more susceptible to disease or environmental  in the period before roots develop optimal mycorrhizae.  Answers to these questions might suggest completely different  7  means  manipulating  of  mycorrhizal fungi than simply trying to  select the optimum fungus. To  time, only 72 fungal species  this  (Castellano, in press)  have been tested in outplanting trials and only about 34 species have  actually  formed  communication). used  in  a  Only  variety  mycorrhizae  (Danielson,  personal  a few of the 72 species tested have been of  situations.  These include Pisolithus  tinctorius, mixed (a euphemism for soil inoculum) and Rhizopogon vinicolor.  There  ectomycorrhizal  are  approximately  5000  species  of  fungi on 2000 species of woody plants (Marx and  Shafer, 1989) and if the within-species variation is considered, there  are  potentially tens of thousands of fungi that could be  evaluated in a variety of circumstances.  It seems clear that if  more information is to be gathered about the biology and ecology of  ectomycorrhizal fungi, then many more trials need to be done  and  should not be confined to extreme circumstances that  these  will  tell  us  little  ectomycorrhizal  about  the normal biology and ecology of  Furthermore,  fungi.  since  only a very small  number  of fungi has been investigated,  making  general conclusions about the application of mycorrhizal  it seems premature to be  inoculation. If  starts  one  certain  normal  from  the  the  ability  to  then  the  problem  becomes  previously which  assumption that in  reforestation situations, ectomycorrhizal fungi  have  consistently.  well-supported  This  mentioned  they grow.  influence seedling performance,  greatly  is  not  how a  diversity  to  achieve  trivial  a positive result  exercise,  given  the  in fungi and the conditions in  The previous discussion has suggested several  8  areas  that  need  objective  of  selecting  fungi  with  for  not  an  outplanting  only  useful  solve  this  problem.  The  Once  outplanting  trials and then improve them  to getting growth responses but also to  information  ectomycorrhlzae. in  to  this thesis was to examine previous approaches to  regard  collecting  exploration  on  the  biology and ecology of  the fungi were selected, they were used  trial  that was conducted where these fungi  might normally grow. The  performance  experiment  involved virtually all  aspects of applied mycorrhizal research.  Roots were examined to  determine roots  mycorrhizal  and  Because of the large number of  status.  confidently  of colonization.  isolation  population  isolation from  to  ability  of  important  that  possible.  were  ectomycorrhizal  streamlined  site.  seedlings.  fungi  types and the  to  facilitate  the  necessary to get a representative fungal  given  infect  adaptation  at  a  about  Fungi were isolated from the field, and  techniques  large—scale  those  this  examined, new techniques were needed to make observations  quickly level  of  to  The Given  specific  fungi were screened for what  growing  we know about the conditions,  it is  growing conditions in a screening process mimic  the place where growth is to be altered as closely as Traditional  screening  procedures have used testing  conditions that may include very wet growing conditions, buildup of  gases,  variations  unusual that  substrates,  may  select  anaerobic  conditions or other  against fungi adapted to the very  conditions in which they must grow. Inoculation  on  a  scale  large  enough  to  do  a  reliable  evaluation with highly variable natural growing conditions  9  requires  that a fairly large amount of inoculum be grown.  Many  problems are involved with the culture of ectomycorrhizal fungi,  and a new culturing technique was developed that is particularly applicable Moderate  to  the  amounts  inoculate  tens  production of moderate amounts of inoculum. of inoculum are defined here as sufficient to  of  thousands  of  seedlings.  Inoculation in a  commercial  nursery  inoculation  must not be too expensive or cumbersome if it is to  be used routinely. assessment  also  presents  certain  obstacles,  since  For that reason, this study also included an  of  two  different  thesis  can  be  methods  of  mycorrhizal  fungus  inoculation. This  selection  and  evaluation  outplanting  trials.  case  evaluation  study  untested  or of  fungi,  techniques  roots  of  virtually a  of  new  viewed as a protocol for the  ectomycorrhizal fungi for use in  Development  description new  therefore  of this protocol included the  certain untested  fungal in  species previously  outplanting  trials,  a  technique for culturing ectomycorrhizal for  the examination of ectomycorrhizal  and an evaluation of two different methods of application  of fungal inoculum. Since these topics are distinct, they will be presented in the following autonomous sections: 1.) In Vitro Growth of Ectomycorrhizal Fungi on Dilute Agar. 2.) A  Comparison  of  Two  Inoculation  Techniques  Ectomycorrhizal Vegetative Inoculum in a Commercial Nursery.  10  for  3.) A New Low Toxicity Stain Useful for the Examination of Mycorrhizae. 4.) The Selection and Evaluation of Ectomycorrhizal Fungi to Enhance the Performance of Seedlings Planted Under Normal Reforestation Conditions.  2. OBJECTIVES  The specific objectives of this thesis research are as follows: 1.) To try to improve the performance of Engelmann spruce and lodgepole pine seedlings in the nursery and in the field by inoculating seedlings in the nursery with ectomycorrhizal fungi isolated from the site where the seeedlings will be outplanted; 2.)  To compare the effectiveness of local isolates of ectomycorrhizal fungi with fungi from distant and different sources;  3.) To observe the behaviour of inoculated fungi on the roots of seedlings in order to determine how they behave in the nursery and the field and to compare their behaviour to that of volunteer fungi; 4.) To compare two methods of applying fungal inoculum,  1) by  injection into the root plug and 2) by applying the inoculum to the plug surface; 5.) To try to improve the culture of the fungal isolates used in this study; 6.)  To evaluate a new low—toxicity stain for examining  11  ectomycorrhizae; 7.) To develop a protocol for the design of experiments involving field trials of trees inoculated with ectomycorrhizal fungi so that sufficient information is collected in future studies that experiments may be compared and patterns in the behaviour of inoculated fungi may be determined,  if they exist.  12  3. LITERATURE REVIEW  In  growth  vitro  Perry,  ectomycorrhizal  Molina and Aniaranthus  fungi  on dilute agar..  (1987) note that different fungi do  jobs for different hosts in different environments and  different that  of  inoculation  strategy  if  with  the  more  fungal  than  mixture  one fungal species is a good is  appropriate.  About  34  ectomycorrhizal fungi have been tested worldwide to this time, outplanting 2000  trials.  some  (1977) estimates that there are over  of fungi mycorrhizal with Douglas—fir alone.  species  reasons  Trappe  in  Many  exist for the lack of variety in inoculation trials, and them were discussed earlier.  of  factor  is  as  Trappe  (1977)  has  However, a major limiting  stated,  “it  is  the common  experience of mycorrhiza researchers all over the world that many fungi grow poorly or not at all in the pure-culture methods tried so far.” At  least  two major types of variations can be made in culture  techniques;  to the nutrient composition of the medium and to the  physical  substrate.  nutrient  composition.  (1982).  Much  Considerable  emphasis  For a good summary, see Molina and Palmer  less work has been done on the physical component  of  the  do  much better in one type or another  Fungi  has been placed on  medium, even though it has been noted that certain fungi (Molina and Palmer, 1982).  are generally grown in small quantities on agar plates, or  in large or small quantities in liquid and particulate substrates (vermiculite still  or  with  agitated,  or  without or  peat).  aerated  liquid culture often grow poorly,  13  The liquid medium may be  or not.  Fungi inoculated into  or not at all.  To help  overcome fungi  this,  a  separate “fuzzing out” procedure, to give the  a headstart,  is often employed  (Molina and Palmer,  1982).  Still, some commonly found genera of mycorrhizal fungi,  including  such  cannot be  groups  routinely  as  Lactarius  Russula,  grown  culture.  in  and  Other  Gomphidlus  species  like  Amphinema  and  Cenococcura  geophilum tend to be slow growing and  culture  techniques  used  byssoides  indurate. The  undergone  few  adaptations  today to  are  deal  characteristics of ectomycorrhizal fungi specific nutrients or other symbiont). technique Kenney, to  was  used  tinctorius. growing  outlined  new,  and have  with  the  special  (slow—growing, requiring The particulate substrate  by Moser in 1958  (Marx and  Marx and Bryan in Marx (1981) made improvements  1982).  Moser’s  widely  essentially  not  technique  and  since  then, this technique has been  in inoculation trials, particularly with Pisolithus Limited forays have been made into using grain as a  medium (Takacs as reported in Marx and Kenney, 1982; and  Park,  1971),  spawn  production  which  is  essentially  technique  (Stamets and Chilton,  1983).  developed  an old commercial mushroom by  James Sinden in 1932  In spite of repeated admonitions to  investigate more of the ectomycorrhizal fungi available, the rate of  investigation  Marx  into  new  cultural  techniques has been slow.  and Kenney (1982) took for granted that,  “Once pure culture  inoculation techniques have been perfected, the value of a fungus should  be tested over a wide range of environmental conditions.”  This  recommendation,  then  figure out how to grow them seems to have been overshadowed  to  select the fungi one wishes to use and  by emphasis on selecting fungi easily grown in culture.  14  As  researchers easily  start  cultured  to realize the limitations of the handful of  fungi,  It is perhaps time to renew interest In  improving cultural techniques.  Two  Techniques For Inoculation of Vegetative  Fungal Inoculum.- Many techniques have  been  Ectomycorrhizae  developed  inoculation of ectomycorrhizal fungi in greenhouse situations.  These have  Riffle  Maronek  and  banding,  slurry  been  subdivided The  (1982).  dips,  into  categories  basidiospore  and  by  are broadcast,  inoculations, pelletizing  ectomycorrhizal seedlings and roots, and others.  these  techniques  require  nursery  categories  seed,  (broadcast,  for the  banding  and  Most of  pelletizing seeds)  that the inoculum be applied before or during seeding.  Application  of  inoculum at this time has several advantages in  that  incorporation  have  developed to any great extent.  of the inoculum is easiest before seedlings  in the planting procedure, assembly  it is easiest to incorporate into the  line nature of planting and it is easiest to utilize a  mechanized Spore  If an extra step is needed  procedure  inoculation  at is  this time  (Riffle and Maronek, 1982).  effectively  most  accomplished  by  incorporating spores into the growing medium or somehow assuring intimate  contact  with the root system. However, Castellano and  Trappe (1985) have had very good success with applying spores in an  aqueous  boom.  slurry from a watering can or commercial irrigation  Such a technique has obvious benefits in economy and ease  of application. Use  of  mycelial  inoculum is further complicated by problems  with survival of the mycelium until infection occurs.  15  Marx  failed  (1980> using  the  achieve  grain  culture  infection  of ectomycorrhizal fungi  technique  of Park and Takacs (Park,  The grain became heavily infected with saprophytic fungi  1971). and  to  bacteria  only  three  weeks  after  inoculation.  There is  often up to an eight--week lag between germination and short root development, have  to  that  and  it is probable that the fungal inoculum would  remain  peat  viable  moss  Melin-Norkrans  at least that long.  and  vermiculite  nutrient  (MMt4)  wetted  (Schenck,  excellent growth of several fungal species. suffers  inoculation.  technique  This substrate also  The  amounts  of  inoculum  used  in this About  to be necessary for good inoculation in a bareroot of  6 to 12% by volume have proven effective in  nurseries  (Marx  as  reported by Riffle and Maronek,  techniques developed for inoculation of mycorrhizal fungi adequate  in many regards.  behavioural  probable different mycelial infection survival and  would support  These are not small volumes of inoculum.  1982).  of  modified  large, ranging from O.27L/m to 2.16L/m.  Rates  container  are  are  seems  nursery.  The  1982)  with  from attack by saprophytes unless the MMN is washed off  before  1L/m-  Marx (1980) found  that  However, given the wide variety  characteristics some  inoculation  types  of  in  mycorrhizal  fungi,  it IS  fungi would perform better with  styles.  In  particular, application of  inoculum at a time when the seedlings are receptive to could  be  particularly  and infectivity.  Fortin,  Fortin,  helpful  in  terms of fungal  Boyle, Robertson and Salonius (1987)  Gaulin, Jomphe and Lemay (1988> have both  reported some success with top applied mycelial slurries, though  16  infection  rates  are  generally  not  as  high as with in:iected  inoculum,  However,  very  this  and  success achieved so far suggests that this  way,  very  simple  mycelial spores the  the  technique  inoculum of  few  fungal species have been tested  warrants  further  investigation.  If  could be applied as easily as the Rhizopogon  Castellano  effectiveness  and Trappe  of  (1985>, without reductions in  inoculation,  it  would  be a great step  forward in mycelial inoculation technology.  A New  Low  Toxicity  Ectomycorrhizae.— ectomycorrhizal percent  Stain  There roots  mycorrhiza  to  are  several  such  colonization,  for  Useful  as  the  Examination  reasons  for  of  staining  to make it easier to determine  to highlight the fungal component of the  assist description and to help distinguish types  based on differential staining. The  Phillips  staining assist  roots in  has  been  Boese,  improving the  approach  (1970) applied  levels  This  1982).  (Daughtridge,  However,  Hayman  evaluating  (Wilcox,  of  and  of  method  to  clearing and  to ectomycorrhizal roots to ectomycorrhizal colonization has  been  improved  upon  Pallardy and Garrett, 1986) with the goal  estimates vigorous  ectomycorrhizal  of  handling  and  colonization.  disruptive nature of the  staining process renders identification of the mycorrhizal types very  difficult and one must resort to hyphal characteristics to  distinguish large is  types  numbers  very  confidently  1990).  When examining  of roots on container plug-size root systems,  impractical  short roots,  (Roth,  to  it  examine hyphal characteristics of all  and in fact, some of the most useful  17  characteristics extramatrical  for  distinguishing  types  are  hyphal appearance, mycelial strand appearance and  morphological  gross  mycorrhizal  Visser, 1990).  appearance  mycorrhizae  of  For this reason,  (Danielson and  Danielson recommends that roots  be examined intact and unwashed. More  detailed  infection  examination  can  Griffiths  be  and  achieved  Parkinson,  examination,  of  hand  the  detail  description  for  Occupational  describes  to  Furthermore, nitrogen  the  blue  the  by  carefully, potential  as  with  stained  trypan  blue  in  Good hand sections can reveal much (1986)  in his guidelines for The Canadian Centre  carcinogen and teratogen.  a and  heating,  sulphur  (Danielson,  and Safety (MSDS record number 26619)  eyes  upon  and  recommended done  Health  mounts  even more precise root  of ectomycorrhizal fungi.  trypan  irritating  For  required by Agerer  the  whole  1984).  lactophenol are recommended. of  with  sections  to confirm mycorrhizal  roots  assumed harmful by skin contact. releases highly toxic fumes of  it  compounds.  Danielson  It is  The  slide heating technique  (1984) may be quite dangerous if not  and if many roots are examined in this manner, for  some  kind of poisoning from trypan blue is  probably high. Other  stains  ectomycorrhizal Pianese and  111-B  Martius  and  is  fungi  recommended  include  for  staining  of  cotton blue, chiorazol black E,  stain  (chlorazol, malachite green, acid fuchsin  yellow)  and the Conant quadruple stain (safranin,  crystal violet, fuchsin  routinely  fast green and gold orange  harmful  (Wilcox,  1982).  Acid  if swallowed, an irritant to skin and eyes  breathing the dust is dangerous.  18  Malachite green is a skin  and  eye  irritant  extremely should when  be  and produces toxic fumes.  Crystal violet is  irritating to eyes and contact with skin and clothing avoided.  Any of these dyes can be used safely, but  examining a large number of roots,  it can become extremely  tedious to continually don and doff protective clothing and make trips  to  staining  the fungi,  fume  hood.  Without examining every dye used in  it becomes quite apparent that there could be a  use for a low—toxicity dye suitable for staining fungi.  19  The  Selection  Enhance  the  and  Evaluation  Performance  of  of  as  burning  broadcast  (and  mixed  Planted  accumulated  ectomycorrhizae  characterized  Ectomycorrhizal  Seedlings  Reforestation Conditions.— The behaviour  of  knowledge  often  or  decrease  Mycorrhizae  may  organisms.  Litter  mycorrhizae. down  Some  cellulose  the  or  stimulated  or  others  may  of  such  site.  the  by  rhizosphere or  decrease  fungi may be able to break  inhibit  hosts  the  rate  of litter  for long periods of time or short  species in forested areas may help maintain  fungi and sometimes not.  increase  Dead  Slash  The same non-crop  as Alnus spp. may at times act as a reservoir for  ectomycorrhizal may  be  ectomycorrhizae.  increase  reduce mycorrhizal Inoculation potential.  layer  best  Organic matter may  reduced  may  ectomycorrhizal  without  Non—crop  species  the  Ectomycorrhizal fungi have been found to persist  soil  periods.  formation  extracts  and  decomposition. in  be  of  burning) may reduce or increase  the  to  Normal  contradictory.  mycorrhizal inoculation potential of a site. stimulate  Under  reforestation can  in  and  Fungi  wood  ectomycorrhizae  or decrease the inoculation potential of a may  or  Removal of the litter  as  serve a  as  very  an  excellent  poor  one.  reservoir for  Coal spoils may be  deficient  in  excellent  review of the wide variation in reported effects, see  mycorrhizal  inoculum  or abundant in it.  For an  Roth (1990). The  seemingly  contradictory  outcome of several factors,  results  described above are an  including the extreme variability In  behaviour both between and within species of ectomyeorrhizal  20  The ectomycorrhizal fungi, as noted earlier, vary widely  fungi. in  5uch  Important  characteristics  as  pH preference, drought  tolerance,  ability  compounding  the  variability  found  interaction  between fungus and tree at the within-species level  variability  is  there  the  This  tremendous  be  natural  certainly  nitrate  and  other  ways.  within the fungal symbiont is the  the  extremely  could  that  noted  within  Trappe,  hypothetically, conditions  reduce  variability  and  (Castellano  to  tree species and specificity of  Furthermore,  1985). high  could  many  in  exist  sufficiently  natural  many  microsite forests and  situations  where  different over a few metres  fungal population might change dramatically. cumulative  and  compounded variability almost  accounts for a large portion of the range in response  in outplanting trials summarized by Castellano (in press)  and Trofymow Early  guidelines  be  would  and van den Driessche (1991). developed  for the selection of fungi that  most useful in pure culture synthesis and inoculation  (as opposed to inoculation with soil or spores) already tried to take  into  fungi.  account  the  diverse  behaviour  of ectomycorrhizal  For example, Molina (1977) suggested the major criteria  for the selection of fungi are: 1) Ease of isolation, 2) Growth rate in pure culture, 3) Effectiveness as inoculum, 4) Effects on host growth and vigor, 5) Ecological adaptions and ecotypic variation, 6)  Interaction with other microorganisms,  7> Host specificity.  21  Molina fungi each  goes  are  on to stress that,  “many species and ecotypes of  closely adapted to their particular habitats, and so  fungal  isolate  must be tested on its own merits”.  recommendations  have  almost  today.  obvious  been  repeated  so  often  These  that they seem  Certainly one must be able to isolate,  grow and inoculate fungi before they may be used in pure culture synthesis. of  The  fungi  noted  exacting  6 and 7) are not new either, for as Hatch (1937)  (5,  from  criteria designed to deal with the specificity  Rommell  in  “mycorrhizal fungi are often more  (1930),  their  site  requirements  which they are associated”.  Even though researchers have known  about fungal specificity for some time, with  in  outplanting  than are the trees with  trials.  Part  it has seldom been dealt of  the  reason  is  that  researchers are limited to working with fungi that meet criteria 1  to  3.  The  mycorrhizal  state  fungi  is  of technology related to manipulation of not  so  advanced that any fungus can be  cultured  and  improved  before mycorrhizal research can advance much further.  In  inoculated.  addition,  specificity fungus  in  will  requires Points  it  is  This is something that needs  one thing to say that there is tremendous  ectomycorrhizae  do  to be  well  in  a  and  given  another  to predict which  situation.  This obviously  detailed ecological information for individual fungi. 3  and  4  also  suggest that the fungi used should form  abundant mycorrhizae, yet the need for high colonization has not been useful  established to  (Stenstrom  have  ectomycorrhizal  some  fungi,  and  Ek,  guidelines it  1990). for  the  So, while it is selection  of  appears that some of the guidelines  are self—evident, some need additional technology before they  22  can  be  applied,  assumption. place  relatively  handicap  who  notes,  small  Southeastern and  The  which the technological limitations  on mycorrhizal research is best illustrated by Castellano press)  (in  and one guideline may not be based on a sound  geographic  United  fungi  regions  with  is  concentrated Northwest  (Pacific  extremely  an  limited  tinctorius  (Pisolithus  Trofymow  vinicolor)”.  research  in and  States), on a few host plant genera (Plnus and  Pseudotsuga)  mycorrhizal  “most  and  group  of  Rhizopogon  and van den Driessche (1991) also note,  “very few outplanting studies have been conducted with seedlings inoculated  with  other species of fungi” (other than Pisolithus  tinctorius).  To offset our inability to adequately evaluate the huge number of  ectomycorrhizal  for  the  fungi,  selection  of  researchers have suggested criteria  outplanting sites to try to insure that  “positive” results are obtained. to  date,  conditions. where  the  vast  These  mycorrhizal  majority  Of the outplanting trials done were  conditions  conducted  under  extreme  are now described as situations  inoculation might be warranted and have been  defined by Castellano (in press) as: 1) afforestation, 2)  introduction of exotic species,  3) environmentally stressful sites, 4) reclamation of mine spoils, 5) rehabilitation of sites  where  there  is  a  change from  vesicular arbuscular mycorrhizae (yAM) to ectomycorrhizae, or some other such radical change. The contradiction inherent in these suggestions is that in  23  most cases  (except option 3), planting of inoculated stock would  be  done in circumstances far beyond the normal ecological range  of  the  fungus  being  circumstances  only  tested reinforce  observation  that  symbiont.  Inoculation  relatively between  virtually  little the  and  to  fungus  trials the  any  trials  conducted  already  symbiont in  is  such  in  such  well—documented better  than no  circumstances  do  expand our knowledge of the relationship and  its  normal  environment.  Similar  anomalies occur if we restrict our observations to fungi that we can  grow easily, or fungi that grow well in the nursery, or any  other  artificial  limitation  designed  to  result  in  easily  applicable commercial technology. This suggests that one approach which would be advantageous to learning to  about  use  as  possible, in  the behaviour of ectomycorrhizal fungi would be  many  mycorrhizal  regardless  pure  culture  important  but  as  will  inoculation trials as  (by implication, techniques for isolation and  in  Hunt  in  of their ease of isolation or growth rate  culture need to be improved). is  fungi  The effectiveness of the inoculum  terms of application to commercial nurseries, has demonstrated, the types of fungi that  (1989)  grow in a nursery and the degree to which they will infect  roots  can  conditions.  be  greatly Fungi  affected  by  manipulation  of  growing  should not be excluded from field trials at  this early stage of understanding because they do not do well in the artificial and easily manipulated environment in a nursery. Fungi vigor,  may be evaluated In terms of their effect on growth and but  the  response  of a seedling in any season or a few  seasons may not give a true indication of the potential of the  24  fungus  (Smith,  1985).  become  further  complicated by our lack of understanding of the  complete  The objectives of any fungal evaluation  ecophysiological  function of the symbiosis (Kropp and  Langlois, 1990).  Furthermore, the normal situation for trees is  that  several  there  are  species  of  fungi  on  a  tree  root  and Visser, 1989; Roth, 1990), even at a young age.  (Danielson Molina  and Trappe (1984) caution that inoculation with only one  fungus  should  be  considered  as only one approach to applying  mycorrhizal technology. The then  of evaluating the effect of mycorrhizal inoculation  task appears  inoculation may  to is  as  follows:  up  in  to determine if mycorrhizal  to trees when the beneficial effect  beneficial  show  not  be  sets of climatic circumstances, the  all  fungal isolate may not match the tree strain, when the microsite conditions  may not match the fungal strain or tree strain, of  presence  the  fungus  might  manifest  itself  in  the some  unlooked-for way, the other fungal symbionts necessary to give a complete  tree  a  that  infect  complement  may not be present, and new fungi  seedlings shortly after outplanting might mask the  effect of the inoculated fungus. diversity variety  is of  anticipated of  the  probably  this  survival  of  documented  growing  conditions.  situation in 1937 when he noted, is  remain  mycotrophy,  large scale evaluation of many fungi under a  carefully  forestation  The only way to deal with such  and root  Hatch  “The science  apparently still in its infancy and it will  so  until  of  the  our knowledge of tree nutrition, of influence  symbionts  has  of been  environment widely  upon  the  explored.  The  obvious need today is for precise information on the influence  25  of different species of mycorrhizal fungi upon the growth of our most important trees planted in a wide variety of habitats. problem, even  therefore,  several  amount  of  is  not  individuals.”  information  relatively  on  one which may be solved by one or Today, we have accumulated a large behaviour  the  of a few fungi in a  small number of environmental circumstances This  situations).  The  (extreme  has done little to expand the understanding  of the intricacies of the plant-fungus relationship and it seems that little has changed since Hatch’s seminal paper. To evaluate how far we have come in collecting the information Hatch  that  suggested  examination  of  and  press)  most  if  reforestation not  species, sites  with we  ectomycorrhizae  does  literature.  requires  Fortunately,  a  thorough  Castellano (in  (1991) have recently  The discussion so far has suggested that we must be  concerned  situations  necessary,  Trofymow and van den Driessche  this.  done  the  was  studies  want  in  to  normal  situation  include  done  really  in  normal  reforestation  understand the dynamics of  reforestation situations.  is  A normal  defined here as reforestation that  afforestation,  the  introduction  of exotic  reclamation of mine spoils or other severely disturbed or  changes  ectomycorrhizae,  or  from other  VAN  similar  mycorrhizal radical  species  changes.  to  If the  studies involving extreme situations are discounted, then only a few relevant studies remain. of  this  nature  that  I  much  each  determining  how  mycorrhizal  fungus  I have reviewed all of the studies  could  find.  study  inoculation.  discussed below.  26  I  was  advanced Several  concerned  with  the technology of studies are briefly  and  Castellano of  species  Trappe  hypogeous  In  (1985).  this  experiment,  fungi were spore—inoculated onto several  tree  species,  were  inoculated onto two provenances of Douglas-fir.  formed  including  mycorrhizae  subsequently,  one  several  growth  trial.  The  description aspect.  Site  not  are  in  seven species of hypogeous fungi that  with of  Douglas—fir  these  two  characteristics  site  several  fungi  in  the  Two fungi  nursery  improved survival and  of seedlings in an outplanting  for the outplanting trial is given a minimal  the  form  of  location,  elevation,  slope and  specific descriptions of the sources of inoculum  given and no attempt is made to relate the environment  of the source of inoculum to that of the outplanting trial. years the  after fungi  greater of  outplanting,  It  indigenous  was to  approaches. general  even though indigenous fungi also colonized  not the  It  indicated site.  used  were  given, for  that  the  study  inoculated fungus was included  some  useful  were at least from the same  inoculation  potential,  and it for  given  about  biogeoclimatic is the  these  were  insufficient  to use as  future studies involving similar fungi.  is  site  This  fungi  though  information  known,  if  climatic area and rudimentary descriptions of the study  guidelines  soil  the seedlings inoculated with one of  The inoculated fungus persisted on the roots  seedlings,  roots.  Two  had greater root collar diameter, greater height and  survival.  the  area  and  the  Little  outplanting site in terms of  indigenous species of plants and fungi, factors.  Since these things are not  difficult to assess or begin to define a typical successful use of this type of fungus.  The site  was previously covered with red alder, which was cut and burned.  27  is  It  not stated if herbicides were used, how intense the burn  was,  what  Yet,  the  species  were  impression  circumstances  left  on the site  created  by  not  (if any) and so on. noting  any  extreme  is by default that this was a more or less normal  reforestation situation. Stenstrom address  and  Ek  ecotypic  This experiment seems to attempt to  (1990).  variability in that it involves several fungi  that were isolated from similar latitudes and not too dissimilar longitudes  and  used  an  in  collection is  from  generally the same species of tree as was  outplanting  trial.  sites were not given.  described  being  as  composition  (sic).  described.  It  Site  a  However,  details  of  the  The soil of the planting site  glacial vegetation  till and  deposit with a sandy site index are also  was not noted if the types of fungi used in the  inoculation trial were found on the site, except that indigenous mycorrhizae  appeared  inoculated fungi. the  inoculated  outplanting.  to  be indistinguishable from some of the  Some description is given of the behaviour of and  The  other mycorrhizal fungi on the roots after inoculated seedlings were smaller in size at  outplanting, but soon passed the uninoculated seedlings in size. The to  levels 25  of mycorrhizal infection at outplanting were low:  in  all  cases,  yet  statistically  5  significant growth  improvements were found. The paper  study by  features,  described above contrasts nicely with a subsequent  Stenstrom, such  as  Ek site  and Unestam (in press), description  and  in which most  description  behaviour of the fungi out in the field, are similar.  of the  The major  difference between the studies is that in the more recent one,  28  the fungi  (except one) came from exotic locations.  characteristic  of  the  mycorrhizal  formers”  fungi  readily  that  conditions. a  fungi used is that they were “assertive which  of  inoculated  form abundant mycorrhizae under artificial  roots  seedlings  recover  infected grew  even  after  furthermore,  showed  little  these  were  fungi  nursery, caution  is an appropriate term to describe  In this case, many of the inoculated seedlings had  number  not  The unifying  by  the  applied  fungus.  The  more slowly in the nursery, and did three sign  effective  at  years of  in  the  recovering.  forming  field,  and  Even though  mycorrhizae  in  the  they had little effect in the field, which serves as a against choosing fungi based solely on their ability to  form abundant mycorrhizae. Richter and Bruhn (1989). Several fungi were isolated from the same tree species and same general area in which the outplanting trial  would be conducted,  similar type of environment.  but not necessarily from the same or The result was that the isolate of  Laccaria  bicolor  survival  on a “droughty sandy” site, came from a moderately wet  site.  This  (R. Mre.) Orton, which significantly improved  isolate was one of the faster growing ones and was  most successful at forming mycorrhizae in the greenhouse.  There  are rudimentary descriptions of the sources of the fungi and the outplanting site, though a slightly more detailed description is included growth.  for This  circumstances.  the  source of the Laccaria bicolor that improved was  It  done  to  highlight  the  paradoxical  was not indicated if the successful fungus,  Laccaria bicolor, was present naturally at the outplanting site.  No  description was given of the behaviour of fungi on the roots  29  after outplanting. Amaranthus very  and  harsh  inoculum  site  were  holes.  Perry, that  plantation  are  site  inoculum,  appeared  achieved  Included  the  Improvements in survival on a  (1989).  by  to be mainly devoid of fungal  transferring  very  good  soil to the planting  detailed descriptions of the  and because of its similarity to the source of authors  describe the inoculum source site  well.  The  though  not much detail is reported in the paper.  both on  roots  also  of  the  seedlings were thoroughly examined, For instance,  controls and inoculated seedlings had Rhizopogon vinicolor them  roots  after  had  extreme  outplanting,  on site  and As  abiotic,  could such  also  it was not reported what the  them at outplanting.  reforestation. and  but  almost  This appeared to be a very  beyond  the  realm  of  normal  the authors point out, many factors, biotic  could  be  involved with the transfer of soil and  have led to the differences in survival, so studies  as these may be of little use in dealing specifically with  the mycorrhizal question. Bledsoe and Tennyson (1982). This is an inoculation trial done in  north  probably were  on  formed  outplanting balanced biomass growth  Washington  State, where fungi from what was  a considerably different climate in western Washington  inoculated  planted they  central  by of  a  onto  Douglas—fir  harsh dry site. mycorrhizae  sites a the  were  in  outplanted  effects were reported.  that  were  then  The fungi were selected because nurseries  quite  description  seedlings  well  of  the  and  grew  well.  The  described but this is not source  of inoculum.  The  seedlings was reduced and no other The roots became colonized by an  30  indigenous fungus that was different from the inoculated fungus, but  unidentified.  that  The  root observations were quite useful In  the inoculated fungi  (Hebeloma crustullnlforme (Bull.: St.  Amans) Quelet and Laccaria  (Scop.:  laccata  Berk.  Fr.)  & Br.> did not grow onto new roots on the outplanted seedlings. Loopstra, fungal  Shaw  and  Sidle (1988). This study evaluates three  isolates,  two  of  location  on  confirmed regimes  that in  continent as the outplanting site. the  the  inoculated authors  the  which came from the same approximate  trees  nursery  with  fungi  conclude,  grown tended  but  under to  The study  normal high fertility  grow  faster  than  trees  with lower fertility regimes.  The  “we caution against sweeping generalizations,  unsupported  by  inoculation  with ectomycorrhizal fungi will necessarily improve  seedling the  quantitative,  performance.”  well—documented  This  site—specific data, that suggest  is more or less a restatement of  observation  that  not  all  fungi improve  growth in every situation. Kropp,  Castellano  inoculated planted slope,  with  on  separate  only  similar  for  affect  the  in  all  Trappe  Cenococcurn  vegetation,  described  and  geophilum  sites and  (1985).  Western  and  hemlock was  the seedlings were  to represent “a variety of aspect,  soil  types.”  However, the sites were  terms of elevation and rainfall, which were sites.  inoculation  The different planting sites did not effect  reported,  which  was  that  inoculated seedlings had greater leader growth than uninoculated seedlings.  There were no differences between seedlings heavily  colonized  by  colonized.  No other growth data is given,  Cenococcum  geophilurn  31  and  those  moderately  in particular, how  the  of  heights  Non-mycorrhizal  the  seedlings  seedlings  at  compared  outplanting.  became infected with Cenococcum from  the site. and  Heidmann trial an  Cornett  (1986).  outplanting site in this  The  presented a quite “normal” reforestation problem. area  open  within  a  young  forest  restocked because of poor nursery stock.  It was  was not properly  that  The site was described  with  brief notes on elevation, slope, vegetation and soil.  soil  description  material.”  No  series  were  litter  layer  Oregon. sparer  “silt  other  and  such  of  of  the  the  spores  since  the  The mycorrhizal but the  This is a point of concern,  inoculated seedlings seem to show the same levels of  colonization inoculated  as  the  controls.  with  the  duff  Some  summarize  from  inoculum sources were even  rates after growth in the nursery were given,  of fungi were not described.  The  tinctorius  outplanting site.  types  rate.  as humus form, pH or soil  Pisolithu5  descriptions those  derived from basalt parent  Two types of inoculum were used: screened  (duff)  than  barns  details  given.  The  infection  was  The  growth  Nevertheless,  the  seedlings  had a significantly higher survival  effects  occurred  that  are  difficult to  because of interactions with a fertilizer treatment.  rnycorrhizae  on  the roots were not described after time in  the field, so it is not clear what the fungi were doing. Marx  in  General.  ectomycorrhizal discussion has  rather  of  No  review  of  outplanting  trials  with  without  some  Don Marx and Pisolithus tinctorius (“Pt”).  Marx  inoculurn  consistently  would  be  reported  performance after inoculation with Pt.  32  complete  improvements  in  seedling  The normal situation for  bareroot seems  nursery  to  be  terrestrls. great  the  almost  improvements  great  in  the southeastern United States  complete  domination  in  Higher  survival  of  quality  action  Cordell  and Clark  greater  than  planting sites tend to not have as  of  Pt  is not clear.  with  have  high  level,  nutrient  reported  For example, Marx,  (1988> reported that when Pt colonization was certain  a  Despite many, many trials,  then  correlated with level of colonization. one  Thelephora  and performance of outplanted  a response to Pt inoculation. mode  by  When this fungus is replaced by Pt, there are often  seedlings.  the  seedlings  that  levels.  high  improvements  in growth  The site used was a good  Even though many researchers  fertility  reduces  ectomycorrhizal  development,  Marx  resulted  more mycorrhizae at three different pH levels, and  the  in  mycorrhizal  acidity  than  formerly fungal  an  Even  holding stress  by  increased  nitrogen  levels.  agricultural  the  field  However,  though  capacity, over  that  higher  the  The  and  this  was  reportedly  It Pt  not discussed in had good moisture  the seedlings underwent considerable drought period  of  the  experiment,  response to inoculation.  internal  site was  may have been lacking in  point  site  plantation  water  which  may have  Walker, West and McLaughlin  ((1982), described in Marx, Cordell and Clark, lowered  fertility  N  development was affected more strongly by soil  inoculum.  detail.  reported  (1990)  (1988)], reported  tension in seedlings with abundant Pt.  appears that many factors contribute to the effectiveness of inoculation,  colonization inoculum,  at  and  these  include  outplanting,  the  the  presence  extent or  of  absence  the fertility and moisture holding capacity of the  33  root of  site,  the  climatic conditions over the time of the study, soil  acidity and inoculum potential of the site. not  be  effective. Pt  (Castellano,  inoculation extreme  growth  only  included,  inoculations have  46o of the time in outplanting  more  literature  exists,  most  of  but  trials  it  described  in  more  entirely  paid  only  are  in press).  project.  reality  the most extreme conditions to  Considerably  almost  research  all  applied  circumstance  outplanting  the  when  been  seedling  Summary.  been  Even  has  improved trials  it  possible to accurately predict when Pt inoculation will  is  which  Because of this,  ectomycorrhizal  deals  with the type of  previously.  The  types  since of  the  made  is  projects undertaken have in Often,  the general geographic area of the  origin with little description of specific site conditions. vocabulary,  to  describe  on  inception of this  little attention to ecotypic specificity.  mention  work  or less normal reforestations has  published  The  on  sites  in  a  way  that  The  conveys much  ecological significance, seems to be lacking. Hardly  ever  inoculated  is  there  any  evaluation of whether the fungus  is actually found naturally at the outplanting site.  This may not be a critical factor for selecting the fungi for an outplanting  trial, as in the one case where the environments of  the  fungal  inoculum  was  found that the fungus from the most disparate site improved  growth. general it  is  Other  were compared to the outplanting site,  evidence  suggests  that  it  from  the same  area are better than fungi from further away.  However,  the  fungi  interaction of the fungi and their behaviour in the  field that will give clues to the mode of action and yield a  34  procedure be  that  is  given  types  for selecting appropriate fungi.  a fungus found at a site may improve performance if it a headstart in the nursery, or it may be that certain  of  sites,  For example, it may  fungi consistently perform better on certain types of  so  these  fungi  should be introduced regardless of the  indigenous fungi. Another general  apparent is  the  in ectomycorrhizal research in  of types of fungi evaluated in normal  paucity  When planting in extreme situations, any fungus is  situations. than  better  shortcoming  none,  increasingly  but  in  normal  situations,  where  it  is  evident that inoculation potential is likely to be  quite high, the crux of the matter may be to have an appropriate Yet certain researchers repeatedly test the same fungi  fungus.  “similar”  in come  perhaps, somewhat surprisingly,  and  up with inconsistent results.  that  conditions  described Most  conditions,  (e.g.  soil,  This may be due to the fact are  microclimate)  often  not  well enough to ascertain if they are in fact similar.  singularly  indigenous  missing  are  ectomycorrhizae  attempts  any  at  a  site  with  to  correlate the  the success of a  particular fungal inoculum. In  spite  numbers  of  of  published  the  assertive  on  techniques  techniques  for  ectomycorrhizae a  few  define  fungi, the  little  few  success  with the relatively small  ectomycorrhizae, to  improve  inoculation  ectomycorrhizae. can be summed up as,  using  a  few  situations  results with the above.”  almost  The  no  work  is  and culture  literature  on  “a few people working with  techniques, working diligently to that  might  give  positive growth  It is not surprising that the  35  ectomycorrhizal years  after  symbiosis has not been fully exploited, even 60  Hatch remarked on the enormous difficulty involved  with accurately characterizing mycorrhizal relationships.  36  4. EXPERIMENT ONE IN VITRO GROWTH OF ECTOMYCORRHIZAL FUNGI ON DILUTE AGAR’  Ectomycorrhizal culture,  liquid  small  amounts be  not at all  means  to  grow  very  slowly  in ordinary  (Stevens, 1981; Marx and Kenny,  nutrient  of mycelium.  used  considerable by  or  often  Agar-solidified  1982).  can  fungi  media may be used to produce  Other substrates such as peat moss  produce  amounts,  greater  but  this  adds  bulk to the medium and makes inoculation difficult  other  than  incorporation  of  the inoculum into the  growing medium. We have had good success with culturing fungi in a semi—liquid medium The  made thixotropic by including agar at low concentration.  technique has several advantages.  Initial growth is faster  compared to standard liquid culture and a separate “fuzzing out” (Molina  and  growth  is  will  liquid  the  culture, We  surface  without  Several  isolates  reasonably of  the  frequent  well.  medium  Faster  shaking or addition of  that  grow very poorly in  The fungi often grow best (Fig.  3), whereas in liquid  the colonies frequently settle near the bottom.  (Marx, were  per flask.  *  grow  1982) procedure is not necessary.  compared two types of media: modified Melin—Norkrans  formula media  sustained  fragments.  glass  near  Palmer,  1969) with no agar added or with 3g/L agar.  (MMN) The  placed in O.25L Erlerimeyer flasks at a rate of O.1L All flasks were autoclaved for 20 minutes and  Published in Mycologia,  82(4),  37  1990, pp.  526—527  allowed  to cool.  4mm  a  agar  Each inoculation consisted of the addition of  plug  isolates  fungal  E-strain,  from an agar-plate colony. were used,  Hebeloma  including Cenococcurn geophilum Fr.,  crustiliniforrne  (Scop.:Fr.)  laccata  Twelve different  (Bull.>  Quelet,  Laccaria  Cke., Amphinema byssoides (Fr.) 3. Erikss.  and others. inoculating  The  it  that  would were  cultures  plug  float  on  shaken  minute  (Boyle  et  liquid  cultures  was placed gently in the agar flask so top  of  the agar.  continuously  al.,  Liquid  100 reciprocations per  at  (Intermittent  1987).  (no agar)  gave similar results.)  shaking of the  The agar cultures were  allowed to rest undisturbed until growth had begun on the top of the  medium, then shaken vigorously once, and allowed to rest at  room temperature for one month. To  evaluate  drained  on  rinsed  with  growth,  filter  cultures  paper  were  heated  to  boiling and  in a Buchner funnel with suction and  0.2L of boiling water.  Mycelium was air-dried for  two days and dry mass was determined. Eleven  of  the 12 isolates grew faster in dilute agar than in  the liquid medium (Table I) that  differences growth  of  (Fig.  4).  A paired t-test indicated  were highly significant  (P=0.004).  The ratio  rates in agar to those in liquid ranged from 0.4x to  7x. We  have  used  ectomycorrhizal inoculate  this  technique  fungi.  seedlings  commercial nursery.  in  Resultant  to  grow cultures  numerous species of have been used to  a variety of applications,  including a  When dilute agar cultures are fragmented in  a blender, they can be injected through a large bore needle or  38  pipette.  We note that it is difficult to separate the agar from  the  fungal mycelium, using this technique.  our  inoculation studies show that separation of the agar medium  from  However, results of  the mycelium is not necessary to achieve good formation of  mycorrhizae. Centrifugation has  proven  stratify,  to  can be used to facilitate separation, but this be  a  bit  messy.  The fungus can be caused to  but it is difficult to remove all traces of the agar.  Accurate mycelial concentration can be determined by subsampling the mycelium and washing with boiling water as outlined above.  39  Table I  Mycelial Mass of Ectomycorrhizal Fungi After One Month of Growth in Liquid or 0.3 Agar MMN  Isolate  Fungal Species  Dry Mycelial Mass (g) Dilute Agar Liquid  A14  unknown  0.0340  0.0053  A18  unknown  0.0789  0.0435  Al 8 B  unknown  0.0098  0.0218  A21  unknown  0.1356  0.128  A2 9 B  Amph I nema byssol des  0.0553  0.0086  A31B  unknown  0.0877  0.0292  A46  Suillus sp.  0.1265  0.0533  E01  E-strain  0.0052  0.0028  C.g.  Cenococcum geophi lum  0.0217  0.0197  Hecr5  Hebel ama crustul ml forrne  0.0202  0.0040  Hecr8  Hebel oma crustul ml £orme  0.0188  0.0031  S238  Laccaria laccata 0.0713  0.0100  Mean difference=0.028 SD difference=0.027 Paired—sample t=3.566 (Degrees of freedom=ll) P=0.004  40  5. EXPERIMENT TWO  TWO INOCULATION TECHNIQUES FOR VEGETATIVE MYCORRHIZAL INOCULUM INA COMMERCIAL NURSERY  Application  of  ectomycorrhizal fungal inoculation technology  is dependent not only on performance of the inoculum but also on cost  effectiveness.  Marx  uses peat moss and vermiculite as a substrate, which means  that  it  must  planting.  This  application, growing  be  if,  season,  additional applying  The fungal culture technique perfected •by  incorporated does  not  and  it  manipulation spores  not  growing medium before  does  inoculation is desired over the require  during  a  certain  planting.  The  amount  of  technique  of  in an aqueous suspension is very flexible with  regard to timing, cheap and easy. of  the  allow much flexibility in timing of  example,  for  in  Work with slurry applications  vegetative inoculum suggests that this type of inoculum does need  (Boyle,  as much technology to apply as was generally believed Robertson  have  tried  that  it  mycelial  surface  Salonius,  mycorrhizae,  slurry.  However,  this  have  done.  1987).  Fortin et al,  (1988)  application of mycelial inoculum and found  produced  inoculated been  and  but  not  relatively  as well as injecting few  fungi  have  been  way, and few trials under different conditions If  slurries of vegetative inoculum could be  applied in a manner similar to to that used for fungal spores,  41  it  would  can  inoculation be  grown  sporocarps), fungi can  the  Vegetative  inoculum. spore  increase  opportunities  inoculum  in  anytime quality  grown  and  has some advantages over  also  consistency  vegetatively  experiment  vegetative  it  (so it is not dependent on collection of  more quickly than spores This  using  that it can be pure culture or mixed,  that are difficult or be  for  can be controlled, some  impossible to obtain sporocarps for and vegetative inoculum may infect  (Marx and Kenney,  1982).  consisted of applying slurries of vegetative  inoculum to container—grown seedlings by Injection into the plug or  by  squirting  inoculum  on  top  of the plug.  An important  difference from all other trials is that the slurry used in this trial  consisted  of  culture  described  removed  from  the in  fungus  suspended  Experiment  One.  in the dilute agar  The nutrients were not  the growing medium before inoculation, which is a  departure from virtually all other procedures in use today. combination  of  dilute  The  agar, no washing and top application of  fungi represents one of the least labour-intensive approaches to inoculation of fungal mycelium yet tried.  MATERIALS AND METHODS  The experiment was conducted in the research greenhouse at the Heffley Reforestation Centre, about 20km north of Kamloops, B.C. The  research  portion  of  there.  The  greenhouse was treated the same as the commercial the  nursery,  Heffley  except for the experiments conducted  Reforestation  Centre nursery is somewhat  different from many nurseries because it does not use  42  slow-re1ease  fertilizer in most stock types; the growing medium  used is mainly coarse peat moss to facilitate aeration of roots; and watering Is reduced, but compensated for by frequent misting to control temperature.  These steps have created an environment  that is much more conducive to the formation of mycorrhizae than was  present  (Hunt,  under the previous and more typical growing regime  1989).  Two species of trees were used, Engelmann spruce and lodgepole pine.  The seed used was from the appropriate provenance for the  planting  site.  The fungi were grown on the dilute agar and MMN  described in Experiment One.  medium  fragmented  in  injected.  Both  with  5mL  pipetter  of  a blender for 15 to 45 seconds to allow it to be the  slurry  was applied with an Oxford  The irioculum was applied by (1)  throughout  of  The  SA). A length of tubing connected the pipetter  to a lmL pipette.  top  spruce and pine seedlings were inoculated  slurry.  (Model  mycelium  The dilute agar slurry was  the plug.  the  plug or  injecting the  (2) squirting the inoculum on  In either case,  the body of the pipetter was  kept below the injector in order to avoid siphoning. were  grown  block, the 1130  in  947  pine  been  PSB  cavities/m, were  grown  313a  schedule shown  (4.4:1  styroblocks  in used PSB 211  v:v).  (1989)  (240 cavities per block,  The growing medium was peat and  Fertilizer was applied according to  shown in Appendix III. by Hunt  (198 cavities per  Beaver Plastics, Edmonton, Alta.) and  ) styroblocks. 2 cavities/m  vermiculite the  used  The spruce  This fertility regime has  to increase the level of mycorrhizal  colonization and the number of types of mycorrhizae on the roots  43  over those using slow release fertilizers. growing  regime  about  were  started  can  be found in Hunt  (1989). Both tree species  weeks old at the time of inoculation and had  eight develop  to  Other details of the  short  roots.  The inoculum was applied to  approximately 50 seedlings in each block, excluding the two rows of  cavities around the perimeter of the spruce blocks to reduce  edge  effects.  end  rows  trees  There were fewer pine seedlings so only the two  and  one  inoculated was  area  was  marked  inoculum  was  side row were excluded. The exact number of counted and recorded, and the inoculated  with  applied  felt to  pen  20  and  plastic  blocks for the spruce  surface-applied  and  surface—applied  and eight blocks injected)  treatments species R.  and 16 blocks  injected)  (10 blocks  (eight blocks  for the pine.  used for each tree species.  Each  Eight  Two of the fungal  used on the pine were an E-strain isolate obtained from  M.  Keith  were  10  markers.  Danielson Egger)  mushroom  (probably  the anamorph of Wilcoxlna mikolae:  and an isolate of Suillus tomentosus taken from a  collected  near  the  study  area.  Two of the fungal  species used on spruce were the same Danielson E-strain isolate, and  an  isolate  Heffley  of  Amphinerna byssoides collected by me from a  Reforestation  E-strain  (0188),  crustiliniforme described  in  Centre  seedling.  Cenococcum (Hecr—8),  Appendix  I.  geophilum  did  not  One  (A188—2), and Hebeloma mycorrhizae  and are  treatment was the dilute agar  medium  used  fungus  growing medium), which was also injected or top applied.  There  were  to culture the fungi  form  The other fungi used,  two  separate  (referred to hereafter as the  controls  for  the  injected and top  applied treatments, but they were identical in that they both  44  had no treatment done to them. of  trees  counted same  the other treatments and they were marked off and  as in  the  way.  The controls had the same number  same  After  manner and treated in the nursery in the  inoculation,  the  blocks  were  placed in a  randomized arrangement. The  experimental  treatments  and  technique.  Squares  missing  This data  block  and  variable.  program  and nested  Layard’s  homogeneity to  do  was  used  called  UBC  Genlin:  was  for  most  of  the  A General Least  because  with  more  one  than  independent  test.  Normality All  was  evaluated  analyses  were  using  the  checked  for  variance and normality, but these data were not  disregard  done  it could handle  of variance was tested using Bartletts  tests.  of  useful  ANOVA results.  In the cases where the data  for the randomized block ANOVA were non-normal, also  inoculation  multiple comparison tests for randomized  designs  Homogeneity  Kolmogorov—Smirnov  used  is  with  crossed  that  program  analyses  were  Analysis of Variance Program by Malcolm Greig and James  Bierring.  and  treatments  The  statistical  design was nested with blocks nested within  with  the  Kruskal—Wallis  the analysis was  non—parametric  ANOVA.  Multiple comparisons were done using Tukey and Bonferroni tests.  45  RES tJL T S  Spruce.—  Both  ectomycorrhizae The  results  differences  of in  inoculation on  the  the  techniques  abundant  seedlings for two types of fungi used.  analyses  the  produced  levels  are  of  shown  in  The  II.  between  colonization  application methods were significant (p<0.05)  Table  the two  for E—strain.  The  mean level of colonization by E—strain for the injected E—strain treatment was 71% (SD± 30) and for the top—applied treatment was 58%  27).  (SD+  was  treatments Amphlnerna  The  level  mean  0.9%.  byssoldes  The for  the  was  69%  treatment  was  68% (std. dev.  by  injected and  21)  26).  in  the  other  colonization  of  Amphinerna  for  of  byssoides  the top applied  The mean level of infection  Amphinema byssoides in the control and fungus growing medium  treatments  was  mycorrhizae treated levels were The  dev.  E—strain  level  mean  treatment  (std.  of  on  27.5%. an  Thelephora terrestris—like fungi formed  average  47%  of  the  roots that were not  with one of the fungi and 16% on the others. of  The total  colonization (total of all species of fungi present)  not different between injected and top-applied treatments. inoculated  levels  of  treatment, were  not  fungal E—strain  fungi  Thelephora but  the  apparently  excluded  Thelephora and the  became significantly different by fungal levels of Thelephora by application method  significantly different within the Danielson E-strain  treatment, even though the levels of E-strain within the treatment,  were significantly different.  In spite of  significant differences by inoculation technique in colonization  46  Table II  Comparison of In:jection Into the Plug Versus Top Application of Ectomycorrhiza]. Mycelial Slurry to Two Month Old Engelmann Spruce Treatment Parameter  Top Applied  Injected  p ANOVA  400  390  A. byssoides myc or r hi z a e (96)  36.6 32.7  35.9 33.2  0.06  E—strain mycorrhizae  18.3 34.4  15.6 29 . 0  0.07  (96)  Thel ephora mycorrhizae  32.7 30.3  30.3 29. 4  0.54  (96)  82.4 20.6  82.1 21.5  0.91  1.56 0.39  1.62 0.37  0 . 06  15.5 3.77  15.6 3.51  0.70  Root Collar Diameter (mm)  3.33 0.41  3.32 0.40  0.86  Root Mass  0.90 0.27  0.95 0.28  0.15  Abundance of Mycorrhizae  2.58 0.60  2.60 0.60  0.79  Root to Shoot Ratio  0.60 0.19  0.60 0 . 19  0.96  Dickson Quality Index  0 . 39 0.12  0.41 0.12  0.24  n  Total mycorrhizal colonization (96) Shoot mass  (g)  Shoot length  (cm)  (g)  The top number is the mean, the number underneath is the standard deviation. The p value in the table is from ANOVA.  47  levels  E—strain  by  measured  mycorrhizae, none of the growth parameters  significantly  were  affected  inoculum was  by how the  applied. The results of the pine trial are shown in Table III.  Pine.— The  affected  significantly level and  of by  E-strain the  pine roots were not  on  application  method.  The mean  of infection for the injected treatment was 62% (SD for  the  the  top—applied treatment was 72% (SD 25).  levels  techniques.  A51  of  significantly  (Suillus  different  were  tornentosus)  (p=0.056)  for  the  21)  However, marginally  application  two  The mean level of A51 on the injected treatment was  (SD±. 12) and for the top—applied treatment was 0.76% (SD  3.55%  The  3.00). were for  levels  colonization  not any  between  levels  total  of infection (all species of fungi)  different  between injected or top—applied treatments  treatment.  There were significant growth differences  injected and top-applied treatments.  These differences  could not be attributed to one specific group such as the fungal treatments, growing medium treatment or controls, but applied to the  subset  average, collar the  10%  all trees.  of  smaller  diameters  top-applied  root  The injected treatments had, on the weights  (p=0.O3),  6% smaller root  (p=0.01) and 10% shorter shoots treatments.  The  Dickson  (p=O.O2) than  Quality  Index  is  significantly (p=0.Ol) higher in the surface-applied treatment.  48  Table III  Comparison of Injection Into the Plug Versus Top Application of Ectomycorrhizal Mycelial Slurry to Two Month Old Lodgepole Pine Treatment Parameter  Top Applied  In:Jected  p ANOVA  n=  284  318  S. tomentosus mycorrhizae (96)  0.90 6.23  0.23 1.64  0.12  E—strain mycorrhizae  17.4 30.0  18.7 33.9  0.11  (96)  Thelephora mycorrhizae  77.7 29,6  75.2 33.0  0.07  (96)  98.3 7.74  96.3 10.9  0.07  0.91 0.29  0.90 0.29  0.61  18.5 3.21  17.5 3.19  0.02  Root Collar Diameter (mm)  2.96 0.46  3.12 0.51  0.01  Root Mass  0.50 0.17  0.55 0.18  0.03  Abundance of Mycorrhizae  2.38 0.66  2.30 0.70  0.40  Root to Shoot Ratio  0.61 0.57  0.64 0.23  0.46  Dickson Quality Index  0.17 0.06  0.20 0.07  0.01  Total mycorrhizal colonization (96) Shoot mass  (g)  Shoot length  (cm)  (g)  The top number is the mean, the number underneath is the standard deviation. The p value in the table is from ANOVA.  49  DISCUSSION  Contrary  the  to  findings  of  Boyle, Robertson and Solonius  (1987)  who  worked  with Hebeloma longicaudum and Fortin et al,  (1988>  who  worked  with  applying  the  E—strain  fungal  and  inoculum  Amphinema  application  method  applied  pine,  to  Laccaria  did but  worked  make  both  quite  byssoides.  not it  bicolor,  methods  successfully for  Interestingly,  and  climatic  pine  were  the  any difference for E-strain  did for E-strain applied to spruce,  though in both cases the infections were quite substantial. spruce  of  inoculated  at  The  different times, so the  conditions at the time of application were different.  The spruce was inoculated earlier than the pine and the inoculum was  older by the time the pine were inoculated.  that  the  fungus  had  It is possible  started to grow after the blenderization  process, and so actually increased in vigor by the time the pine was inoculated. the  success  slurries for  of  grown  Apparently, some fairly subtle variables affect these  techniques,  but  in  general, mycelial  in dilute nutrient agar can be used as inoculum  some fungi without washing away nutrients, whether injected  into a plug or top—applied. The behave  A51  (Suillus tomentosus)  differently,  depending  treatment in pine appeared to  on  application  method.  This  fungus was very borderline in its ability to form mycorrhizae in the  nursery.  forming  The  limitation  that  kept  it  from vigorously  mycorrhizae in the nursery may or may not be related to  the different response to inoculation technique.  50  One could  speculate it  was  the  that the A51 isolate had a short life expectancy once released Into the environment, so intimate contact with  roots  However,  other  growing been  might  have  Increased  explanations  conditions  suitable  near  are  the  the  Inoculation  possible,  for  success.  example, the  surface of the plug may not have  for Suillus tamentosus.  More study is necessary  to answer this question. No  significant  spruce, Either  differences  in  growth  were  evident in the  even though there were differences in infection levels. application of  objective  the  trial.  then  colonization, advantages  technique  circumstances.  If  If the desire is to achieve maximum into  injection  some  for  fungi,  the  be used, depending on the  could  or  the  in  growing  some  as  medium  yet  undefined  object is just to get fungi to grow on  the  roots, and ease of application is the primary concern,  the  inoculum  and/or  might  has  be  top  applied.  circumstance—variable,  and  then  Again, this is species-  the  limitations  of  both  techniques need further exploration. one interesting result is that the injected treatments in pine had  smaller  root mass,  root collar diameters and shoot lengths  than the top-applied treatments. attributed injected  to  one treatment.  controls  were  These differences could not be The means of the top—applied and  grouped  closest  measurements, except root collar diameter. injected  and  top-applied  for shoot weight, collar  diameter.  for  all  The means of the A51  treatments were contiguously grouped  but not for shoot length, The  together  root weight and root  most consistent source of the variation  between injected and top—applied treatments seems to come from  51  the  E—strain  A51  treatment,  and  fungal growing medium treatments, though the  in general,  has spreads between the means of the  injected and top-applied treatments. growing  medium  fungus effect.  treatment  seem  The differences within the  to indicate that this is not a  The injected growing medium treatment was bigger  than the control, and the top—applied treatment was smaller than the  control  in  for  reversed  measurements.  length and shoot mass measurements, but.  shoot the The  root  collar  and  diameter  root  weight  fungal treatments followed the same pattern  having larger shoot length and mass measurements and smaller  of root  collar  treatments. treatments,  diameters  for  The  that  fact  the the  injected  versus  top—applied  inoculum was applied to both  just in different manners, seems to suggest that it  just a nutrient effect.  was  not  one  would have expected the same response in growth, regardless the  of  application  occurred  from  injected  and  But,  this  However, some dripping frequently of  bottoms  tubes  that  had  the inoculum  could be related in some way to the effect.  if it were an effect that was altered by dripping from the  tubes,  one  might  top—applied in  However, fungus the  the  method.  If it were a nutrient effect,  treatment, three  it  expect  out  to  where of  be there  more  pronounced  was  little  in the  dripping.  four measurements, the top—applied  growing medium treatment is more similar to the means of controls  than  is  the  injected  fungus  growing  medium  treatment. Other growth  possibilities of  some  are that the growing medium promoted the antagonistic  organism,  or  that  carbohydrate-rich growing medium encouraged the growth of  52  the  organisms  that  tied up nutrients, but that this effect somehow  did not occur when the inoculum was surface applied. be  if for example, the growing medium on the surface  i:’ossible,  desiccated possible  This could  and that  did not promote the growth of organisms. the  injected  growing  medium  itself  It is was  antagonistic  to  process  was  disruptive in some way such as the transmission of  disease  or  greater  similarity  applied  or  resolution  seedling  disturbance  injected of  this  roots,  of roots.  between fungus puzzle  the  and/or  that  the  injection  The absence of an obviously controls  growing  and  medium  either the top treatments makes  almost impossible, without further  study.  53  6. A LOW-TOXICITY STAIN FOR EXAMINING ECTOMYCORRHI ZAE  It  is  often  ectomycorrhizae Hartig  net.  useful to  to  stain  facilitate  Microscopic  the  fungal  observation  of  portion  the  of  mantle or  examinations of fungi in root tissue  commonly use traditional stains, such as Trypan Blue and Aniline Blue,  which  When  are  examining  exposure taken.  to  carcinogenic and/or teratogenic and/or toxic. large  dye  numbers  can  be  of  quite  roots on a daily basis, the high, even if precautions are  The risk of exposure is particularly high when preparing  dyes from powders or when heating slides to drive off bubbles or set stains.  The safety procedures that should be used with such  stains  be time—consuming when shifting repeatedly from low  can  magnification  observation  magnification  with  clearing  staining  several  and steps  separating  requires use  on  hyphae  et  less  al.  to  and  disturb  Hayman, the  root,  virtually  the  plug.  staining  (1986)  uses  Acid  power  require  1970)  rhizomorphs The  high  procedures such as  and  which  makes  useless  for  technique  of  Red 112 as the dye and  It could probably be adapted for  plugs to take advantage of intact extramatrical  rhizomorphs,  distinguishing probable  on  stain  staining  (Phillips  manipulation.  intact and  Some  severely  hyphae  types  Daughtridge  stain.  and  extramatrical  without  different  but the efficacy of the technique for types  is  not  evaluated,  and it is  that the loss of colour would cause more dependence on  microscopic carcinogen,  characteristics. toxic to breathe,  Also,  Acid  Red  112  is  a  ingest or touch and produces toxic  54  fumes  on  heating  (Canadian Centre for Occupational Health and  Safety, MSDS record no. As  a  blue  244572).  safe, convenient alternative, we have successfully used food  colouring  manufactured  by  Specialty  Brands  (65  International Boulevard, Suite 206, Etobicoke, Ontario, M9W 6L9) to selectively stain the fungal portion of ectomycorrhizae. stain  is  fairly  months.  It  having and  readily  and persists at least for several  available  and consistent in quality,  meet Food, Drug and Cosmetic Act  to  is  is  lightfast,  The  (FD&C) requirements  also presumably quite benign, though Miller and Nicklin do cite some minor reactions to ingestion of the primary  (1980)  dye contained in this food colourant. The  product  Ribbon  used  Food  in this method is the blue dye in the Blue  Colour  manufacturer  Preparation.  indicate  that  <6% a subsidiary colour. as  Brilliant  42090  and  Discussions  eriogluacine.  is  The main colouring ingredient is known  also  Conn  commonly  (1977)  known  describes  Its Colour Index is  1.  to  it  staining uses to those of Alphazurine A, C.I. for  Aiphazurine  A  the  the dye is >85% total colour, with  Blue FCF or FD&C Blue No. it  with  are given.  histologists  as  having  as  similar  42080, but no uses  Staining Procedures  (1973) also  does not list any uses for either dye. The  dye  dilution per is  5Oml placed  is in  prepared  for use in staining ectomycorrhizae by  70% lactic acid at a rate of about 20 drops of dye  of lactic acid. in  The whole mycorrhiza or thin section  a drop of the staining solution on a slide.  The  slide and stain are heated over an alcohol lamp until the lactic acid just about boils.  This sets the stain and drives off  55  bubbles.  The stain may be drawn off with absorbant paper.  is  particularly  to  the  thin  useful if small amounts of wax are attached to The molten wax can be drawn off without  section.  disturbing the sections.  overly KOH  solution  The  stain  or  other  colour  solutions  on  is  the  green.  Slides  months)  by  This  More stain, clean lactic acid,  mountants, can be put on the specimen. pH—dependent,  specimen  so  putting  different pH  can change the colour from blue to  be preserved semi-permanently (for several  may  sealing  the  edge  of  the  coverslip  with  clear  fingernail polish. This stain is useful for most unpigmented or lightly pigmented fungi.  The  stain  pigmented  darkly  is  not  visible,  or may not be taken up by  fungi such as Cenococcurn geophilurn.  There is  also some variablity in how lightly pigmented hyphae take up the For  stain. may  This  example,  Endogone  seems  to stain very brightly.  be a useful characteristic for helping to distinguish  fungi. Figures No.  12  and 13 show a fungal mantle stained with FDA Blue  Figure 7 shows an E-strain Hartig net stained the same  1.  way. The  simplicity  associated  with  of the  the stain  procedure make  and the negligible hazard  this technique ideal for the  researcher examining large numbers of ectomycorrhizae in detail, for  unsupervised  student use and for general application where  special safety equipment is unavailable or inconvenient to use.  56  7. EXPERIMENT THREE  THE SELECTION AND EVALUATION OF ECTOMYCORRHIZAL FUNGI TO ENHANCE PERFORMANCE OF SEEDLINGS PLANTED UNDER NORMAL REFORESTATION CONDITIONS  It  been  has  situations, (Marx, are  adequately  any  mycorrhizae Danielson  1977).  better  than  established are  in  than  in  extreme  no mycorrhizae  has countered with “some fungi  (1988)  others”  better  that,  reforestation  situations.  The  question  that needs to be answered before mycorrhizal fungi can  be  for  used  “which ask  many  “which”,  there  are  (1988) many  unsolved mysteries such as: are  early  stage and late stage successional fungi and  nursery  fungi  be selected on this basis,  types  of  why can’t we get  putatively late or multi-stage fungi to grow on  stock and do nursery fungi continue to offer advantages  when  field  fungi colonize the roots.  suggested in the Introduction. specific over  points out that before we can  truly  nursery even  Danielson  fungi?”  there can  inoculation in normal reforestation situations is  from  ecotypic  is  Other unknowns are  They include such things as:  specificity,  is the fungal inoculum left  a mature forest suitable for seedlings, do different  fungi become more active as environmental conditions change, much  how  colonization  does  it take to affect growth,  how  can seedling  performance be improved by introducing exotic fungi and how long does  it  take  for  a  seedling  complement for a given site.  57  to  acquire the optimum fungal  These  unanswered questions suggest some areas that need to be  better  addressed  in  that  established  mycorrhizal  research.  ecotype variability exists  It  has  been  (Trappe and Fogel,  1977) but what is not clear is how important this variability is to  seedling  determine  or  tree  this  may  descriptions that  two  performance. result  Part  from  are  the  failure to  inadequate  (see Literature Review). ecosystems  of  ecological  It is easy enough to say  different  when  they  are  greatly  different, but the manipulator of ectomycorrhizal fungi needs to know how big a difference is relevant to the mycorrhizal fungus. A  precise  trial  description of ecotype is needed in each outplanting  to  resolve  this  question.  reforestation  situations  potential  at  normal  inoculum  for  clear  that  studies  things as the inoculum  seedlings and others as outlined above.  It seems  these questions will be answered only by conducting  in  normal  reforestation  mycorrhizal  such  afforestation  further  such  sites, the suitability of residual fungal  consider as  include  Problems related to normal  studies  areas.  Recommendations  to  inoculation in some extreme circumstances  at  should more  not  normal  be  mistaken  sites  are  to mean that of  no  value.  Questions related to the behaviour of fungi on the host such as; how much colonization is needed to affect growth, how many types of can of are  fungi be  normally  inhabit a root and so on as outlined above,  addressed only by careful observation and documentation  the mycorrhizal fungi in defined situations. problems  inoculate  Finally, there  relating to the inability to isolate, culture and  many fungi.  Many questions will remain unanswered as  long we are dealing with only a small fraction of the diverse  58  population of ectomycorrhizal fungi. For  the  ectomycorrhizal inoculation component of this thesis  research,  a  mortality,  site was selected that typically had high seedling but  encountered  by  characterized  within  in  detail  V.  J.  details  of  the  Identification Land  Region”,  normal  fungi  using  the  in  the  range  of  area.  conditions The site is  ecological classification  Kraiina as the basis for site description. approach are included in “A Guide to Site  and  Interpretation  Management  comprehensive  This  the  mycorrhizal  of  approach The  was  for  the  Kamloops  Forest  Number 23, February,  Handbook  1990.  biogeoclimatic classification system covers  many variables but overlooks the critical ectomycorrhizal fungal population. the  of  Therefore,  fungi  study  the  guideline  present on seedling roots occurring naturally in  area. for  this study also includes a description  This type of information will serve as a good  applying  the  result5  of  this  study to other  situations. fungi  The derived  selected  for  evaluation  from a variety of sources,  in this study have been  in order to address what has  been demonstrated, and is suspected about ecosystem specificity. Fungi have been selected based on their presence on the roots of seedlings by  in the study area, but the fungi used are not limited  this criterion, since exotic  distant where appear in it  (defined here as meaning from a  location and therefore, most probably, other  site  from a situation  characteristics are different as well)  fungi  as likely to perform well outside their normal range, as (at least, at this early stage of investigation).  Iwan Ho  (1987) demonstrated that there can be significant within—species  59  variation  important  in  isolates  physiological characteristics, between  from the field or from the nursery, so fungi were also  isolated from the nursery. based  their  on  used  slow-growing. light  colonization  For  in this study vary from very fast to very  Seedlings  intentionally  fungi were not selected  perceived ease of commercial application.  fungi  example,  In addition,  by  outplanted in the field had heavy and inoculated fungus.  the  avoided  applying  This study has  previous conceptions of what a  good fungal inoculum should be. included  Finally, descriptions  of  in  the  the  observations  are  detailed  behaviour of the fungi after outplanting.  Even  if significant seedling growth effects are not found,  type  of  observation  behaviour  be  of  valuable  understanding  in  specific fungi  Observations involving some destructive sampling in  but  sufficient  will  serve  outplanting  trials,  information  that  behaviour  of  so  will  first growing  seedlings were planted so that several  years of observation could be made.  approach  the  in defined ecological  study were made after the possibly critical,  season, more  action  and  situations. this  may  this  as  a  at  least  contribute  ectomycorrhizal  or  model  fungi  a  It is hoped that this protocol  basic  for  future  minimum amount of  to the understanding of the can  be gleaned from each  trial, and the technology of mycorrhizal fungal inoculation will be advanced in a systematic and comprehensible way. The  issues  of  culturing  and inoculation of fungi have been  dealt with in Experiments One and Two of this thesis.  60  MATERIALS AND METHODS  Selection.—The  Site problem  for  seedlings sites  site  presents  a  typical reforestation  the area in that it is hot and relatively dry with  sometimes  suffering  up  to 70% mortality on similar  (Gary Hunt, personal communication). two  (1988)  The site was logged  years prior to planting but had received no special  treatment such as burning or scarification. Site  Description.—  The  nursery  work was carried out at the  Heffley Reforestation Centre and the outplanting site was in the Shuswap  Highlands,  08’W).  Heffley  8km  northeast  Reforestation  of Barriere  Centre  is  (51.16’N_12O.  about 20km north of  Kamloops and Barriere is about 36km north of Heffley Creek. As  has  been  discussed  in  the  Introduction and Literature  Review,  it seems that one of the most critical factors that may  affect  the  interpretation  ectomycorrhizae came  from  of  outplanting  trials  with  is the description of the site where the fungus  and where the outplanting trial was done.  It may be  that forest sites are so diverse that they defy description on a scale large enough to be of any value in predicting the behviour of  inoculated  mycorrhizae.  However,  the  British  Columbia  Ministry of Forests uses a hierarchical ecosystem classification system that allows description to a very specific site level. As well, the system provides all the information necessary for many types of grosser comparisons.  Classification is based on a  61  broad  range  characteristics including vegetation, soil and  of  geographic characteristics. was  place  recently mapped using a new version of the ecosystem system  classification area in  The area where the field study took  (Lloyd et al,  used  1990).  by  the Ministry of Forests in the  In addition, the site was classified  detail by the author and Gary Hunt based on existing mapping  and  as  procedures  outlined  in  Lloyd  et  al,  1990.  The  classification procedures consist of determining such factors as elevation, texture,  aspect,  slope,  soil  type,  pH,  this  information  forest cover and types of understory plants.  described  as  classified,  the  variant  level.  as  classification,  the  site  has  been  and supposed climax forest.  site was determined to fit into the category  IDFdk2. which  interior  Douglas—fir,  type  this  zone.  The is  the  which The  “IDF”  refers  coarsest is  “dk”  specifically “dk” means dry cool. of  Once  it is possible to infer some other characterisics of  outplanting  described  Based on  it is possible classify the site to the level  the site such as general climate  in  material, soil  humus form, soil moisture regime, soil nutrient regime,  soil  The  parent  to  category.  the  zonal  IDF means  the predominant climax forest refers  to  a subzone rating,  The “2” refers to the variant  the subzone and refers to a very specific mix of vegetation,  soil  moisture  material  and  regime, soil  aspect,  successional  nutrient regime.  Thus,  stage,  parent  this classification  system can result in very site specific information about sites. If  this information is not sufficient to describe sites for the  purpose  of  describing  the behaviour of ectomycorrhizae,  probable that it will not be practical to do so.  62  it is  Lloyd  et  al,  1990  indicate  that the climate of the IDF is  continental with warm dry summers and cold winters. moisture Frosts about  deficits  are  common  Substantial  throughout  the growing season.  are common in June and late August.  The “dk” subzone is  in the middle of the IDF zone in terms of temperature and  moisture regimes and is very common in the study area. represents  it even  though  subzone.  a  normal  seedling  As such,  reforestation situation for the area,  mortalities  can  be  very  high in this  The specific site description is shown in Table IV.  TABLE IV Detailed Description of Outplanting Site Elevation— 1150m Aspect- SW Slope— 5 to 18% Soil  Type- Orthic Humo—Ferric Podzol  (Canadian System of Soil  Classification) Parent material— glacial till Mineralogy- mixed Soil Texture- sandy loam Coarse Fragment Content— 77% (average for top O.64m) Humus  Form-  F horizon about 2cm, H horizon about 15cm: Humus at  site  described  was  disturbed  about  Humi—fibrimor  150m  by  from  (after Bernier,  Annual Precipitation (mm)— 568  the 1968)  (623—843)  Growing Season Precipitation (mm)— 221  63  logging  (185—313)  but  was  site and was a  Annual Snowfall  (cm)— 222  Mean Annual Temperature  (178—264)  (C)- 4.1 (2.5-5.9)  Mean Growing Season Temperature  (C)- 11.1  (9.7—13.4)  Mean Minimum January Temperature  (‘C)- -11.9  Growing Degree Days  (953—1266)  Frost Free Period Biogeoclimatic  (>5C)— 1133  (days)— 95  Zone-  (-13.5-—1O.6)  (62—132)  Cascade Variant,  Dry  Cool Interior Douglas-fir  Zonal Site  (IDF dk2)  Supposed Climax Forest- Douglas-fir Soil Moisture Regime—  Submesic,  borderline subxeric  Soil Nutrient Regime- Medium Soil pH— Ae 6.0  (5.1 in CaCl,), Bf 5.2  (5.3 in CaCL.)(The Bfl  measurements were repeated four times on  two  different pH meters and same  pattern occurred each time) Observed  Forest Cover—  Predominantly lodgepole pine, small (109&)  Engelmann  uphill  from  spruce  clearcut,  component increasing  Douglas-fir downhill Observed  Understory-  pinegrass kinnikinnick spirea  (8%),  (20%  of  (8%),  twinflower  saskatoon  Prince’s pine  (3%),  ground  (8%),  (5%), lupine  (1%), soopolallie  (10%),thimbleberry (5%)  64  cover),  Observed  Mycorrhizae  On natural lodgepole pine seedlings to  years old): Suillus—like  5  observed others  (60% of  mycorrhizae), E—strain (30%),  (10%)  natural  On  (1  years  spruce  old):  E—strain  seedlings  Arnphinema byssoides  (30%),  others  (1 to 5 (50%),  (20%)  The common names given here correspond to the scientific names shown in Appendix 3 of Lloyd et al,  The  data  methodology  in  Table  outlined  1  in  were Lloyd  description is based on Bernier  1990.  mostly et al.  mycorrhizae  regenerated roads  on  (1990).  the  The humus form (1981).  The observations  roots were made by digging up ten naturally  seedlings  in  cutblock and along the edges of  the  in the immediate vicinity (within about lOOm for pine and  within 2km for spruce) examined 1990).  of the planting site.  The seedlings were  in the spring when the seedlings were planted (May 23, The  analyses T. M.  (1990).  using  (1968) not Klinka et al.  The climatic data is from Lloyd et al. of  collected  percentages  were  shown  are  crude  estimates.  done as per the Forest Soils Manual  The pH  (1987-88) by  Ballard.  Collection suitable  of  Isolates.—  To  increase the odds of getting a  isolate, several collection strategies were used.  The  first strategy was to collect sporocarps from similar sites in  65  immediate  the  outplanting  site.  vigorously from  vicinity  Sporocarps  few  a  (within  of  kilometres)  the  were collected from under young  growing seedlings, both natural and planted and also  mature  stands. were  sporocarps fruiting.  Unfortunately,  collected  were  the two seasons in which  very dry and there was little  This necessitated isolation from mycorrhizae.  Again,  young vigorously growing seedlings were selected from within the immediate all  vicinity of the site to about 38km distant.  isolates  were  biogeoclimatic moisture the  resulted  were  in  Husted  to  encourage  received  a  potential conditions  isolates  in the nursery  of fungi from other sources G.  Hunt  (Heffley  Site 10, Comp 10, RR#3, Kamloops, B.  M.  (Kananaskis Centre for Environmental  Danielson  (Department B.  and  Centre Ltd.,  University  Columbia,  nutrient  mycorrhizal growth and this has  These included isolates from Dr.  R.  Research,  slopes,  Heffley Reforestation Centre uses a growing  Finally,  tested.  Dr.  similar  a wide variety of mycorrhizal fungi  Reforestation C.),  from  Isolations were also made from seedlings in  The  1989).  (Hunt,  and  regimes.  modified  regime  taken from the same or very nearly the same  zone  nursery.  However,  C.). lot  of  Calgary, Forest  Calgary, Alberta) and L.  D.  Sciences, University of British  Certain fungi were selected because they had of  nursery  of  attention inoculum  in this study.  from  so  they  other researchers as good were evaluated under the  The specific sources of the isolates  are shown in Appendix I. Isolation sporocarps  Techniques.— was  that  of  isolation  The  Molina and Palmer  technique (1982).  used  Initially,  small pieces of sporocarp tissue were placed on MMN (Marx,  66  for  1969)  and potato/dextrose agar plates, both with tetracycline.  to  seemed  be  consistently  potato/dextrose tetracycline concern,  was  revealed  different  mycorrhizae.  that  The  Danielson,  (1982),  Larsen (1978).  and  the  procedure  these methods. water.  Tween  placed  were  and without  tried for isolating from  (1984) and  These techniques are all quite similar  finally  used is an amalgamation of some of  Fairly large pieces of root were rinsed clean in  a  beaker  Sunlight  with about 300ml of water and one drop of detergent and stirred with a magnetic  dish  for five minutes. in  95%  hydrogen  removed  the  contamination was not a  Griffiths and Parkinson  The short roots were all removed and  ethanol.  They  remained  seconds up to about five minutes. 30%  with  so  Then, approximately 15 short roots were cut off and  in or  stirrer  MMN  techniques tried include those of Molina and  and  placed  Trials  bacterial  techniques  Zak  tap  dropped.  the  so the inclusion of the antibiotic was later stopped.  Several  Palmer  agar  on  better  Growth  at  peroxide five  for  second  5  in ethanol for a few  Then the roots were placed in to  45 seconds.  intervals.  The roots were  It was felt that treating  each type of mycorrhiza for varying periods of time would reduce the  risk of selecting against any particular fungus.  roots to  30  Benomyl  were mm. as  rinsed in cold and  placed  (3C) sterile distilled water for 15 on  but  MMN  or PDA with streptomycin and  per Danielson, Griffiths and Parkinson (1984) or on  MMN and PDA without Benomyl. slant,  The short  this  was  later  Initially, one root was placed per changed  to  two roots per plate.  Plates were easier to use for cleaning up colonies and observing  67  colony  morphology.  dropped, There  not  possible  Parafilm  to  to  it  sterile. Initially,  are  ruin is  Some  the  and antibiotic were dropped.  and  contamination.  extremely  the  to  ectomycorrhizal  and  described viable water in  in by  for  some  Any spore-forming  Colonies that looked like  fungi were transferred and if the new then  the isolate was saved on plates,  sterile distilled water under refrigeration, as Marx  at  and  least  technique.  which  and  grew only after as long as six weeks.  possible  pure,  growing  of the  over the long incubation  plates had to be watched closely.  appeared  Many  insure that plates are truly  were cut out and discarded.  slants  slow  cultures  important  isolates  Plates were poured as thickly  colonies  culture  isolates, the PDA was  many  effects of drying and wrapped with  drying  fungi  can  so  Benomyl  the biocides.  reduce  contaminants  making  to be a marked decrease in the success of  delay  ectomycorrhizal  period,  the  seem  without  isolation as  later  and  did  After  Daniel three  (1976).  Some cultures remained  years using the sterile distilled  A slight modification was made to the method  cores were taken from the agar plates.  A thin-walled  hollow glass tube with a firm rubber bulb on one end was used to suck up cores for transferring. the  glass  plugs  from  not  being pulled up too far. large  (1982) work  supplemented  about 5mm above the cutting end to prevent the  tube  transferring Palmer  A small stricture was placed in  numbers  note  well  of  This is a useful tool for  plugs efficiently.  Molina and  that the cold water storage technique does  for  this one.  all  fungi  so  the  other  two  methods  Plates with thick agar and wrapped with  Parafilm kept for several months, and served as a good source of  68  working  material  for  starting  cultures  or  for  small scale  inoculation trials. Prescreening suggested  of  Hutchison  and  Malloch  (1988)  four criteria for determining which cultures isolated  mycorrhizae  from  Cultures.—  might  actually  be mycorrhizal fungi.  These  included the absence of conidia, slow growth in culture, absence of  cellulase  and  foolproof,  not  Wilcoxina fungi  produce  determined.  most it  This guideline is  since some ectomycorrhizal fungi, e.g.  grow quite rapidly and some ectomyeorrhizal  cellulase. of  Furthermore,  ectomycorrhizal  the complete range in fungi  has  not  been  To avoid erring on the wrong side, cultures were to  discarded reliable became  however,  mikolae,  characteristics  be  the absence of pectinase.  only  if they produced conidia, since this is the  indicator of a non-mycorrhizal fungus.  impossible  to  However,  use all the isolates in pure culture  synthesis, so some of the faster growing Isolates were not used, and  isolates that appeared to be duplicates were also not used.  69  Culture  Near—Pure  synthesis  culture  was  system  apparatus  was  designed.  moistened growing mixture, bags  and  very  thin  autoclaved plastic,  Seedlings  were  reported  sterilized minutes  in  The pots were filled with  placed in Fisher autoclavable plastic  for  Look oven bags, which are  one hour.  were  autoclaved  germinated  separately for one hour.  aseptically using the method of Zak  Molina and Palmer,  Seeds were surface  1982).  in 3096 hydrogen peroxide for times ranging from five to  one  non—quantified about  base of the  The hole in the bottom of the pot was  with silicone rubber sealant.  sealed  The  a five—inch clay pot filled with a 4 to 1 by volume  peat-vermiculite mixture.  (as  After many trials, a new pure  Synthesis.-  30  hour  evaluation  minutes  lodgepole  pine  germination  at  of  and  five—minute of  the  surface  and  plates,  spruce.  shorter  was  Longer  times  Based  on  it was decided that  sterilization  Engelmann  rates  intervals.  optimum for times reduced  resulted  in  higher  contamination. When  the  flow  hood  oven  bag was attached to the lip of the pot with tape to form a  pots with  chamber  (See  moisture  and  clay  pot  plastic throughout substrate  had cooled, they were each planted in a laminar  1).  dissolved  and  the  some  pot.  procedure,  for molds.  The chamber was not sealed in that  nutrient could pass through the porous  presumably,  this  4cm deep tray.  germinants and the open end of the Look  Figure  and bag  three  gas could diffuse through the  Careful as  the  monitoring  was  needed  growing medium was a good  After planting, the pots were placed in a  In later trials the tray depth was changed to  70  the  same  as  depth  the  polyethylene  supported  subirrigateci  by  pots by  filling  The  (13cm).  a  wooden  the  tray  tray was made of  frame.  The  with water.  pots  were  The pots were  placed in a growth chamber, and on an open bench with additional incandescent  and fluorescent lighting.  The day lengths were 16  hours on the open bench and 21 hours in the growth chamber. temperature on  in  The  the growth chamber was 18C and the temperature  the bench was ambient, which was typically around 2OC.  trees  in  photoperiod  the  growth  for  the  chamber  bench  grew  much  faster,  The  and  the  seedlings was also increased to 21  hours/day. The  experiment  fertility used  in  practise in  the  tested  levels. the  Only  final  runs.  for  were tested  trees  per fungal isolate.  lOOug/L, consisted  of  some isolates from spruce were tested  (one pot) at each fertilizer level,  the  in  and  5Oug/L  (lodgepole pine) was  their ability to form mycorrhizae.  trees  concentration  one tree species  trial, though spruce and pine were used in  However,  nursery  39 fungal isolates at three different  urea  thus nine  The fertility levels were based on N  water Oug/L  two  used to flood the trays, and were of  weeks  N. after  The initial fertilization planting  the seedlings,  followed by another urea fertilization after three weeks. this, Shur  Three  After  the plants were fertilized every two weeks with Greenleaf Gro  20—20—20  water-soluble fertilizer.  This is a fairly  complete fertilizer that also has boron (0.O2), copper chelated  iron  and  (0.05%).  zinc  (0.106), manganese  (0.O5),  (0.O5), molybdenum (0.0005%)  The application rates were based on the N  71  already described.  rates  The fertilization procedure consisted  of dissolving the appropriate amount of fertilizer in sufficient water to fill the tray to the top. to  stand  medium  for  was  Watering  but  moisture  after  stress  slime  hydrophobic  and but  solution.  two  began to form on the pots.  was  The  pots  were kept moist at  months, an attempt was made to increase  allowing  by  This  die,  long,  If the nutrient  accomplished similarly, except tap water was used  waterings.  not  too  nutrient  of  first,  two days and then siphoned off.  left  was  instead  The tray was filled, allowed  a  would  the  mistake,  pots  to  dry  more between  as the dry peat moss became  rewet only very slowly.  The plants did  some were nearly dead and did not recover by the  end of the experiment.  The growing medium was unsuited to this  type  The peat—vermiculite mixture was chosen  of  manipulation.  because  it  is  used in the nursery where the next phase of the  experiment was conducted. The  germinants were transplanted on October 3,  Eight  weeks later,  growth  developed,  the  at  about  dilute  inoculum fine  inoculated.  1988.  1988 the trees in the  By this time, the roots were  with a number of short roots.  The trees  open bench were inoculated from January 3 to January 5,  the  1989,  were  chamber  quite—well on  on December 5,6 and 8,  4 and 5,  was  agar  12 weeks of age. technique  prepared  by  The inoculum was grown using  described  in Experiment One.  The  treatment in a blender until it was  enough to pass an 18 gauge needle.  The different cultures  varied  in resistance to disintegration and the blendering times  varied  from 15 to 45 seconds.  These were well within the times  described by Boyle, Robertson and Salonius  72  (1987) as having  little  effect  separate very  on  the  growing  difficult,  cultures  especially  alcohol  safety  previously  viability.  medium  uncontaminated.  burning flow  inoculum  in  Attempts were made to  from the fungus, but this proved since it was necessary to keep the  The  blender  cup  was sterilized by  the cup while rolling the cup in a laminar  cabinet.  The  sterilized  blender cup was sealed with  metal  aluminum  foil.  The mycelial slurry was  into a 5OmL syringe through an 18 gauge needle.  sucked  Filling  the syringe this way greatly reduced the likelihood of blockages during the inoculation procedure. inoculate  To  the  through squirted  the  bag  into  seedlings,  covering  the  the  the  syringe  pot  and 2OmL of inoculum were  was simply poked  growing medium (See Figure 2).  The bag was  with 70 ethanol before injecting and the hole was sealed  wiped with  a piece of masking tape after inoculation.  The medium and  low fertilizer treatments were reinoculated on February 10,  1989  and  the high fertility treatments were reinoeulated on February  14,  1989.  The seedlings were reinoculated because preliminary  examinations procedure  revealed  was  changed  few  mycorrhizae.  slightly  The  inoculation  for the second inoculation in  that a sterile pipette was poked through a small slit in the bag and  the inoculum was placed well down into the growing medium.  This bottom  was  done  of  the  because most of the roots were located near the pots.  The  pots  received 5mL of inoculum the  second time. The 1989,  high—fertility  treatments  were  harvested  the medium—fertility treatments on March 24,  low-fertility treatments on March 28,  73  1989.  on March 23, 1989 and the  The roots were  examined  under  them  a  tray  of  mycorrhizal  were  hand sectioned after allowing the root to dry  in  for  about  30  a dissecting scope after rinsing and submerging water.  seconds  Any  short  roots  that appeared  and then embedding it in paraffin.  The  partial  drying of the root was necessary to allow good adhesion  to  the  paraffin.  it  on  the  end  microscope with  a  of  slide  hot  longitudinal The  The root was embedded in paraffin by placing a  drop  of  paraffin  that was stuck to a  and then heating the paraffin around the root  needle.  The paraffin was cooled in cold water and  sections were taken with a hand-sharpened scalpel.  sections were placed in a drop of stain  heated drawn  to  melt any clinging paraffin.  off  lactic  with  acid  slices.  was  a  piece added  Longitudinal  length  (see Section 6) arid  The melted paraffin was  of absorbant paper and more stain or before  sections  placing  a  coverslip over the  allow observation of the whole  of the root to avoid missing small spots of colonization  as described by Danielson, Griffiths and Parkinson (1984). None  of  the  general procedures of fixation, dehydration and  clearing need be used for routine examination of ectomycorrhizae by  this  method.  paraffin of  to  Individual short roots are simply embedded in  immobilize them for sectioning.  The whole process  sectioning and staining a root takes a few minutes and it is  possible (1986)  to  see  most  of  the mantle views required by Agerer  for complete description of ectomycorrhizae.  Trial Inoculations in the Nursery.— Seven fungal isolates were inoculated of  1988.  and A29  onto  eight week old spruce seedlings,  The isolates used were A18,  (Appendix I).  in the spring  A18B, A19, A20, A21, A22  Ten seedlings were inoculated with each  74  isolate. colony  The inoculation procedure involved fragmenting a large from an agar plate in a blender as per Danielson, Visser  and Parkinson  (1984),  except that the colonies were chopped in a  Waring blender and the mycelial suspensions were not centrifuged or  washed.  Ten millilitres of suspension were inoculated into  the plugs of each tree.  The roots were examined on November 29,  1988 when the seedlings were eight months old. Preparation the  for  early  trials  described  different  for  Inoculum for the Nursery.— The fungi selected  nursery  technique  of  of  using  grown  in Experiment One.  periods.  as .January,  the  were  the  dilute  agar  The inoculum was grown  of the isolates were started as  Some  1989 because they were slow-growing.  Several  slow—growing isolates were started well before the pure  culture  synthesis  were complete.  trials  Cultures that became  contaminated were discarded and replaced by new cultures, so the inoculum  final various  inoculum The pack  used  ages. for  all  took  It these  cultures  in  a  of  of cultures of  two months to prepare  reasonably large scale inoculation trials.  The  contents of several jars were fragmented in a  blender  and mixed together.  for  seconds.  colonies  minimum  consisted  were grown in batches of dilute agar in 75OmL hot  jars.  30  cases  in  most  This  The cultures were first fragmented  seemed  cases,  so  more than adequate to break up time  was reduced to 15 seconds,  keeping in mind the work of Boyle, Robertson and Salonius which  seemed  to  suggest  resulted in better inoculum. blender  (model  Connecticut)  31BL92,  that  shorter  fragmentation  (1987) times  Fragmentation was done in a Waring Waring  operated at low speeds.  75  Products,  New  Hartford,  Boyle, Robertson and  Salonius wet  of  weight  slurries  were  with  it  good success with inoculations of 2?6 by  fungus  used  washing paper  found  (1987)  water.  to  determined  lOOmL  of  by  concentrations of the  The  taking  SOmL sample and  boiling water on a piece of filter  a Buchner funnel with suction.  in  a  The wet weight of the  filter  paper  filter  paper on the same apparatus until the free moisture lust  had  disappeared. manner  The  and  sufficient paper.  filter  paper was weighed five times in this  it was determined that 5OmL of sample would provide mass  The  to  mask  mycelial  concentrations Salonius  been previously determined by suctioning the  in  the moisture fluctuations in the wet slurries  were  diluted  give  excess of those used by Boyle, Robertson and  (1987).  inoculum was grown in 500mL or 600mL batches.  The  to  different  numbers  concentrations  of  batches  of the  of  within  fungus  each  fungus  There were and  the  each batch varied.  The  batches were fragmented, combined and then subsampled and washed with  wet weight. made for  water to determine the concentration of fungus by  boiling  The  inoculum was divided in half and the volume was  with distilled water to 5L for the spruce and up to 4L  up the  pine.  The dilutions with water were made lust before  inoculation and were different for pine and spruce because there were  fewer  inoculum  in  pine  seedlings.  percent  The  final concentrations of the  wet weight of mycelium of total weight of  inoculum are shown in Table V. Most 25,  of  1989.  spruce  the spruce seedlings were inlected on May 24 and May The  Hecr  on June 15,  8 and A51 treatments were applied to the  1989.  The inoculum was placed in storage at  76  2C  until  June  27  and  June  28,  1989,  when  the pine were  Inoculated.  TABLE V CONCENTRATIONS OF INOCULUM USED IN THE NURSERY INOCULATION TRIAL  Isolate  &mycelial conc. 9  as wet weight/tot,  weight  A188-2 (Cenococcurn geophilurn): 3.36 spruce, 4.1% pine R947 (Danielson’s E—strain): 4.8% spruce, 6% pine 0188  (Husted’s E—strain):  A29  (Arnphinema byssoides):  A51  (Suillus tornentosus):  Hecr-8  6.15% spruce, 4.8% spruce,  7.89% pine 6% pine  not measurecl* on spruce or pine  (Hebeloma crustuliniforme):  not measured on spruce or pine  *_ Additional concentrate was added to these two isolates just before the trees were the inoculated, and mycelial concentrations were not calculated. The concentrations would have been the same order of magnitude as the other cultures.  77  At  this to  added the  time,  some  freshen the older inoculurn.  ectomycorrhizal  storage, preparing  additional Hecr 8 and A51 cultures were  fungi  were  though  sterile  the  dilute  agar cultures for injection.  Inoculation  was  applied  There were  in the E0188 culture.  were inoculated with 5mL of slurry.  with  connected  an the  was applied by (1) (2)  procedures were not followed in  of Seedlings in the Nursery.- Both the spruce and  seedlings  tubing  evident in the inoculum after  even  just a few small mold colonies  pine  Little growth of mold or  Oxford  pipetter  The slurry  (Model SA). A length of  pipetter to a lmL pipette.  The inoculum  injecting the mycelium throughout the plug or  squirting the inoculum on top of the plug.  In either case,  the body of the pipetter was kept below the injector in order to avoid siphoning. PSB  The spruce were grown in used and unsterilized  313a styroblocks  Beaver  Plastics, PSB  used  211  styroblocks. v:v).  (198 cavities per block,  Edmonton, (240  Alta.)  cavities  per  947 cavities/ma,  and the pine were grown in block,  1130  cavities/m)  The growing medium was peat and vermiculite  (4.4:1  Fertilizer was applied according to the schedule shown in  Appendix  III.  This  fertility  regime  has been shown by Hunt  (1989) to increase the level of mycorrhizal colonization and the number slow can weeks  of  types  release  of  mycorrhizae on the roots over those using  fertilizers.  be found in Hunt  (1989).  Other details of the growing regime Both tree species were about eight  old at the time of inoculation and had started to develop  short roots.  The inoculum was applied to approximately 50  78  seedlings  around  in  the  each  block,  perimeter  of  excluding  the  the two rows of cavities  spruce  blocks  to  reduce edge  fewer pine seedlings so only the two end  effects.  There  rows  one side row were excluded. The exact number of trees  and  were  inoculated was counted and recorded, and the inoculated area was marked  with  applied  felt  to  pen  and plastic markers.  20 blocks for the spruce  Each inoculum was  (10 blocks surface—applied  and 10 injected) and 16 blocks  (eight blocks surface-applied and  eight  the  fungus was  growing  diluted  injected fungus  were  for  medium  as  top applied.  growing for  pine.  One treatment was the  used to culture the fungi.  same  the  or  controls  The  injected)  blocks  medium  the  media  This medium  with fungi and was also  This treatment is referred to as the  treatment.  There  were  two  separate  the injected and top applied treatments, but they  identical in that they both had no treatment done to them. controls  treatments  manner  the  had  and  they  treated  and  same were  in  number marked  the  of  trees  as  the  other  off and counted in the same  nursery  in  the same way.  After  inoculation, the blocks were placed in a randomized arrangement. Evaluation  of  preliminary  Nursery  examination  trees  was  taken  objective  was  to  10  possible  before  Trials.-  The  on September 14,  seedlings were given a 1989, and a sample of  from each block on November 3,  leave  the  examination,  trees as  1989.  The  in the nursery as late as  there  is some evidence that  mycorrhizal development proceeds most rapidly in the fall, after buds  are  November  set 3  were counted.  (Hunt,  sampling,  personal the  communication).  During  the  surviving trees in the treated area  Trees for mycorrhizal assessment were randomly  79  selected,  based  on their numerical position in the block and a  table of 6/49 Lottery numbers.  in  The remaining trees were wrapped  put in boxes and placed in cold storage at 2C,  cellophane,  according to standard operational storage procedures. The  sample  storage over  trees  were  placed  in a refrigerator at OC,  the  next  deterioration  several in  the  in  bags and put in  from which they were examined  months. quality  plastic  There was not any noticeable of roots over this period.  The  roots were initially examined by submerging the intact plug in a tray of water and looking at it under the dissecting scope. root  was then washed and re-examined.  longer  submerged,  as  was  it  The  Later, the roots were no  difficult  to see extramatrical  hyphae when they were wet.  The washing stage of observation was  discontinued,  removed  as  rhizomorphs, estimation  of  this  which  made  the  extent  extramatrical  observations  more  hyphae  difficult.  and The  of colonization of each plug by the  various fungi was dependent to a large extent on observations of extramatrical  hyphae.  Since  several  of  the  experiment  (E-strain,  Thelephora  terrestris  byssoides)  have-  indistinct  mantle  quite  fungi and  structure  in this Ainphinerna  at  low  magnification and it was not feasible to examine each short root under  high magnification, the extramatrical hyphae were used as  indicators of the extent of colonization by certain fungi. example, stiff  the hyphae of E-strain are thick,  (Figs.  E-strain  5,  6 and 7).  mycorrhizae  can  For  reddish—coloured and  Even under low magnification (7x), be  quite  accurately separated from  those of Thelephora terrestris and other fungi by the presence  80  of  these  hyphae.  The hyphae of Arnphinerna byssoides are fine,  pale yellow and tend to form dense mats.  They can be seen quite  easily  but lose much of their  when  they  are  dry  (Fig.  10)  distinctness when wet. The  types  fungal  approaches. apparent  The  types  were  identified using a synthesis of many  general  methodology consisted of identifying  from  intact  plugs  at low magnification (7x).  Minor uncertainties were dealt with by examining whole mounts in lactic  for  acid  detailed  mantle  evaluation  of  hyphal  and  root  the  characteristics.  was  More  accomplished using the  longitudinal  section  short  were  examined  microscopically, and this revealed  not  possible  to separate the different types of  that  roots it  was  mycorrhizae examined was by low  Hundreds of  with absolute confidence unless each short root was  under high power.  ambiguity, more  technique described earlier.  than  as it was common for short roots to be infected one fungus.  magnification  carefully,  as  Even when examining sections, there  it  as was  Roots tentatively identified under  being non-mycorrhizal were examined very frequently  found  that  rootlets  with  sloughing epidermal cells and root hairs had Hartig nets.  Since  it was not possible to examine each short root in detail,  it was  felt as  that the approach of relying on gross characteristics such extramatrical  power  microscopic  could  be  achieved  particular Appendix II.  hyphal appearance, augmented by frequent high examination on  the  was  scale  as accurate an approach as of  features of each fungal type  this  experiment.  The  identified are shown in  The estimates of percentage colonization were made  in increments of 10%, except for a spot colonization of for  81  example, around 10 mycorrhizae, observations  Other  shoot  diameter, and  length,  estimate  an  made  of  which is described as seedlings  on  included root collar  air dry shoot mass, air dry root mass  the  relative number of short roots.  The  of the relative number of short roots is based on a  observation  scale of 1 to 3, with 1 being few and 3 being many.  Determining  root weights presented a problem because some of the hyphae  the  (especially  Amphinema byssoides) bound the root so tightly that  it was difficult to get rid of all of the growing medium and the fungal hyphae. in  wide  a  washing The  Several techniques were tried,  stream  water  from a sink with much agitation,  under a narrow jet, and drying and shaking (very poor).  technique  that  an  on  roots  of  including washing  was  finally used consisted of placing the  approximately half-inch mesh and washing with the  spray from a Clarke Little Laser power washer. set  near  slight  lowest  fan.  growing used  the  This  medium.  setting and the nozzle was adiusted to a  method  It  roots  overall. lost  monitored intact.  in  this  The  considerably  less  and it appeared that most remained  root  than  number of short  but individual short roots were  process,  of  if  it appeared to be a satisfactory  were not made of the  spraying,  amount  away all of the fungi and  also strip away the root cortex,  However,  Counts  during  stripped  would  too vigorously.  method  The pressure was  any  lost  generally  technique  which  seemed  to  be  required manual  manipulation of the root. Two  statistics  were  derived  These  were  Index  (Dickson, Leaf and Hosner,  the  root  to  from  the  measurements taken.  shoot ratio, and the Dickson Quality  82  1960 as cited in Hunt,  1989).  Both  of  these  measures  morphological  balance.  used  are  indicators of seedling  The Dickson Quality Index is calculated  as: dry weight/height—diameter ratio Outplanting  as  Trials.-  The  +  shoot-root ratio.  outplanting  trial  was  randomized  block  treatments  that produced mycorrhizae in the nursery,  growing by  medium  design  with  the  treatments,  at  random.  except A51 The  fungal  (Suillus tomentosus),  treatments,  almost  all  the  the  top  toraentosus) cases,  (Suillus  applied  and  Trees were selected evenly from  injected blocks.  The A51  (Suillus  treatment did not generate heavy infections in most  and seedlings were selected for this group based just on  presence  heavily  or  absence of the inoculated fungus.  infected A51  before  outplanting  specific  The  A51  colonized by E—strain and Amphinerna byssoides when they  both  Inc.  except  were selected  correspondingly treated trees were very  placed into cold storage.  with  the fungus  The outplanted seedlings for all of  were  the  The three fungal  all had very high levels of the inoculated fungus,  tomentosus),  heavily  blocks.  treatment and the control were each represented  30 seedlings per block.  since  five  done in a  trees  (Suillus tomentosus)  so  could  the  behaviour  be monitored.  of  Six of the  trees were identified the fungus on those  The trees were all marked  double—faced write-on aluminum markers from Neville Crosby of Vancouver, B. trees  were  C..  planted  within  each  block in a randomized  pattern generated by a double randomizing program written by me. The  identity  of each tree could therefore be determined by its  tag  and  position.  its  The blocks were laid out such that one  half of each block was pine and the other half spruce.  83  The  trees  were  deviations tree  planted for  on  an  approximately  and  the  grid with many  The spruce were all planted by one  obstacles.  planter,  lm’  pine  were  all planted by another.  placed each tree in the approximate planting position,  I  to insure  that the order was correct. The seedlings were planted on May 22 and  1990.  23,  conducted.  On  July  12,  1990,  a  mortality  check  was  It was assumed that trees dying within the first six  weeks were dead or near dead on planting. Examination  of  Seedlings  height from the ground, survival.  tops  and  the  Outplanting  Trial. On  1990, the outplanted trees were measured for total  September 13,  and  From  incremental height, root collar diameter  Miscellaneous  forms  of damage, such as broken  multiple tops, were noted.  This work was carried out  by  Heffley Reforestation Centre employees under the supervision  of  Hillary  from  each  exhumed the  by  root  were  MacMillan. treatment Dr.  collected  and  1990,  four trees  in each block were selected at random and  T. M.  systems  On October 12 and 13,  Ballard and me,  taking great care to keep  as intact as possible. dead  trees  noted  Tags from dead trees in  the  earlier field  evaluation were confirmed by tag and position. The the  exhumed trees were examined in the laboratory, using much same  procedure  as  described earlier.  The roots were not  washed before examination under the dissecting scope. gently  washed,  required.  during  examination,  with  a  They were  squirt bottle as  The new roots could be distinguished because the plug  shape could still be seen and it was possible to determine which roots were growing away from the plug.  84  This distinction could  not be made very clearly after the roots were washed. of  mycorrhizae  short  roots  and  was  The types  their relative proportion of the total new  determined.  The new short roots consisted of  those on roots egressing from the plug. The  relative  difference  between  the  growth  and  types of  mycorrhizae on roots pointing down versus those growing from the side of the plugs were noted. The  weight  same  of needles was stripped off the leader from  each tree and combined by treatment for each block. were  air  Inc.  of Richmond,  the  dried  S.  U.  and B.  The needles  sent for analysis to Pacific Soil Testing C.  A standard sample of pine needles from  Department of Commerce National Bureau of Standards  was sent with the field material as a reference sample. Statistical Procedures.— The field plots were established in a randomized a  block design and the nursery trials were arranged in  nested  design with styroblocks nested within treatment.  field trial experiment had five blocks, block. four  There trees  evaluation. years.  were  per The  with four treatments per  30 trees per treatment per block,  treatment remaining  per  block  trees  The  but only  were harvested for full  will  be examined in future  There were two species of trees, but these were treated  as separate experiments. In the nursery trials, there were eight inoculation treatments (including the control) inoculum each spruce  was  applied by two methods.  combination and  for each of the two tree species and the  of  inoculum  and  Ten blocks were used for application  method in the  approximately 50 trees were treated per block.  pine was treated similarly, except only eight blocks were  85  The  treated per combination of  inoculum and application method.  Statistical analyses were done after consultation with Malcolm Greig  of  the Computing Science Department,  Kozak of the Faculty of Forestry,  U.  B.  C.  U.  B.  C.  and Dr. A.  The prograril that was  for most of the statistical analyses is called UBC Genlin:  used  General Least Squares Analysis of Variance Program by Malcolm  A  Greig  and  could  handle  James  Bierring.  missing data and do multiple comparison tests for  randomized  block  independent  variable.  Bartlett’s the  and  regard  variance.  to  recommended underlying  where the  more  than  one  test.  deviations  The ANOVA tests are very robust normality and homogeneity of  from  for that  the  “Because of the poor performance of  multisample  testing  the  be  former of  assumptions  for  were  states:  Malcolm Greig confirmed this.  variance homogeneity, and the robustness of analysis  variance  data  with  Normality was evaluated using  tests.  values are very small.  for  checked  designs  Homogeneity of variance was tested using  Layard’s  (1984)(page 183)  tests of  nested  A. Kozak said that ANOVA was still applicable even if  Bartlett Zar  and  Kolmogorov-Smirnov  with  This program was useful because it  homogeneity not  used  the of  among  performed latter.”  means, as  All  a test of the analyses  variance and normality,  to disregard ANOVA results.  it is not  were  but these  In the cases  data for the randomized block ANOVA were non-normal,  analysis  was  also  done  Kruskal—Wallis  with  the  using  Tukey and Bonferroni  non-parametric ANOVA. Multiple tests.  comparisons  were  done  Mortalities was also analysed using contingency tests on  the SAS statistical package.  86  RESULTS  Pure at  Culture  all  Synthesis.- The pine trees grew reasonably well  three  the  of  fertility  levels  used.  All  of the  unfertilized seedlings developed a distinct purplish tinge. plants  exhibited  needles  were  almost  unusual  very  entirely  long  wye  mentioned previously, moss  growth  in that the  (up to 10cm) and the short roots were  shaped,  even  when  not mycorrhizal.  As  it was not possible to allow the high peat  medium to dry and stress the trees and then rewet  growing  it through the porous pots.  There were 30 control seedlings and  of them became mycorrhizal, so the system was tight enough  none to  characteristics  The  stop  contamination  by ectomycorrhizal fungal spores in the  surrounding environment. Few isolates formed mycorrhizae, anything  so it was not possible to say  the effects of the fertilizer treatments on the  about  ability  of  obvious  with the small number of infections that occurred.  E—strain  the  fungi  isolate  vigorously.  A  to  from  form mycorrhizae.  R.  Hebelorria  Danielson  formed  No patterns were  mycorrhizae most  crustuliniforme isolate obtained from  Gary Hunt developed mantles on a few mycorrhizae, roots  were  formed  infected.  unusual  branched, Suilloid consistant  but  The  looking  A51  roots  (Suillus tomentosus)  isolate that  but only a few  were  swollen  and  much  the mantle, which is normally a major feature of  mycorrhizae, with  The  Harley  was and  very  weakly  Smith  developed.  (1983)  where  This  is  nutrient or  physical stresses are reported to cause poor mantle development,  87  but Hartig net may be normal. E—strain  Two  technique,  synthesis  mycorrhizae B-strain and  isolates formed mycorrhizae in the pure culture  much  0188  appeared  in colour, Trial  but  more  came  the  from  isolate  vigorously  than  Danielson formed  the  E—strain  0188.  from the Harrop Nursery near Nelson, B. C.  on agar to be very similar to Danielson’s isolate  rate of growth and hyphal characteristics.  Inoculation  in  the  Nursery.—  Of  the  seven  fungal  isolates used in the nursery inoculation trial, only isolate A29 (Amphinerna  inoculated  seedling was  an  isolate  isolates that  byssoides)  from  were  to  from the  formed  It  caused  each  become virtually 100 colonized.  This  the  field  detectable  mycorrhizae.  Other  nursery.  Arnphinema byssoides  did not form levels of colonization background  against  levels of Amphinema  byssoides in the nursery. Observations lodgepole  from  pine  Inoculation  seedlings,  crustuliniforme,  Amphinema  of Nursery Seedlings.— On the  the  Cenococcum geophilum,  and  byssoides  E-strain  Hebeloraa  0188 from  Harrop did not form any mycorrhizae and no further analyses were done are which  on these treatments. shown came  10—year-old the  Table from  The isolate A51  sporocarp tree  treatment,  under  (Suillus tomentosus)  a  vigorously  growing  formed mycorrhizae intermittently in and  less  in  the  surface  applied  Even though the level of colonization was low, there  significantly  mycorrhizae  VI. a  planted  injected  treatment. was  in  The results for the other treatments  on  other treatments.  (p<0.01)  the  A51  more  treated  Suillus  type  seedlings than on any of the  The mean percentage of A51  88  tornentosus  (Suillus  Table VI  Observations on Lodgepole Pine Seedlings Grown in the Heffley Reforestation Centre Nursery for 4 Months Following Inoculation With Ectomycorrhizal Fungal Mycelium at 2 Months Treatment Parameter  A51  E—strain R947  Fungus Growing Medium  Control  n=  146  160  149  144  S. tomentus—like mycorrhizae (96)  2.06 a 8.75  0.00 b 0.00  0.07 b 0.82  0.10 b 0.93  <0.01  E-strain mycorrhizae  0.95 b 5.98  67.0 a 24.2  0.17 b 1.22  0.00 b 0.00  <0. 01  (96)  Thelephora rnycorrhizae  92.8 b 13.0  32.3 a 24.4  95.1 b 10.6  89.0 b 14.8  <0. 01  (96)  98.2 a 8.17  99.7 a 3.95  97.5 a 9.58  93.5 b 13.4  <0.01  0.98 0.29  0.87 0.27  0.88 0.30  0.87 0.30  0.03  18.7 2.96  18.1 3.23  17.7 3.58  17.6 3.08  0.30  Root Collar Diameter (mm)  3.02 0.51  2.99 0.47  3.11 0.47  3.06 0.52  0.58  (g)  0.52 0.17  0.49 0.16  0.53 0.19  0.54 0.18  0. 55  Abundance of  2.35  2.39  2.30  2.31  0.90  Root to Shoot Ratio  0.59 0.62  0.59 0.18  0.65 0.26  0.68 0.49  0.31  Dickson Quality Index  0.19 0.07  0.18 0.06  0.19 0.07  0.19 0.07  0.58  Total mycorrhizal colonization (96) Shoot mass  (g)  Shoot length  Root Mass  (cm)  p ANOVA  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukey’s and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA.  89  type  tornentosus)  0.07% for the fungus growing medium treatment.  versus  formed  R947  whether The  of mycorrhizae in the A51 treatment was 2.06%  mycorrhizae  virtually  every tree inoculated,  the inoculum was injected into the plug or top applied.  mean  level  E-strain  R947  treatments. There  on  E-strain  of E—strain on blocks treated with Danielson’s was  67%,  including  and  injected  top applied  The mean level for the other treatments was 0.37%.  were significant differences between the injected and top  applied treatments, and these are discussed in Experiment Three. The  control and A51  all  heavily  infected  colonization  had  rate).  roots  short a  (Suillus tornentosus) treated seedlings were  of  the E—strain treatment.  injected  (p<0.01)  colonization  treatments.  The  and  Thelephora  terrestris  (91%  mean  Thelephora terrestris colonized 32% of the  significantly  mycorrhizal  with  A51  The control treatment  lower level of  (sum  all  (59o lower)  fungi)  (Suillus tonientosu,s)  of total  than the other  treatment  (combined  top applied) had significantly greater shoot mass  (13%  larger  on average) than all other treatments.  mass  was corroborated by the shoot length of the A51 treatment,  which  was greater on average,  other  treatments.  in  growth  only  one  growing regard 93.5%  (but not significantly), than all  No other significant differences were found  statistics marginally  medium  or  indices derived from them.  treatment  was  There was  significant difference between the fungus  treatment  and  the  control  and that was with  to the total colonization by all fungi. (std.dev.  The higher  The control was  13.4%) colonized and the fungus growing medium 97.5%  (std.dev.  9.5%)  colonized.  relatively few other fungi present on the roots,  90  There were  other than  inoculated  those  infections  of  than  type  inoculated  (A51).  to  Thelephora  terrestris was Mycelium  next  Tomentella-like  roots  a  Hartig  net.  fungi  The second most  at an average colonization  occupied  O.28 of the total  and unknown types constituted O.119o.  colonization formed  There were light  of Suilloid fungi, mainly with different  atrovirens Melin (MRA—like),  l.26.  short  the  fungus  radicis  Thelephora terrestris.  (O.36 mean)  morphology dominant  and  was  questionably  dense  mantle—like  The  called  infections  of  mycorrhizal.  but  layer  The MRA-like It often  rarely had a distinct  volunteer  fungi  (fungi that  colonize trees without benefit of inoculation) appeared grouped. That  when a particular type of volunteer fungus was found,  is,  it  was  more likely to appear again in the same styroblock than  in  the  general  previously culled  and  the  growing  seedlings  significant  The  styroblocks  may have harbored inoculum.  during  deformed  population.  so  season,  had  The seedlings were  any dead, diseased or  were removed from the block.  differences  between  been used  There were no  treatments with regard to the  number of seedlings lost over the growing season. On  the  spruce  seedlings, the Danielson E—strain isolate and  the Amphinema byssoides isolate formed abundant mycorrhizae with virtually Table VII.  every  tree  inoculated  The other isolates did not form detectable levels of  colonization.  There  were  Arnphinerna  byssoides  in  inoculated  trees  a  approximately Arnphinerna  and the results are shown in  had  99%  byssoldes  of  the  also  all  of  much  high the  higher  levels  treatments,  of  volunteer but  the  colonization rate, with  treated seedlings being infected with  versus 75% of the control seedlings.  mean  91  The  Table VII  Observations on Engelmann Spruce Seedlings Grown in the Heffley Reforestation Centre Nursery for 5 Months Following Inoculation With Ectomycorrhizal Fungal Mycelium at 2 Months Treatment Parameter  A51  E—strain R947  Fungus Growing Medium  Control  n=  200  200  200  190  p ANOVA  A. byssoides mycorrhizae  68.5 a 23.6  9.12 b 19.7  28.8 c 27.4  26.1 c 27.7  <0.01  ()  E—strain mycorrhizae  0.75 b 4.47  64.7 a 29.6  0.55 b 4.28  1.35 b 7.61  <0.01  (%)  Thelephora mycorrhizae  10.8 a 18.6  21.7 b 22.5  42.2 c 29.2  52.4 c 28.4  <0.01  ()  Total mycorrhizal colonization (6)  80.1 a 19.5  96.3 b 10.0  72.0 a 24.7  80.5 19.3  <0.01  Shoot mass  1.68 a 0.38  1.47 b 0.37  1.60 a 0.39  1.62 a 0.36  <0.01  16.3 a 3.64  15.0 b 3.54  15.3 3.70  15.5 3.32  0.02  Root Collar Diameter (mm)  3.36 0.40  3.27 0.43  3.36 0.39  3.31 0.39  0.38  (g)  0.93 0.29  0.88 0.27  0.98 0.27  0.90 0.27  0.12  Abundance of Mycorrhizae  2.68 0.54  2.63 0.57  2.58 0.66  2.47 0.61  0.11  Root to Shoot Ratio  0.58 0.22  0.61 0.17  0.64 0.19  0.57 0.17  0.11  Dickson Quality Index  0.40 0.12  0.39 0.12  0.43 0.12  0.40 0.12  0.16  (g)  Shoot length  Root Mass  (cm)  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukey’s and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA.  92  percentage the  of roots infected by  Arnphinerna  bys3oides  Arnphinema byssoides was 68% in  treated  trees and 27 .5% mean for the  control and fungus growing medium treatments. the  The difference in  percentage of infected roots was significant  (p<O.Ol).  The  mean number of roots infected with E—strain in the E—strain R947 treatment  was 716 versus O.79&  mean level of volunteer E--strain  for the other three treatments. of  (sum the  all  types) was significantly (p<O.Ol) higher  E—strain  There  The total level of colonization  R947  treatment  than  for  (24%)  in  the other treatments.  were significant differences between the injected and top  applied treatments, and these are discussed in Experiment Three. The  seedlings  tops  and  treated  were with  seedlings the  treated with Arnphinema byssoides had the largest significantly E-strain  were  differences  R947.  and  heavier than those  The Amphinema byssoides treated  8% taller, and 7.5% heavier than the average of  three  other  taller  treatments.  between  There were no other significant  measured statistics or the indices derived  from them. There  high levels of Thelephora terrestris colonization  were  in most of the treatments, in  mean  16.2%  level in the two treatments with introduced fungi was  and  fungus  the  than  mean  level  in  growing medium was 76%.  (p<O.O1). the  The mean level of Thelephora terrestris was 31%.  the pine.  The  though the levels were not as high as  MRA—like,  The differences were significant  There were several other types of volunteer fungi on  spruce. those  the control and treatment with  E—strain appeared on about 0.9% of the roots other inoculated  with  E-strain.  Other types,  including  Tomentella—like and unknown, constituted only 0.2% of  93  the  total are  types were  too  higher than  mycorrhizae.  given in Appendix II. sporadic  pine  non—inoculated Thelephora smaller  The infections by these fungi  to be able to detect trends in grouping.  proportion (18) the  Detailed descriptions of the different  A  of the spruce roots was non-mycorrhizal  roots.  A typical colonization pattern with the  roots  was  terrestris  to  have  Amphinema  byssoides  or  growing on the bottom of the plug with a  area of non-mycorrhizal roots near the top, where there  might  be a small colonization of some other species of fungus.  There  were  no  significant differences between treatments with  regard to the number of seedlings lost over the growing season.  94  Observations July 12, given  1990  a  on  Seedlings After One Season in the Field.— On  (about seven weeks after planting), the trees were  preliminary  examination  might have been dead on planting.  to  try to distinguish which  The early part of the growing  season was very wet and only one tree, a pine control, was dead. There  is  an  which  is  eight  rainfall  Environment  Canada  kilometres  southwest  of the study area.  in Barriere was 2.84 times higher  in May,  1990 and 2.5 times higher  1990.  In  July,  respectively. normal.  August  were  temperatures  and  6.9%,  than normal  (101.9mm) than normal in June,  September,  9.3%  (95.9mm)  The  and  11.0%  1990,  the mean daily  higher than normal,  In September the precipitation was 8mm or 25% of  Mortalities rose considerably by the time the seedlings  harvested  were  weather station at Barriere,  and  this  may have been related to the hot dry  weather during late summer. Most  of  seedlings  the  parameters  were  measured  about 15 weeks after outplanting  on  all  outplanted  (September 13,  1990)  and these results are shown in Table VIII for lodgepole pine and Table XII for Engelmann spruce. mass  measurements weeks  19 are  shown  Observations of mycorrhizae and  were gathered from seedlings collected about  after outplanting (October 12 and 13, in  Table  IX  1990) and these  for lodgepole pine and Table XIII for  Engelmann spruce. The  majority  typically  grew  of  the roots on both pine and spruce seedlings  straight  proportion (about 10%) plug.  There  from any plug.  were  no  down  from the plug with only a small  of the roots growing from the side of the long lateral roots growing horizontally  At most, there were individual short roots or  95  small on  bunches of short roots growing in cauliflower—like clumps  the sides of the plug.  side often  often  had  one  different  The clumps of roots growing from the  type  of fungal colonization and this was  from the predominant colonization on the roots  growing down. Observations the  E-strain  (6%  difference  (Suillus  on  Pine  Seedlings. The root collar diameter of  R947 treatment was significantly (p<0.0l) smaller between  than  means)  the  control.  The  A51  tomentosus) treatment was significantly (p=.03) taller  (6% difference between means) than the control  (Table VIII).  Table VIII  Observations on Lodgepole Pine Seedlings Inoculated With Ectomycorrhizae in the Nursery and Grown For 5 Months in the Field (Field Measurements) Treatment Parameter  A51  E-strain R947  Fungus Growing Medium  Control  n=  120  142  143  145  10.8 2.98  10.14 3.16  10.4 3.31  9.91 3.60  0.14  29.1 a 5.00  28.5 5.26  28.7 4.85  27.4 b 5.03  0.03  Root Collar Diameter (mm)  4.02 0.66  3.94 a 0.66  4.04 0.70  4.18 b 0.67  <0.01  Proportion Living  0.96 0.20  0.96 0.20  0.94 0.24  0.97 0.18  Incremental Height (cm) Total height  (cm)  p ANOVA  0.68  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukey’s and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA.  96  It  should  between  noted  be  blocks  adjacent  to  apparently  for  one  that  there  were significant differences  every parameter measured.  another  and  quite  similar,  the  died  by the time the seedlings were exhumed  fungal  fungus  counted  treatments.  tomentosus)  (Suillus  five  they were  yet  different enough to cause more differences in growth  than  A51  The blocks were  Few lodgepole pine seedlings had  treated,  (Table VIII).  six E—strain R947 treated,  growing medium and nine controls died.  as  dead  only  Five  Trees were  if they had a complete absence of green  needles. Thirteen pine  different types of ectomycorrhizae were found on the  roots,  with an average of 2.75 types per tree.  of types per tree ranged from zero to five. types the  The roots with zero  had no discernible root growth from the plug, tree  appeared  tops  The number  even though  live and well in most of these cases.  There were significant differences between the treatments in the numbers had  of fungi per tree.  significantly  types/tree The  vs.  The fungal growing medium treatment  (p=0.04) more types of ectomycorrhizae  2.20  types/tree)  than the control  (3.05  (Table IX).  A51 treatment was virtually identical to the fungus growing  medium  with  significantly  regard  to  different  the from  number of types per tree, but not the  control.  The E—strain R947  treatment was halfway between the control and the fungus growing medium in the number of ectomycorrhiza types on the root. The In  behaviour of the inoculated fungi was extremely variable. many  tornentosus)  cases,  mycorrhizae  formed  with  the  A51  (Suillus  treatment appeared to have died after outplanting.  There were often large patches of dead roots on the plugs, but  97  Table IX  Observations on Lodgepole Pine Seedlings Inoculated With Ectomycorrhizae in the Nursery and Grown For 5 Months in the Field (Laboratory Measurements) Treatment Parameter  A51  E-strain R947  Fungus Growing Medium  Control  20  20  20  20  Number of Types of Mycorrhizae/tree  3.00 1.17  2.70 0.98  3.05 a 1.00  2.20 b 1.06  Shoot mass  2.38 a 0.67  2.11 b 0.84  2.18 b 0.62  1.80 b 0.55  0.06  (g)  1.15 0.32  1.03 0.41  1.16 0.43  1.14 0.43  0.74  Root to Shoot Ratio  0.50 0.14  0.52 0.19  0.56 0.30  0.66 0.23  0.31  n=  Root Mass  (g)  p ANOVA  0.04  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukeys and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA. these were rarely present on the other treatments. where  patches  missing,  of root were dead,  which  suggests  that  In the cases  the Suilloid types were often  the  dead patches may have been  colonized by Suilloid types.  However,  there were also instances  where  growing  from  Suilloid  Suilloid of these  type  an  were  the  were identified on the egressed  (type 10) than  (other with  types  types  (Fig.  Thelepl-iora  roots.  Three  roots, and one  9) was the most common volunteer fungus terrestris,  which is discussed below)  average colonization level of 7.44&.  Another Suilloid  (type 4) was the second most common volunteer, with an  98  colonization (Fig.  level of 4.69%.  was  8)  treatments) It  fungus.  on  1.75%  The third Suilloid type  of  the  egressed  and  was  indistinguishable  was  not  possible  roots  from  to determine,  the  (type 12)  (mean of all inoculated  in the few cases  where this fungus was growing on the inoculated seedlings, originated Suilloid  from the field. type  fungus  on  However, the  if it  in at least one case,  the  plug was growing vigorously and  appeared to be growing onto egressed roots. The that  situation was different with E-strain, had  in that every tree  been inoculated with E-strain had E-strain colonizing  egressed  roots.  egressed  roots  The levels of colonization by E—strain on the (side  and bottom) varied from 20 to 100%.  volunteer level of colonization for E—strain types,  on all other  Table X Volunteer Fungi on Egressed Roots of Lodgepole Pine Grown in the Field For Five Months With Different Mycorrhizal Treatments Fungal Species  Percent Infection of Egressed Roots  Thelephora terrestris (Types 2 and 11) (mean excludes E-strairi treatment)  69.83  Suilloid  (Type 10)  7.44  Suilloid  (Type 4)  4.69  Suillous tomentosus (Type 12)  1.75  E—strain (mean only includes uninoculated treatments)  3.83  MRA (Type 1)  3.69  Type 3  3.06  Tomentella-like  (Type 8)  2.00 99  The  treatments,  was  with a similar drop  (oil  3.836.  There were a few other types of fungi  level of colonization, Type 1  type—3.06%)  and  Type  8  (MRA—3.69%), Type 3  (Tomentella—like--2%).  The  levels of volunteer fungi on pine are shown in Table X. The on  fungus that had the highest overall level of colonization  egressed pine roots was Thelephora terrestris-like.  cases,  the  appeared  Thelephora  to  be  terrestris—like fungus from the nursery  healthy on the plug and to be growing directly  onto the egressed roots  (both side and bottom).  discrete  Thelephora  colonies  non—infected (Fig.  control from  roots  and  the  of  from  the  fungus  nursery  growing was  not  were  plug  In 5 out of 40 cases  15).  In many  In other cases,  separated  by zones of  and the nursery Thelephora  (the 40 cases consisting of the  medium growing  treatment),  the Thelephora  onto egressed roots.  In  addition, constant differences appeared in the morphology of the Thelephora  found  on the egressed roots,  were described as two different types. on  only  new  frequently type  was  a  fungus, the  shorter  longer  mycorrhizae  mycorrhizae  and  rarely  (though cystidia were still present). separate  nursery,  formed  One type, which appeared  with white blotches of cystidia.  covered  formed  blotches  roots,  to the point that they  then with  type it  an  of  Thelephora  from  had  that  were  The other the  white  If the long type that found in the  was by far the most common type of volunteer average colonization level of 16.25.  All of  fungi found and the levels of colonization are described in  Appendix II. No  significant  differences were found in the nutrient levels  in the tissue samples.  The results are shown in Table XI.  100  Table XI  Foliar Nutrient Analyses For Lodgepole Pine Grown in the Field for Five Months With Different Mycorrhizal Treatments Treatment Nutrient  A51  E-strain R947  Fungus Growing Medium  Control  5  5  5  5  1.63 0.19  1.63 0.21  1.74 0.28  1.60 0.27  0.63  0.20 0.02  0.20 0.02  0.22 0.02  0.20 0.01  0.28  Calcium ()  0.25 0.06  0.29 0.04  0.26 0.06  0.26 0.05  0.76  Magnesium  (6)  0.13 0.02  0.13 0.01  0.14 0.02  0.12 0.02  0.30  Potassium  (9)  0.64 0.07  0.67 0.05  0.72 0.08  0.66 0.07  0.27  (ug/g)  5.20 0.45  5.60 1.14  6.20 1.79  5.80 0.84  0.68  54.0 7.84  46.2 4.09  57.6 12.7  53.0 7.25  0.08  87.4 26.8  92.6 26.0  92.2 22.3  86.6 14.0  0.88  294 19  327 66  328 86  328 68  0.81  11.8 2.28  12.6 1.95  13.8 3.03  14.4 5.08  0.41  n= Total Nitrogen Phosphorus  Copper  ()  (6)  Zinc (ug/g) Fe  (uglg)  Manganese Boron  (ug/g)  (ug/g)  p ANOVA  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukey’s and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA.  101  Observations on Spruce Seedlings. seedlings  was  much  higher  The mortality of the spruce  than of the pine, averaging 23.5%.  The mortalities varied significantly (p<0.O1) by block from 8 to The mortalities varied by treatment from 21 to 27% (Table  50%. XII)  were  and  treatment diameter  not significantly different.  The E—strain R947  had significantly (p<0.Ol) smaller  (6.4%) root collar  (Table  XII)  than the fungus growing medium treatment  and control. The  E-strain  R947 and A29 (p<O.O1)  significantly XII)  (Amphinema byssoides) treatments had  smaller  (12%) height increments  than the fungus growing medium and the control.  inoculated  with  significantly treatment  Arnphinerna  (p=0.025)  (Table  significantly  XII).  different  byssoides  taller All  (6.5%) of  by block.  were  the  Seedlings  tallest  the parameters measured were The root mass of the fungus  nearly  A29  treatment  A29  (Table  The E-strain had the greatest root to shoot ratio and it  was  though  and  than the E—strain R947  growing medium was significantly greater than the control XIII).  (Table  the  significantly different  (p=O.O6)  (Table XIII).  The  (Amphinema byssoide5) had the greatest shoot mass  difference was not quite significant  (p=O.O9).  The  treatment also had one of the smaller root to shoot ratios,  which was in keeping with its larger top.  102  Table XII  Observations on Engelmann Spruce Seedlings Inoculated With Ectomycorrhizae in the Nursery and Grown For 5 Months in the Field (Field Measurements)  Treatment Parameter  A29  E-strain R947  Fungus Growing Medium  Control  n=  121  125  116  122  11.0 a 3.68  10.72 a 3.30  12.2 b 2.89  12.5 b 3.00  26.2 a 5.00  24.6 b 5.26  25.7 4.85  26.0 5.03  0.03  Root Collar Diameter (mm)  4.47 0.80  4.32 a 0.82  4.63 b 0.85  4.61 0.81  0.06  Proportion Alive  0.78 0.42  0.77 0.42  0.79 0.41  0.73 0.46  0.57  Incremental Height (cm) Total Height  (cm)  p ANOVA  <0.01  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukey’s and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA.  Nine spruce  different roots.  types  of  fungi  were identified on egressed  The mean number of types overall was 1.69/tree.  There  were  types  of fungi present by treatment.  significant  ) differences in the number of 76 (p=O.01 The E—strain R947 and the  Amphinerna byssoides treatments both had 1.4 types/tree versus 2. 0  types/tree  (Table XIII). four;  in  four  for  control and fungus growing medium treatments  The number of types per plant ranged from zero to cases  no root growth occurred, even though the  tops looked alive.  103  Table XIII  Observations on Engelmann Spruce Seedlings Inoculated With Ectomycorrhizae in the Nursery and Grown For 5 Months in the Field (Laboratory Meaurements) Treatment Parameter  A29  E-strain R947  Fungus Growing Medium  Control  20  20  20  20  Number of Types of Mycorrhizae/tree  1.35 a 0.75  1.40 a 0.67  2.05 b 0.94  1.95 b 1.10  0.02  Shoot mass  3.85 0.92  3.12 0.70  3.77 1.03  3.57 1.15  0.09  (g)  1.89 0.69  1.68 0.41  2.09 a 0.90  1.52 b 0.53  0.04  Root to Shoot Ratio  0.49 0.14  0.63 0.35  0.56 0.21  0.44 0.14  0.06  n=  Root Mass  (g)  p ANOVA  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukey’s and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA. The  mean  E-strain the  level  of  E-strain  colonization on new roots of the  R947 treatment was 886.  other  by,ssoides  treatments on  the  was 14.5&.  The mean level of E-strain on The mean level of  Amphinema  new roots of the Amphinerna byssoides treated  trees  was 84, while the mean for the other treatments was 15.  These  two  (other fewer  fungi  than than  were  by  far the most common volunteer fungi All  Thelephora).  1% of the roots.  were  non—mycorrhizal.  them  are  summarized  the other types are present on  Only 4.38 of the new short roots  The types of fungi and the statistics on in  Appendix II.  fungi are shown in Table XIV.  104  The levels of volunteer  Table XIV volunteer Fungi on Egressed Roots ot Engelmann Spruce Grown in the Field For Five Months With Different Mycorrhizal Treatments Fungal Species  Percent Infection of Egressed Roots  Thelephora terrestris (Type 2) (mean excludes the A. byssoides and E—strain treatments)  43.38  Amphirieriia byssoldes (Type 3) (mean excludes the A. byssoides and E—strain treatments)  20.89  E—strain  14.50  (uninoculated treatments)  Other Types  <1.0  level  mean  The  Thelephora  of  on the egressed roots of the  control and fungus growing medium treatment was 439o and the mean on the E-strain and Amphirserna byssoides treatments was 3.  level  Seven  5%.  (out of 41)  treatments  did  not  of the control and fungus growing medium  have any Thelephora on the egressed roots.  All of the Danielson E-strain treated trees had E—strain growing on the new roots and all  (except one, where a large piece of the  root  the Ainphineina byssoides treated trees had  was  missing)  Amphinema E—strain to  of  byssoides on  100%.  growing  on  the new roots.  The level of  new roots of E—strain treated trees ranged from 40 The  level  of  Arnphinema  byssoides on new roots of  Arnphinema byssoides treated trees ranged from 30 to 100%. There and  was  one significant difference  this is shown in Table XV.  105  in tissue nutrient level  The control had a significantly  (p=O.02)  lower calcium level than the E—strain treatment.  Table XII  Foliar Nutrient Analyses Engelmann Spruce Grown in the Field For Five Months With Different Mycorrhizal Treatments Treatment Nutrient  A29  E—strain R947  Fungus Growing Medium  Control  5  5  5  5  1.18 0.14  1.31 0.16  1.29 0.19  1.23 0.10  0.44  0.21 0.02  0.24 0.01  0.23 0.04  0.20 0.01  0 11  Calcium (%)  0.32 0.04  0.41 a 0.06  0.38 0.09  0.29 b 0.03  0.02  Magnesium ()  0.12 0.01  0.13 0.02  0.14 0.02  0.11 0.01  0.09  Potassium (%)  0.80 0.09  0.83 0.05  0.84 0.16  0.76 0.09  0.67  (ug/g)  4.60 0.55  5.20 0.45  5.00 0.00  5.00 1.00  0.37  40.8 4.97  43.6 5.13  46.2 7.79  39.6 8.26  0.50  71.0 19.3  105.2 40.3  82.2 12.8  87.8 30.8  0.26  n= Total Nitrogen Phosphorus  Copper Zinc  Fe  (p6)  (6)  (ug/g)  (ug/g)  Manganese Boron  (ug/g)  (ug/g)  p ANOVA  .  262 34  327 48  272 73  267 58  0.26  11.4 1.52  12.8 1.48  11.0 2.45  10.6 1.52  0.22  The top number is the mean, the number underneath is the standard deviation and means with different letters were determined to be different based on Tukey’s and Bonferonni’s tests with p= 0.05. The p value in the table is from ANOVA.  106  DISCUSSION  Near-Pure culture  Culture  synthesis  ectomycorrhizae. needed  Synthesis Technique.techniques  have  fairly  rigorous  A  keep  to  any  Many different pure  been  growing  developed  isolation  system  to  study  procedure  from  aerial  Is  spore  contamination,  which can be a major problem in or near forested  areas..  (as  Melin  first  person  seedlings and  in  use  a  in  Molina  Also,  to  cited  Molina and Palmer,  in  the  quite  simple  Hacskaylo  flask.  1982) was the  approach  of growing  (1973), Marx and Zak  (1965),  all added modifications to this technique.  (1979b)  several other, more radical, techniques such as synthesis  solid agar  Palmer,  1982),  Ashford, been  (Pachlewski and Pachlewska as cited in Molina and growth  in  pouches  1986> and in petri plates  developed.  Peterson  (Grenville,  (Wong and Fortin,  and  1988) have  (1937) anticipated the problems with the  Hatch  techniques mentioned here when he identified the shortcomings of pure culture synthesis apparatus as: •  partial  (2)increased  of  (3)accumulation  pressures  products  (4)saturated substrates.... These  abnormal  “(l)excessive humidities...  (in  carbon  of  cases  some  dioxide.... toxic)....  (5)10w radiation intensities  growing conditions led Molina and Palmer  (1982)  to remark that positive synthesis results confirm the ability of the  fungus  to  produce  inconclusive.  Melin  overcame  of  Palmer  many (1982),  mycorrhizae  made  these the  more  several problems, complex  difficult it is to keep it intact.  107  but  negative results are  elaborate  apparatus  that  but as noted by Molina and the Also,  apparatus,  the  more  when  evaluating large numbers of fungi,  difficult that in  to set up many such devices.  some the  it becomes logistically There is ample evidence  fungi which might grow in the nursery might not grow  unusual conditions found in most pure culture synthesis  devices.  Therefore,  some effort was put into developing a new  pure culture synthesis device. The  pure  useful  synthesis technique developed was not very  predicting  for  nursery.  which  fungi  grow  might  well  in the  Of the five fungi that formed mycorrhizae in the pure  culture Two  culture  synthesis,  E—strain  only two formed raycorrhizae in the nursery.  isolates  were used in the pure culture synthesis  trials and one formed mycorrhizae vigorously while the other was much  less  nursery  vigorous.  This  behaviour  was  paralleled  in the  study, where the vigorous isolate from the pure culture  synthesis produced many rnycorrhizae and the other produced none. The  E-strain  cultures  characteristic ornamented  fuzzy  hyphae.  appeared  appearance  and  E—strain  similar  unique  large  with  a  diameter,  The fact that one isolate grew well and the  other did not may illustrate ecotype one  very  isolate  was  not  (or possibly species, since  identified  to  species level)  variation. One  weakness  screening the  fungi  of  the  pure  culture synthesis technique for  fungi for use in outplanting trials, was that some of which did not form mycorrhizae were almost certainly  capable of doing so under some conditions.  The weak development  of the mycorrhizae suggested that the seedlings were not allowed to  grow  long enough after inoculation.  only about 2.5 months after inoculation.  108  The seedlings grew for Molina  (1979) grew  seedlings  for  evaluating isolates  six  under  their mycorrhlzae. may  regulating plastic  months  fluorescent  levels  in part,  and  ethylene.  roots  Solution  objectives  of  technique  this  developing  used  in  this  addition  of  some  its advantages.  of  build—up  a  was  problem  a  of  experiment  has  like  the  main  technique.  The  one  synthesis  new  gases of  been improved by the  glass cover with a membrane filter, without losing  maintenance requirement  The dichotomous branching of  suggested  of  from difficulties in  poor gas exchange through the  bag cover and porous pot.  nori-mycorrhizal  before  The lack of colonization by some  also have resulted,  moisture  lights,  (watering, for  The advantages include low cost, easy and  fertilizing  infrastructure  and  good  injecting), protection  little against  contamination. moss  Peat  was  because  system  a of  particularly  poor  growing medium in this  its hydrophobicity problems.  Peat moss was  used in an attempt to mimic conditions in the nursery, but given the  other  was  a  huge  futile  variances between the systems, gesture.  It  seems  environment for screening fungi will the  be  grown.  nursery  there  inescapable  using peat moss that the best  is the environment in which they  The Arnphinerna byssoides isolate selected from  inoculation trial formed mycorrhizae very well, so  is at least some evidence that the nursery might serve as  a suitable screening ground for ectomycorrhizal fungi. In used  spite  of its shortcomings,  as  major  nursery  a  trial.  criterion Four  for  the pure culture synthesis was selecting  the fungi for the  of the five isolates used in the nursery  trial formed mycorrhizae in the pure culture synthesis  109  (Amphinema byssoides did not). in  experiment  this  was  were  previously  ability.  myeorrhiza—forming nursery  Most of the fungal isolates used  available  their  inoculation and some of the fungal  isolates were very difficult to grow, very least,  for  limited number of trees in the  A  for  untested  so it was felt that at the  the fungi used in the nursery trial should be proven  mycorrhizal fungi. Nursery nursery types  The  Trials.-  attempts  inoculate  to  fungi  in the  using a new type of inoculum were successful with three of  fungi  tomentosus), byssoides  and  and  Amphinerna  CE-strain,  byssoides  and  highly successful for two of those  E-strain).  Suillus  (Amphinerna  The inoculum was not washed free of  the nutrients in the growing medium, yet successful inoculations were achieved.  This contradicts dogma with regard to vegetative  inoculation, removed.  which  More  suggests  research  that  is  and so on.  E—strain  as  between  a  Danielson  should  be  if the timing of application is (1988) has previously identified  capable of surviving well over the time  fungus  inoculation  nutrients  needed to clarify how many fungal  species can be handled this way, critical  the  and  infection.  A list of fungi with this  characteristic would seem to have some use. With Amphinerna byssoides and E-strain, mycorrhizae were formed when types  the medium was injected into the plug or top applied. of  potential  fungi  that  commercial  Cenococcurn geophilurn, This  confirms  (personal  the  Two  have received a lot of attention as good inoculants,  Hebelorna crustuliniforme and  failed to form mycorrhizae in the nursery. lack  of  success  in  attempts  by G. Hunt  communication) to get these fungi to form mycorrhizae  110  in  the  nursery  Robertson  and  geophilurn  to  though  using  other  Salonius form  inoculation  (1987)  mycorrhizae  mycorrhizae  were  also in  formed  techniques.  Boyle,  failed to get Cenococcw a in  container nursery, even growth  pouch  assays.  Cenococcum geophilurn is reputed to do well on hot dry sites, but the  container  nursery  may  be  a  major barrier to using this  fungus on seedlings destined for such sites. On  the  pine,  very  few  mycorrhizae,  heavier  shoot  A51 treatment (Suillus tomentosus) yet  treatment  not  this  low>  emphasis growth  on can  by  Amanita  (Scop.) Fr.,  Kummer.  Fr.>  Stenstrom and Ek  large  percent  of  mycorrhizae.  possibility  and Tricholoma albobrunneum (Pers.  colonization.  Significant alterations in  that  Suillus  Danielson (1988) has also reported sporocarps  the  under  trees with few  This may suggest several things, such as some Suillus species do not need to form  large infections to fulfill their role. of  Fr.) Hooker,  (L.:  be brought about by apparently insignificant levels  fruitings  Suilloid  musearia  This illustrates the peril of placing too much  of mycorrhizal colonization.  all  had significantly  weights than other treatments.  Lactarius rufus  the  this  formed  reported similar effects with low levels of colonization  (1990) (but  the  Suillus mycorrhizae,  It may also be that not  on a single root system,  form  the characteristic Suilloid mycorrhiza. Danielson, reported  Visser and Parkinson  failure  (1984)  and Molina  with attempts to inoculate Suillus tornentosus  and other Suillus species into container nurseries. nursery  (1981) both  The Heffley  did have a modified growing regime, but even with that,  colonization was poor.  This work suggests that successful  111  with Suillus may not be impossible,  inoculation and  that it may be worth pursuing further.  just difficult,  It may also be that  apparently low levels of colonization with Suillus fungi are all that are possible and needed. For  the  spruce  seedlings, the Arnphinerna byssoicIes treatment  resulted in seedlings that were tallest and significantly taller than  the  Danielson  Estrain  trees  E—strain  had  smaller  than  the  treated  seedlings  significantly  treatment,  other had  treatments. the  heavier  heaviest  than  findings  findings  of Danielson and Visser same  of  the  the  the  isolate  Husted  reforestation situation.  spruce  grown  study and  involved Jackson  growth but  of the  at  shoots  (1990)  (1988).  and  these  treatment.  were This  but contradicts the Both of these studies  but neither was done in a  The Rusted study involved white  a growth cabinet and the Danielson and Visser jack pine planted on oilsands tailings.  (1983)  also  reported  that  Thomas  E—strain reduced the  Sitka spruce seedlings in the nursery and the field, field  (personal E—strain  The Arnphinema byssoides  E—strain  of E-strain,  normal  in  the Danielson  the lightest shoots and were significantly  confirms  used  while  was  a  partially  communication) from  has  sterilized  reported  that  seedbed.  Hunt  seedlings  with  the same nursery used in this study were smaller  outplariting,  but  grew  better in the field.  The Amphinerna  byssoides treatment had the largest root collar diameters, shoot lengths  and  shoot weights of all the treatments,  including the  controls, while the Danielson E-strain treatment was smallest in each  of  those  categories,  as  well as root weight.  other reported study on the effects of  112  inoculation with  The only  Amphinerna  byssoides  study  Husted  had  of  is  the  white  spruce in a growth chamber  She reported that Amph1nerta byssoldes  (1990).  growth similar to controls.  Danielson  (1988)  failed to get  Arnphinema by.ssoides to form mycorrhizae on jack pine. growth  The an  applied  effects found in this study may be important from point of view,  increasing  or  limiting has  communication)  top  growth.  indicated  that  C. one  Hunt of  (personal  the  problems  at the Heffley Reforestation Centre is limiting top  encountered growth  since there are advantages to either  in certain circumstances. The results are most important  though, because they illustrate that manipulation of fungus in a nursery can change the growth characteristics of seedlings, even in the high—fertility, There  were,  treatment  essence,  in  experiment,  since were  high—moisture environment of the nursery.  the  heavily  three  fungal  control  and  infected  fungal  with  in  treatments growing  Thelephora  this medium  spp.  Each  treatment behaved differently, which undeniably illustrates that the  role  of  ectomycorrhizal  fungi,  in the nursery, cannot be  reduced to the notion that “any fungus will do”. The modified growing conditions in this nursery were effective in  promoting  untreated  formation  terrestris—like fungi  in  the  insignificant, treatment). higher  trees  nursery  of  mycorrhizae  because  the roots of  were heavily infected with Thelephora  fungi,  and  there  nursery  (in  low levels that I hesitate to call  in  However,  light  of  were several other types of  what  inoculation  with  total levels of colonization.  mainly by excluding Thelephora.  113  happened  with  the  A51  fungi resulted in even  The inoculated fungi grew  The effect of altering the  nursery of  environment may be cumulative over time, as populations  fungal  and  the  This  inoculum build up in the styroblocks or the nursery, effectiveness  may  be  of funga]. inoculation might be reduced.  possible,  but  the  styroblocks  used  in  this  experiment were not new and the patterns of mycorrhiza formation suggested  that  inoculum that  in  this  significant brought  be  there  was  already  nursery.  an  accumulation of fungal  Furthermore,  it is quite apparent  alterations to nursery fungal populations may  about  by  relatively simple vegetative inoculation  techniques. This  year  to  variables. the be  the case. be  varieties others.  ready  for  immediate  commercial  depending on climatic situations or other  year,  Observations by 0. Hunt (personal communication) at  Heffley  could  not  since there is a possibility that effects may vary  application, from  is  technology  Reforestation Centre nursery suggest that this may In addition, there are still many more fungi that  tried, of  including  Suillus,  Another  local  field  very  isolates  of Thelephora spp. and  isolates  important  of B-strain, more  avenue  to  explore  is  the  application of mycorrhizal “cocktails” with more than one funga]. species.  If  Amphinema increases growth and B-strain decreases  it, what would happen with good infections of both? be  that  increasing  the  biodiversity  on  It may well  the root systems of  nursery seedlings would stabilize the growth of the seedlings in regard to changing environmental conditions, and thus allow more uniform results for the grower. Field  Trials.—  The  B—strain  treatment  resulted in smaller  (root collar diameter in pine, root collar diameter and height  114  in  spruce) trees both in the pine and spruce.  tomentosus  on  trees  treated  treatments  on  of  largest,  have  had  for the spruce, the  was significantly smaller than other  pine  to  in  the  growth  in  tops of the A51 treatment were  cause  height  It is not possible to say what might mean over the longer term.  morphology differences  and  even  after  mycorrhizae  were  not  in survival, even though the  relatively high mortalities.  The mortalities might  higher if the early part of the growing season  not been so wet.  time.  However,  but not significantly.  been  had  which  the  differences  spruce  species.  significantly heavier and the incremental height was  sufficient  in  On  differences  The  Amphinema byssoides (on spruce)  the  Amphinema byssoides treatment was similar to  E-strain,  marginally  these  their the  treatments.  and  carried on from the nursery as being the tallest  for  increment that  pine)  The A51 (Suillus  the  Some studies have shown that differences first year continue and grow larger with  For example, Marx, Cordell and Clark III  (1988) reported  that over eight years, the growth of loblolly pine planted on an old  agricultural  field  was  positively related to the initial  amount of Pisolithus tinctorius ectomycorrhizae. in  yearly  Pisolithus  basal  area  tinctorius  growth  between  fungi  The difference with  a  lot of  and those with none, grew larger for the  first five years after outplanting. Many  fungi grew onto the egressed roots of the seedlings over  the first summer in the field, though the levels of colonization reached the  by  the volunteer fungi were much smaller than those of  inoculated  on similar sites.  fungi.  There is no other published information  Roth (1990), working at a coastal site in  115  British Columbia, found a similar pattern, where the maiority of fungi  on new roots came from the nursery fungi, but significant  levels  of  field fungi moved onto the roots.  communication) the  general  some  has  made  vicinity  population  that  grow  uva—ursi reports  this study.  seedlings  they  ectomycorrhizal  considerable observations of roots in  of  circumstances,  He has observed that in  remain  in  had  with  the  mycorrhizal  nursery until the roots of  the  harboring plants such as Arctostaphylos  fungus  contact  into  G. Hunt (personal  with  the root plug.  In general,  have varied on the extent of volunteer colonization, by  indigenous  fungi,  shortly  after  outplanting.  Castellano and  Trappe (1985) reported that Douglas-fir seedlings had some roots infected  by  inoculated the  indigenous in  the  and  on  colonized  on  the  western  rotten over  after  two  years, but a fungus  nursery still constituted more than half of  mycorrhizae  nonmycorrhizal  fungi  Kropp  (1982)  planted  hemlock seedlings, both on mineral soil  wood, one  seedlings.  and  seedlings  growing season.  became  nearly  In general,  totally  it seems that  most normal reforestation sites have some inoculation potential. The  extent  outplanted several  to  which  seedlings  factors.  requirement  for  mycorrhizae  on  indigenous in  the  Contact  first  with  colonization, the  fungi  the but  outplanted  colonize the roots of  season  may be related to  root is an obvious first the presence or absence of  seedlings,  and  mycorrhiza all presumably could have some effect. colonization  can  occur  does  the  type  of  The fact that  not eliminate the possibility of  beneficial effects from inoculation, as indicated by the results in  this study, those of Castellano and Trappe (1985) and others  116  discussed in the Literature Review. The  used in the inoculation trials seemed well-chosen,  fungi  because  the more common mycorrhizae from the field were usually  similar  types to those that were inoculated.  an  The pine provided  interesting case, in that the inoculated fungus A51 (SuIllus  tamentosus)  did  significant  growth results, and fungi similar to A51 were among  most  the  common  naturally  on  vigorously  onto  for  tree,  every  Thelephora be  to grow on the egressed roots over the  types  The other inoculated fungi and even the Thelephora that  summer.  grew  not grow very well in the nursery, but yielded  the  the  nursery  seedlings,  egressed roats.  continued to grow  However, this was not so  there were several (17%) cases where the  and  did not grow at all onto new roots.  There may still  some debate over whether Thelephora is a suitable fungus for  the  field,  abundant  but this study, in conjunction with observations of fruitings  of  Thelephora  sporocarps  shows  conditions  of this study.  roots  highly  been  was  that  Thelephora  variable  can  seen grow  while well  collecting under  the  The amount of fungal growth onto new for all treatments, which may have  attributable to microsite differences, genetic differences  between seedlings or presence of fungal inoculum. Some  significant  differences pine  in  results  the  are  not easy to rationalize.  The  numbers of types of ectomycorrhizae on the  would seem logical if the E-strain treated trees had fewer  types, fungus.  since  their  However,  the  roots  were  dominated  by the inoculated  control and the E-strain had the fewest  types, while the fungus growing medium and A51 had the most.  may be possible that the fungus growing medium acted as a  117  It  substrate  which  allowed  better  growth  indigenous inoculum, while injected fungi low  colonization  number  of  fungus  growing  numbers  types.  of  explained  levels> In  the  spruce  medium  had  similar  fungi by  dominated  than  the  and  colonization  by  (except A51, which had  the  root and reduced the  trial, the control and the and  significantly higher  fungal treatments.  This could be  the high colonization rates of the two inoculated  fungi, which may have resulted in the exclusion of other fungi. Another  unusual result was that for spruce, the control had a lower  significantly B-strain levels  tissue  treatment. of  growing  level  than the Danielson  The calcium levels were grouped, with the  the  Danielson  E-strain  medium  treatment  being similar, and the levels of the  Amphinema control  calcium  byssoldes treatment  Danielson  B—strain  treatment was  not  treatment  and  the fungus  and control being similar.  The  obviously out of line, nor was the  treatment.  Further  study  is  needed  to  determine if this is a real effect. There are several ways these results might be interpreted with regard  to  the  importance  of  normal reforestation situations. of  a  fungus  that  can  ectomycorrhizal  inoculation in  One might say that high levels  grow in the field (Thelephora) without  assisted inoculation on nursery seedlings, and if that is  human  not enough, many wild types colonize seedlings rather quickly in the  first year of planting, so inoculation with ectomycorrhizal  fungi even  is  not  though  proportion clearly  important. many  of  the  fungi  Alternatively, one might argue that, grow  onto  the  roots,  the  largest  colonization on egressed roots in the field  comes from the nursery fungus, so the nursery fungus is  118  important. the  seedling  nursery  is in  and  decrease Many  It  in  also quite clear that the type of fungus on the  the  seedling  factors  field.  growth  can  affect  Different  under  exactly  growth both in the  fungi can increase or the same conditions.  other than growth might be altered by mycorrhizal  inoculation  but  mycorrhizal  nursery  the  changes  manipulation  in  growth  illustrate  that  is indeed an important tool in normal  reforestation situations, even though significant differences in survival,  would have had major and immediate importance,  which  were not realized. These say  specific  results are useful, but it is not possible to  whether the growth effects achieved in this experiment will  be reproducible from year to year with the inevitable changes in environmental  conditions.  communication)  suggests  findings  this  highlight with  from  Work  that  by  G.  they may not be.  Hunt  (personal  Therefore, the  study have been stated in a general way to  what they contribute to the technology of inoculation  ectomycorrhizal fungi  in normal reforestation situations.  This study shows that: 1.  Ectomycorrhizal infections, with  two  fungi  can  be  induced  in a nursery with a modified relatively  simple  to  form  heavy  cultural regime,  mycelial  inoculation  techniques; 2.  Different fungi do not affect growth in the same way under the same growing conditions;  3.  It is normal for seedlings to have more than one fungus on their roots in normal reforestation even after a very short time in the field;  119  type  of  situations,  4.  The fungi that infect roots in the first not mask the effects of of  roots 5.  seedlings  the  fungi that  in  the  field  season do  were  on  the  nursery;  Heavy infections by an inoculated  fungus  fungal diversity found on roots after one  may  reduce the  growing  season  in the field; Even  6.  small  ectomycorrhizal  levels fungi  colonization  of  have  may  by  significant  certain  effects on  growth; 7.  Thelephora terrestris has long been cited as an example of fungus  a  that  grows  well  in  the high fertility, high  moisture environment in the nursery and does not grow well in  the  typically  environment  in  lower  the field,  fertility,  lower  moisture  in this study Thelephora often  grew very well in the field; 8.  Local  fungal  byssoides)  isolates  improved  (Suillus  growth  over  tomentosus,  Arnphlnema  nursery  fungi  (a  predominant nursery fungus was Thelephora terrestris-like) but  an  isolate  from  a  distinctly  different  reduced growth, even though the same  (E—strain)  type  area of  fungus is common in the study area; 9.  Inoculation the  level  natural  with of  fungi  in  colonization  colonization  of  the  the  nursery  may  increase  even in nurseries with heavy same fungus or a different  fungus. The first point to note about these general statements is that none of them is,  in fact, general and they only positively apply  to the conditions described in this study.  120  However,  they do  describe for  potential behaviour.  inoculating  fungal  the  offers  potential  mycelium in inoculation trials.  fungi same  fungi  The simplicity of the techniques to easily apply  The fact that different  may potentially affect growth in different ways under the conditions  is  further  interact  with  their  fungi  presence  of  seedlings  more  than  confirmation that ectomycorrhizal in very specific ways.  hosts  one  type  of  confirms other recent work  fungus  (Roth,  on the roots of  1990;  Danielson and  Visser,  1989)  finding  one fungus that completely dominates a root may  the  only  This  is  showing  approach  The  this and suggests that concentrating on not be  to inoculation that should be investigated.  particularly  true  in light of the suggestion in this  study that one dominant fungus can reduce the number of types of fungi  that the  that  colonize type  of  a  There has been much speculation  root.  fungus  on a seedling in the nursery is not  important in natural reforestation situations because fungi from the  field  will  colonize  roots  so quickly that there will be  little carryover from the nursery. nursery even  fungi continue to influence the seedling in the field,  though field fungi move onto roots soon after outplanting.  Other low  This study confirms that the  recent levels  influence  studies of  (Stenstrom and Ek,  colonization  growth,  and  this  with  some  study  1990) have shown that fungi  are  confirms that.  enough to Therefore,  infectivity should not be the sole criterion for selecting fungi to  evaluate in outplanting trials  colonization well  in  the  has high  to  occur). nutrient  nursery can also grow well  (except,  of course, that some  Thelephora terrestris that grows  and  moisture  environment of the  in the typically, comparably lower  121  nutrient  moisture  and  environment  found in field situations.  This very adaptable fungus warrants considerably more study. component  essential  of  every  outplanting trial should be the  identification  and  inoculation  fungi should still be considered,  inoculated levels, The  of  fungus  evaluation  is  present  An  of  in  local  fungi.  Finally,  even when the  the nursery at high natural  or when some other fungus appears to dominate the root.  inoculation procedure may give enough of a headstart to the  inoculated  fungus that significant differences in growth can be  achieved.  122  8. OVERALL CONCLUSIONS  In serve  the as  Introduction, a  ectomycorrhizal  it was suggested that this study might  protocol fungi  for  further  studies  evaluating  in normal reforestation situations.  The  key elements of the protocol are: 1. The use of a comprehensive ecological classification system to describe outplanting sites and the sources of fungal isolates (a suitable system would be one similar system developed by Dr. V.  J. Krajina  British Columbia Ministry  of  (1969)  Forests  2. A description of the types of  to  the  biogeoclimatic  and expanded by the  and others>;  ectomycorrhizae  present  on  the natural seedlings at a site; 3. A description of the types of ectomycorrhizae on  seedlings  at outplanting; 4. A description of the changes that occur to of mycorrhizae on planted seedlings, after  the populations  outplanting,  over  the short and long terms; 5.  The use of a wide variety of fungi  situations,  including  local fungal  in  normal reforestation  isolates, exotic  assertive rnycorrhiza formers, minimalist  (fungi which  to affect growth while colonizing only a small root) mycorrhiza formers 6. Development of  portion  isolates, are of  the  and those in between;  improved cultural and inoculation techniques  for ectomycorrhizal fungi; 7.  able  The assessment of growth and survival  and long terms.  123  over  the  short  In  addition  to  fungi,  several  relate  to  questions  the  protocol for evaluating ectomyceorhizal  questions  were  posed in the Introduction that  selection of the best fungus for inoculation.  These  and the answers suggested by this study are presented  below. Q.  How  important  are the fungi on the roots of seedlings at  outplanting? A.  This study established that fungi inoculated in the nursery  do continue to affect growth after outplanting.  Growth can be  increased or decreased, but the duration of this study was not enough fungi  to  determine  persist,  if  and  those effects persist.  continue to dominate the roots after one  season in the field, even though other fungi A  potentially  inoculated decrease  important  fungi the  The nursery  infect the roots.  observation was that more assertive  CE-strain and Arnphinema byssoides) tended to  diversity  of types of mycorrhizae on seedlings  after one season in the field and a minimalist fungus did not.  tomentosus) E-strain  All of these fungi affected growth, but  decreased it. could  well-suited  to the site,  fungal  It  advantageous  if  the  tree and climate or  the  could  might  fungus  is  it could also be  if the fungus is not welladapted.  population  fungus.  be  The ability to exclude other types of  mycorrhizae  disadvantageous  (Suillus  A diverse  reduce the risk of having the wrong  also  be  possible  to  inoculate  with  minimalist fungi without reducing diversity. Q.  How  long  complement  of  does  it  fungal  take  for  a  seedling to develop the  genetic material necessary to be fully  adapted to its site?  124  A.  While  some  seedlings  had  or five fungi after one  four  growing season, the mean number for pine trees was 2.75 and 1. 69  for spruce.  root  after  that  four  are  one or  growing  was  There is also no guarantee  fungi for the site.  conducted, to  continue  season.  five types is the maximum number,  optimum  the  study  Many trees had only one type of fungus on the  play  it  or that they  At the site where this  seems clear that the nursery fungi  a major role,  even after the first growing  season. Q.  many  How  species  fungi  of  are  found  normally  on  a  particular tree species at a particular site? A.  There  were after  seedlings had  so  four  many  only  has  probably  not  not  relevant  now.  seedlings  young  species  fungi,  approach  to inoculum.  exposure  five  of  one growing season. of  types  will  seedlings  or  this  fungi on several Not all seedlings  but it is probable that more number,  given  the chance of  The upper limit to the number of types  been reached, but perhaps this question is The are  important capable  answer is that even very of  sustaining  diverse  ectomycorrhizal populations. Q.  the  Can  introduction of exotic fungal material result in  improvements  in  performance  in  normal  reforestation  situations? A.  In  this  byssoides  study,  the  local  and Suillus tornentosus)  fungal  isolates  (Amphinema  increased growth, while the  isolate  from  growth.  This occurred, even though E-strain was indigenous to  the  more  the study area.  125  distant  site  (E—strain)  decreased  Does  Q.  diverse fungal population improve performance, as  a  environmental conditions vary? A.  The  inverse  unexpected  infection  fungi  inoculated  by  between successful  relationship  and diversity of mycorrhizal  populations makes this question difficult to deal with in this study.  seedlings  The  had strong growth of inoculated  that  fungus had less diverse fungal populations.  No differences in  survival were found between the treatments but several factors could  been  have well  been  adapted fungi  displaced This  question  The inoculated fungi may have  interacting. to  the  not, and they may have  or  were more or less adapted to the site.  that needs  site  to  be  more  specifically addressed in  future work. some types of mycorrhizae more susceptible to disease  Are  Q.  or environmental stress? A.  This study did not reveal differences in seedling survival,  even has  though  shown,  been  better  than  mycorrhizal root  mortalities in spruce were quite high.  no  in  mycorrhiza,  studies, it  may  generally  others.  specific  probed,  the  be  improved  possible  that all  nutrient  uptake  like or  It may be that unless a tree is exposed to  some specific stresses or competition, the  that any mycorrhiza is  fungi confer some minimal set of advantages,  protection,  possibly  other  Since it  attributes  of  any fungus will do.  different  fungal  As  isolates are  more general attributes of mycorrhizal fungi may  also become apparent.  126  H,  c-i0  CD CD CD  tY .—  I—’-  ‘.0 H-  Z ‘.0 c--  H,  Di (ft (ft CD H c-Ic--< CD  tI  Di (ft (ft CD H c-Ic-• < CD  ‘-<  Pi  Di Z Cl  “  (tJ I (ft c-t H Di  C  H,  CD  i,Q  CD •  0  Dl  ci-  ty CD c-Ic-ICD H  (1)  I-’-  N Di  CD CD  Y Di (1)  c-c-I-  Di (ft  ‘-  CD 0 c-Ic-” 0 Z  Z  H,  I—-  F-  Di  (ft ti  (I) c-IDi  CD (ft c-IDi tY’  c-I0  (ft  Z Q ç (I)  -h  Cl)  FF-  -< 0 C’ H H t3  Dl  c-Itr Di ct  H I—’  Dl  0 I—.’ CD  c-ICD  i-  Q ç  (ft ciDi  ‘-<  t  0  CD Dl H  (ft  F<  CD Di H I-’  c-iZ Di c-I-  u CD (ft c-ICD  (ft C  ‘Q c--  CD H  <  CD  C)  •  0  0 0  c—’i  Di H Dl 0 c-ICD H -. (ft c-I-• 0 (ft  Dl  0  i-(I) 0  i—  Z  Di < CD  Cl)  F-j-, C’ H  I--  c  F-’.  ( c-I-  0 H  Di  CD c-—’ C’  Z CD  C’ H  C’  I--  Di  I.-.  — t1 Di 0 o Di H  Di  0 F-h -Z  (A)  0  ,-‘-  CD 0 c-I-  H,  0  Z  I-’-  F-  Fp-’(ft 3’  CD (ft c-IDi  CD Dl (ft c-—• I—’ c--<  c-I0  c-I<  ‘-“  c--’  Dl  CD  c-I-  0 CD  CD H (ft c-I-  Di H Di 0  0  (ft c-I-  1-’-  fl tY’ Dl H Dl 0 c-iCD H  c-Ic-’Di I—’  CD (ft (ft CD  Di  CD  Ill  CD H  cQ (ft •  i-a-  (ft CD CD Cl  c-itY CD c--H  0 I-h  0 tY CD  Di CD  ‘.  CD  Dl  c-I0  (1) CD CD  Di c-i-  ‘  c-I-  I—  c.Q  H  ( c-cDl Q CD  CD Dl H I—’ -<  CD Cl  i-a-  Cl CD (ft 0 H  Di  ‘—  c-I-  0  CD  i—’ Cl  0  cDl (ft  ‘-—  — FcC a> a>  CD I—’ (A) 0  F-  C1 Di Z  •  (ft  0 ‘V ‘V 0 H c-Ic’Z Z  I—h  0  CD  Di ‘<  c—• c-I-  0 H  ‘.  FF-  H,  ci0  H ‘3 FCD  0 C FDi 1  i-’-  ‘V Di H c-i-  Dl  Z Dl (ft  Dl c--<  c-I-  i—i  •  (I)  c-Ic-’0  F-  0 0 Z Cl  0 H  (ft CD c-I-  Di H  I—’  0 C  F-’-  ‘V Dl H c-i-  ‘-<  Di  Dl H CD  -J  F-  Dl  c-ItY’ Dl c--I-  CD c--’  F-  c-’  c  r Dl c-I-  CD  (ft 0  (ft  (ft CD CD  i-’-  cC)  F-h C  Di Z -<  c—’ tY 0 CD  •  Ct CD  --  (1)  Di c-I-  ci-  c-I0  Di ‘V c-ICD Cl  Cl  Di  -< c—c-I-  H 0 0 c-I(ft  CD  c-I-  Z H, CD 0 c-i-  F—’  c-IC  Dl Cl Di O ct CD Cl  I—a ‘<  Di  c-i-  C  F-  Cl)  I-I,  CD Di 0  Di c-I-  c-I-  CD  ‘tJ  CD X Dl  Di  CT)  D 0  Di  (ft c--• c-ICD  Di  Di c-i-  tY ‘.0 (ft  I-’-  CD CD CD Cl F-’  U  CD H Di c-Ic--  CD  I-I CD  I<  F-  H Di  c-I-  Di  0 tI  N Dl CD  I--  0 0 H H  5l c-I-  CD Cl  Di (ft ( (  Di (ft  c-I-  ‘—4  Z ‘.0 (ft •  F-  CI) CD CD Cl I—’  0  H 0 0 c-I(ft  c-IT CD  i  0  (ft CD CD tl  ‘-•  Z  H,  0 1t  () CD H (ft  Cl c-’< CD H (ft CD  c-ItY CD  0 H,  0  I—-  Dl c-i-  <  0 T (ft Cl) H  CT)  c-I-  F-h H 0  CD  (ft  i-a-  Di H  (p  0  CD (ft c-I-  Q  CD H  C) c-I-  •  0  F-’-  Di c-I-  I--  < CD (1) c-I-  i--  0 H  H,  CD  H  (12  I—’-  Dl c-i-  c-i-  Di H Cl) Dl  Dl  F-’-  (ft  c-I-  H 0 C’  0  c-fi  0  I—’ Di cI-  ‘V 0 ‘V  N Dl i—  --  ‘-< 0 0 H H r  CT) Cl  i•  H,  C’  c-0 (ft  z Di  ‘-<  Cl  CD  ‘-3  c-I.)  CD  F-  Di  <  ‘V H CD  CD  CD 0 Di  0 t (ft  ‘--  Cl c--c-I-  0 0  H CD H, CD H H CD Cl  c-I(ft  1-  i—-  S c-t  CD  <  H-  Di 0 c-t  CD (ft (ft  F-  CD Cl  F-’•  Dl  CD  H  CD H  o c-I-  CD  c-I-  I-’ CD  I—’  c-I0  c-ICD Q  Dl  Di Cl  0 H CD  (ft  ct  i-’  Di c-i-  tY  c-i-  c-IDi Z 0 CD CD  (I)  H 0 z  F-  0  CD  Z  ci-  I-’-  Di c-ICD  F-  Cl 0  CZ (ft  Z  i-t-  0 Z CD  Cl  i-  0  c-I•)  0  0 CD  0 Di  rt  Z’ CD ‘-<  c-i-  Cl  i—’  C  C) H  CD 0 c-I(ft  H,  CD H,  c-I-  c.Q H 0  c-I0  Dl H Cl  H CD  c--. c-I1  Dl c-ICD  .  Cl o  ci0  CD  I<  H,  Z CD  0  0 H  F-h  0 -<  c-iCD Z Cl CD  Di  (Y’ CD  Cl) H CD  c-I-  c—’ Cl  0  H 0 0 c-I•)  Di  C t  c-iCD H Di 0 c-I-  I  C z  0 tY  (ft  Cl o  0  •.  Dl H CD  (1)  CD (ft c-Ic--a()  cO  0 H CD  X  Ci t (ft •  F-’-  Cl c-’c-I-  0 0  CD  UI Di  CD  ci-  t$ Cl CD H  c-ItY  H C)  .0  Cl CD 0 H CD Dl (0 CD  0 H  Z 0 H CD Di (ft CD  F-  0 Di  I—’-  H,  H, H, CD H Ct’ Z ci-  F-’-  Cl  Di ct  ci-  Cl  Di  ri!  z  M-  tS CD  0  tS  Di  c-I-  0 H CD  Di < CD  F-<  H Di I-’ F  Di c-I-  (  Q  (ft CD CD Cl Fc—’-  Di ci-  c-i-  0  Di c-I-  (ft CD H  0  f CD  ct  C  H  F-h  Cr.c  (p c-ICD  0 tY  c-I-  Cl)  CD  rj)  < c— C  0  CD  C)  •  0 H CD  (I> Cl) < CD H CL’ F  (ft CD Cl  H Di  Di (ft 0  Di J  cc-i-  (11  CD (ft c-l c-’0  Q  CD  (ft 0  c-I 0  CD H (11  (ft  Dl  Cl  CD  LO o CD (ft ci  CD  Di (ft  Cl <  (SI c-I  (  i-  c-i  I—’ CD  consistantly  reduce  seedling growth.  One might speculate that  these fungi may confer some general mycorrhizal benefits such as increased  root life, disease protection and increased uptake of  nutrients and water.  An unavoidable consequence of rapid growth  by the fungus is high carbohydrate drain.  Over time, the fungal  population of the root could become more attuned to the site, as other  this  which  It is interesting to note  study were E-strain and Thelephora terrestris, both of  might be considered assertive fungi.  E—strain study be  on the root.  some of the most common fungi to infect roots in the field  that in  establish  fungi  reduced growth.  Yet,  the Inoculated  It was not possible to tell from this  if the volunteer E-strain also reduced growth, but it may  that a reduction in growth is not as critical as the need to  become mycorrhizal as quickly as possible. There study  were with  stated,  two regard  heavy  somewhat  contradictory observations in this  to fungal populations on roots.  infections  As already  by inoculated fungi seemed to reduce  the fungal population diversity on seedlings after one season in the  field.  inoculated  However, mycorrhizae  even  seedlings  began  populations in the field.  to  with  develop  high  levels  of  more diverse fungal  The reduction in population diversity  may  not  but  simply a reflection of the ability of the inoculated fungus  to  get  be  the result of active exclusion,  there  assertive fungi for that  new  fungi  Suillus  first.  This  in general.  may  also apply to early—stage or  There seems to be little difficulty  to colonize in the field. tornentcisus  or outcompetition,  had  The affect on growth  with low levels of infection may  indicate that a low percentage colonization is all that is  128  necessary for some types of mycorrhizae to affect growth. The  reductions  growth  In  caused by assertive fungi and the  increase in growth caused by Suillus tomnentosus describes  Suillus  as  multi-stage  fungi)  (Danielson,  suggest  a possible  strategy for improving the performance of nursery seedlings. the  vigorous  achieving  the  seedlings  late—stage effort  fungi  manipulations  with  early—stage  fungi  establishment the  is an adaptation to  it may be possible to  by assuring that nursery  with  multi—stage or late—stage fungi.  idea  is that many of these multi- and  not  culture and/or inoculate well.  gives  a  have  that  been  multi-  done  and  will  Some  allow inoculation.  description  brief  unsuccessful.  early—stage  about  role  techniques  that  largely  that  do  this  If  be put into finding such fungi that do inoculate,  (1988)  inoculation  for  this  developing  Danielson  been  early—stage fungi  infected with  might  into  need  are  problem  The  of  mycorrhizal infections quickly,  circumvent  or  growth  1988  to  of  some of the  try to get successful  late-stage fungi.  Attempts have  Since late-stage fungi do replace  in the field,  it is interesting to speculate  fungi may play some type of nurse role in the  of later stage fungi.  mechanics  of  More  information is needed  mycorrhizal—fungus succession on tree  roots. While  many  questions  are  raised  here,  this  study  has,  nevertheless,  resulted in some concrete advances in techniques.  For  top—applied  fungi  example, were  lodgepole inoculation  mycelial slurries of ectomycorrhizal  used successfully to inoculate Engelrnann spruce and pine  seedlings  in  a  commercial  nursery.  This  technique was extremely simple and could be adapted  129  to  application  commercial  fungal  the  fungal  of  in  conjunction above.  mentioned  applicable  easily  inoculation useful  of  tools  developed  the  with  These  two  steps  in  simple  that  A new  simplifies the  inoculation  procedures  research,  applied  the  are  enormously  procedure  could constitute two  commercial  a  ectomycorrhizae  investigating  expense.  The fungal culture technique works  ectomycorrhizal fungi.  for  of  synthesis  little  very  culture and speeds up the growth of most of  isolates tested.  fungal  well  was  technique  culture  process  with  protocol  However, since  two  the  they are also  the  culture obstacles  major  diverse  for  populations  and to of  ectomycorrhizae. An and what out  important aspect of this work was the observation of roots mycorrhizae  to  qualitatively and quantitatively  the mycorrhizae were doing.  Many researchers have pointed  growth statistics can convey only a small part of the  that  mycorrhizal  picture.  heavily  the  on  Future  work will have to rely even more  detailed examination of roots.  A new staining  using a low-toxicity dye suitable for the evaluation  technique, of  describe  certain  introduced  features in  researchers  this  to  ectomycorrhizae,  of  developed  and  It is already being used by other  study.  examine  was  ericoid  mycorrhizae and should have a  very high potential application for routine use. These for  developments  are  useful, but in the end,  outplanting trials must be stressed.  ectomycorrhizae outlined  in  is  collected  this thesis,  in  a  the protocol  Unless information on  systematic  way,  such as  it may be a very long time before the  the many questions about ectomycorrhizae in  130  normal reforestation  a H a  a H a a  0  I—  a  rt  (ft  LITERATURE CITED  R. Agerer, 1986. Studies on ectomycorrhizae. II. Introducing remarks on characterization and identification. Mycotaxon 26: 473—492. Amaranthus, M. P. and Perry, D. A. 1989. Rapid root tip and mycorrhiza formation and increased survival of Douglas-fir seedlings after soil transfer. New Forests 3: 259—264. Bernier, B. 1968. Descriptive outline of forest humus-form classification. 7th National Proc. Meeting Soil Survey Committee of Canada, Edmonton. pp. 139-154. Bledsoe, C. S., Tennyson, K. and Lopushinsky, W. 1982. Survival growth of outplanted and Douglas-fir seedlings inoculated with mycorrhizal fungi. Can. 3. For. Sci. 12: 720-723. Boyle, C. D., Robertson, W. 3. and Salonius, P. 0. 1987. Use of mycelial slurries of mycorrhizal fungi as inoculum for commercial tree seedling Can. 3. For. Res. 17: nurseries.. 1480—1486. Castellano, M. A. In Outplanting Press. mycorrhizal inoculated seedlings: a review.  performance  of  Castellano, M. A., and Trappe, 3. M. 1985. Ectomycorrhizal formation and plantation performance of Douglas—fir nursery stock inoculated with Rhizopogon spores. Can. 3. For. Res. 15: 613—617. Conn, H. 3. 1977. H. & Wilkins Company,  Conn’s Biological Stains. Baltimore. pp. 254 and 257. 3.  The Williams  Danielson, R. M. 1984. Ectomycorrhizal associations in jack pine stands in northeastern Alberta. Can. 3. Bot. 62: 932-939. Danielson, R. M. 1988. Mycorrhizae in forestry: The state of the art in land reclamation. In: Atelier canadien sur l’usage des mycorhizes forestrie. en Lalonde Ed: M. and Y. Piche. Departement des sciences forestieres, Universite Laval. pp. 39—42. Danielson, R. M. and Visser, S. 1988. Ectomycorrhizae of jack pine and green alder: assessment of the need for inoculation, oil sand on tailings. Report RRTAC 88—5, Alberta Land Conservation and Reclamation Council, .Edmonton. Danielson, Visser, R. M. and S. 1989. Host response to inoculation and behavior introduced of and indigenous ectomycorrhizai fungi pine grown of on oil sand jack tailings. Can. 3. For. Res. 19: 1412—1421.  132  Danielson, R. M. and Visser, S. 1990. The mycorrhizal and nodulatlon status of container-grown trees and shrubs reared in commercial nurseries. Can. J. For. Res. 20: 609—614. Danielson, R. M., Visser, S. and Parkinson, D. 1984. The effectiveness of mycelial slurries of mycorrhizal fungi for inoculation of container—grown :iack pine seedlings. Can. the J. For. Res. 14: 140—142. Danielson, R. M., Griffiths, and Parkinson, C. IL 1984. Reinstatement of biological activity in severely disturbed soils: Ectomycorrhizae in amended oil sand tailings and subalpine coal mine spoil and in undisturbed jack pine and spruce stands. Alberta Land Conservation and Reclamation Council Report tRRTAC 84-7. lO8p. Daughtridge, A. T., Boese, S. R., Pallardy, S. G. and Garrett, E. 1986. H. A rapid staining technique for assessment of ectomycorrhizal roots. infection Can. 3. Bot. 64: of oak 1101—1103. Fortin, C., Fortin, J.-A., Gaulin, G. Jomphe, N. and Lemay, S. 1988. Large-scale ectomycorrhizal inoculation of containerized—grown seedlings. Atelier canadien In: sur l’usage des mycorhizes en forestrie. Ed: M. Lalonde and Y. Piche. Departement des sciences forestieres, Universite Laval. pp. 115-118. Grenville, D.J., Peterson, R. L. and Ashford, A. E. 1986. Synthesis in growth pouches of mycorrhizae between Eucalyptus pilularis and several strains of Pisolithus tinctorius. Aust. J. Bot. 34:95—102. Hacskaylo, E. 1973. Carbohydrate physiology of ectomycorrhizae. In: Ectomycorrhizae: Their Ecology and Physiology. Eds: G. C. Marks, and T. T. Kozlowski. Academic Press, New York. pp. 207—230. Harley, 3. L. and Smith, S. E. Academic Press, London. 483 p. Hatch, A. Pinus.  1983.  Mycorrhizal Symbiosis.  B. 1937. The physical basis of mycotrophy in the genus Black Rock Forestry Bulletin 6, 168 p.  Heidman, 3. 1986. Effect of various L. and Cornett, Z. 3. nutrient regimes and ectomycorrhizal inoculations on field survival and growth of ponderosa pine (Pinus ponderosa var. scopulorum Engeim.) container seedlings in Arizona. Tree Planters’ Notes 37:15—19. Ho,  1987. I. mycorrhizal 855—858.  Enzyme activity and phytohormone production of a fungus, Laccaria laccata. Can. 3. For. Res. 17:  133  Hunt, G. A. 1989. Effect of controlled—release fertilizers on growth and mycorrhizae in container-grown Engelmann Spruce. West. 3. Appl. For. 4(4): 129—131. L. 1990. Husted, D. Influence soil temperature of on the effectiveness of ectomycorrhizal fungi. FRDA 52—41-109. Final report for Canada—British Columbia Forest Resource Development Agreement; 49 pp. Hutchison, L. 3. and Malloch, W. 1988. D. A verification protocol for cultural isolates of ectomycorrhizal basidiomycetes. In: Atelier canadien sur l’usage des mycorhizes en forestrie. Lalonde Ed: M. and Y. Piche. Departement des sciences forestieres, Universite Laval. pp. 121—124. K., Green, Klinka, R. N., Trowbridge, R. L. and Lowe, L. E. 1981. Taxonomic classification of humus forms in ecosystems of British Columbia. First approximation. B. C. Mm. For., Land Manage. Rep. No. 8. Kropp, B. R. 1982. Formation of mycorrhizae on nonmycorrhizal western hemlock outplanted on rotten wood and mineral soil. For. Sci. 28: 706—710. Kropp, B. R. reforestation.  and Can.  Langlois, 1990. Ectomycorrhizae C. 3. For. Res. 20: 438—451.  in  Kropp, B. R., Castellano, M. and Trappe, A. 3. M. 1985. Performance of western hemlock (Tsuga heterophylla (Raf.) seedlings Sarg.) inoculated with Cenococcurn geophilum. Tree Planters’ Notes 36: 13—16. Loopstra, E. M., Shaw III, C. G. and Sidle, R. C. 1988. Ectomycorrhizal inoculation fails to improve performance of Sitka spruce seedlings on clearcuts in southeastern Alaska. West. 3. Appi. For. 3(4): 110—112. Lloyd, D., Angove, K., Hope, G., and Thompson, C. 1990. A guide site to identification and interpretation for the Kamloops Forest Region. Land Management Handbook Number 23. B. C. Ministry of Forests, Victoria. Marx, D. H. 1969. The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59: 153-163.  134  —  1977. The role of mycorrhizae in forest production. Tappi Conf. Pap., Annu. Mtg., Feb. 1977, Atlanta, Ga. pp. 151—161. Ectomycorrhizal fungus inoculations: a tool for improving reforestation practices. In: Tropical Mycorrhiza Research. Ed: P. Mikola. Clarendon Press, Oxford. pp. 13-71. Variability in ectomycorrhizal development and growth among isolates of Pisolithus tinctorius as affected by source, age and reisolation. Can. J. For. Res. 11: 168-174. 1990. Soil pH nitrogen and influence Pisolithus ectomycorrhizal development and growth of loblolly pine seedlings. For. Sci. 36: 224—245.  Marx, and D. H. Cordell, C. E. 1988. Specific mycorrhizae improve reforestation and reclamation in the Eastern United States. In: Proceedings, Canadian Workshop on Mycorrhizae in Forestry. Ed: M. Lalonde and Y. Piche. Center de Recherche en Forestiere, Biologie Faculte de Foresterie et de Geodesie, Universite Laval, Ste-foy, Quebec. pp. 75—86. Marx, and D. H. Daniel W. 3. 1976. Maintaining cultures of ectomycorrhizae and plant pathogenic fungi in sterile water cold storage. Can. 3. Mierobiol. 22: 338-341. Marx, and D. H. D. Kenney, Production S. 1982. of ectomycorrhizal fungus inoculum. In: Methods and Principles of Mycorrhizal Research. Ed: N. C. Schenck. The American Phytopathological Society, St. Paul. pp. 131—146. Marx, D. H. and Shafer. 1989. Fungal and bacterial symbioses as potential markers biological of of effects atmospheric depostition on forest health. In Proc. National Research Council Workshop Markers on of Air Pollution Effects in Forests, Wilacres Retreat, NC. 25—27, April 1988, NRC, Washington, D. C. pp. 217-232. Marx, D. H. formation 66—75.  and Zak, of slash  1965. B. Effect of pH on mycorrhizal in aseptic culture. For. Sci. 11: pine  of loblolly pine with Pisolithus ectomycorrhizae performance on good-quality a site. South J. Appi. For. 12(4): forest 275—280. Mikola, P. 1970. Rev. For. Res.  Mycorrhizal inoculation in afforestation. 3: 123—196.  135  mt.  Nikola, P. Application 1973. mycorrhizal of symbiosis in forestry practice. In: Ectomycorrhizae: Their Ecology and Physiology. Eds: G. C. Marks, and T. T. Kozlowski. Academic Press, New York. pp. 383-411. Miller, K. and Nicklin, S. 1980. Adverse reactions to food additives and colours. In: Developments in Food Colours—2. Ed: John Walford. Elsevier Applied Science Publishers, Essex. Molina, R. 1977. Ectomycorrhizal fungi and forestry practice. In: Proceedings, Mushrooms and Man: An Interdisciplinary Approach to Mycology. Ed: T. Walters. Linn-Benton Community College. 309 pp. 1979a. Eetomycorrhizal inoculation of containerized Douglas—fir and lodgepole pine seedlings with six isolates of Pisolithus tinctorius. For. Sci. 25: 585—590. Pure culture synthesis and host specificity of red alder mycorrhizae. Can. J. Bot. 57: 1223—1228. Mycorrhizal inoculation and its potential impact on seedling survival and growth in southwest Oregon. In: Proceedings, Reforestation of Skeletal Soils, Medford, OR. Eds: S. D. Hobbs and 0. T. Helgerson. For. Res. Lab., Oregon State Univ. pp. 86-90. Molina, R. and Palmer, J. G. 1982. Isolation, maintenance and pure culture manipulation of ectomycorrhizal fungi. In: Methods and principles of Mycorrhizal Research. Ed: N. C. Schenck. American Phytopathogical Society, St. Paul, Minnesota. pp. 115—129. Molina, R. and Trappe, J. M. 1984. Mycorrhiza management in bareroot nurseries. In: Forest nursery manual: Production of bare root seedlings. Ed: Duryea and T. D. Landis. M. 1. Forest Research Laboratory, Oregon State University, Corvalis. Martinus W. Junk Publishers. The Nijhoff/Dr. Hague/Boston/Lancaster. pp. 211—223. Moss, B., Stribley, D. P. and Le Tacon, F. 1981. Ecology of mycorrhizae and mycorrhizal fungi. In: Advances in microbial ecology. Ed: N. Alexander, Cornell University, Ithaca. New York Plenum Press, New York. pp. 137-207. Park, J. Y. 1971. Preparation of mycorrhizal grain spawn and its practical feasibility in artificial inoculation. In: Mycorrhiza, Ed: E. Hacskaylo, USDA Forest Serv. Misc. Publ. 1189. pp. 239—240.  136  Perry, D. A., Molina, R. and Amaranthus, M. P. 1987. Mycorrhizae, rnycorrhizospheres, arid reforestation: current knowledge and research needs. Can. 3. For. Res. 17: 929—940. Phillips, 3. M. and Hayman, D. S. 1970. Improved procedures for clearing roots and staining parasitic and vesicular—arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55: 158—161. Pirozynski, K. A. and Malloch, D. plants: a matter of mycotrophism. Richter, D. L. and containerized red slurries mycelial 247—258.  W. 1975. The origin of Biosystems 6: 153—164.  land  Bruhn, 3. N. 1989. Field survival of and jack pine seedings inoculated with of ectomycorrhizal fungi. New Forest 3:  Riffle, W. and 3. Maronek, D. Ectomycorrhizal M. 1982. procedures inoculation for greenhouse nursery studies. and Methods and principles of mycorrhizal research. Ed: N. In: C. Schenck. American Phytopathological Society, St. Paul. pp. 147—155. Roth, A. L. 1990. Mycorrhizae of outplanted conifer seedlings on eastern Vancouver Island. M.Sc. thesis, University of British Columbia, Department of Soil Science. Schenck, N. Research,  C. 1982 Methods American Phytopath.  and Principles of Mycorrhizal Soc., St. Paul. 244 pp.  Smith, S. E. 1985. The concept of effectiveness in symbiotic relationships. In: Proceedings, 6th North American Conference on Mycorrhizae. Ed: R. Molina. For. Res. Lab., Oregon State Univ., Corvallis. pp. 146—149. Stamets, and P. Agarikon Press,  Chilton, 3. S. 1983. Olympia, Washington.  The Mushroom Cultivator. pp. 42—43.  Stenstrom, E. and Ek, M. 1990. Field growth of Pinus sylvestris following nursery inoculation with mycorrhizal fungi. Can. 3. For. Res. 20: 914-918. Stenstrom, Ek, M. and Unestarn, T. In press. Negative field E., responses of Pinus sylvestris seedlings following nursery inoculation with mycorrhizal fungi. Can. J. For. Res. Stevens, R. B. Washington Press,  1981. Mycology Seattle, p. 326.  Guidebook.  University  of  Thomas, G. W. and Jackson, R. N. 1983. Growth responses of Sitka spruce seedlings to mycorrhizal inoculation. New Phytol. 95: 223—229.  :137  Trappe, J. M. 1977. Selection of inoculation in nurseries. Ann. Rev.  fungi for ectornycorrhizal Phytopathol. 15: 203-222.  Trappe, J. M. and Fogel, R. D. 1977. Ecosystematic functions of mycorrhizae. In: The Below—ground Ecosystem: A synthesis of Plant-Associated Processes. Ed: J. K. Marshall. Range Soil Dep., Science Series No. 26, Colorado. pp. 205—213. Trappe, J. M. and Strand, R. F. a Douglas—fir region nursery.  1969. Mycorrhizal deficiency in For. Sci. 15: 381—389.  Trofymow, J. A. and van den Driessche, R. 1991. Mycorrhizas. In: Nutrition Mineral of Conifer Seedlings. Ed: R. van den Driessche. CRC Press, Boca Raton. pp. 183—227. Wilcox, H. E. 1982. Morphology and development of ecto- and eetendomycorrhizae. In: Methods and Principles of Mycorrhizal Research. Ed: N. C. Schenck. American Phytopathological Society, St. Paul. pp. 103—114. Wong, K. K. Y. and Fortin, J. A. 1988. Ectomycorrhiza formation on Pinus banksiana roots by Laccaria bicolor variants under aseptic conditions. In: Proceedings, Canadian Workshop on Mycorrhizae in Forestry. Ed: M. Lalonde and Y. Piche. Center de Recherche en Biologie Forestiere, Faculte de Foresterie et de Geodesie, Universite Laval, Ste—foy, Quebec. pp. 161—167. Zak, B. and Larsen, M. 1978. Characterization and J. classification of mycorrhizae of Douglas—fir. III. Psuedcitsuqa + Byssoporia (Poria) terrestris vars. menziesii lilacinorosea, parksii, and 56: sublutea. Can. J. Bot. 1416—1424. Zar, J. H. 1984. Biostatistical Analysis. Inc., Englewood Cliffs. pp. 718.  138  2nd Ed. Prentice—Hall,  The pure culture synthesis chamber also showing trees FIG. 1. and the watering tray. FIG. 2. Inoculation of the pure culture synthesis chamber.  139  showing A dilute—agar culture the concentration of FIG. 3. Cenococcum geophiluni in dilute—agar near the top. FIG. 4. growth culture on the left and liquid medium on the right.  140  FIG. 5. Characteristically coarse E—strain hyphae on the surface root. Bar = 0.4mm. FIG. 6. E—strain hyphae growing from a a of root. Bar severed = 0.3mm. FIG. 7. B—strain Hartig net with characteristic circular shape near the centre. Bar = l5um.  141  F  Exudates Note the FIG. 8. on Type 12 Suilloid fungus in KOH. FIG. 9. Typical violet—coloured exudates. Bar = lOum. brownish—coloured exudates on a Type 10 Suilloid rhizomorph. Bar = 3Ourn.  142  FIG. 10. The typical growth of Amphinerna byssoides extramatrical FIG. 11. Arnphinerna byssoides hyphae on a plug. Bar = 4mm. showing the same vigorous growth on a seedling from the (arrow) field trial.  143  FIG. Type 3 mycorrhiza on pine showing the characteristic 12. in the mantle. oil droplets Blue stain is from FDA Blue No. 1 described in Experiment Three. Bar = lOum. FIG. 13. Longitudinal section of Type 3 on pine showing mantle stained with FDA Blue No. 1. Bar 0.25mm.  144  14. The golden yellow exudates and lactifers of Type 8 on FIG. = 2Oum. FIG. 15. Pine roots growing straight down spruce. Bar from the plug, showing non—mycorrhizal roots near the top and mycorrhlzal further clown. Bar = 4mm.  145  APPENDIX I FUNGI USED IN PURE CULTURE SYNTHESIS and THE NURSERY SCREENING TRIAL  A6:  This  culture was isolated from a Rhizopogon spp.  collected glauca)  under  Douglas-fir  (Pseudotsuqa  sporocarp  menziesii  near Haggard Creek at about 210Gm elevation.  biogeoclimatic  zone was the  var. The  IDFmw2 and the collection site  was about 15km east of the study area. A13:  Culture on  taken from a highly tuberculoid, white mycorrhiza  an  approximately  Collected the  October 14,  MSdm2  south  of  isolated  five-year-old 1987,  biogeoclimatic the from  study  Isolated  pine.  near Sargent Creek at 1300m in zone.  area.  mycorrhizae  This site is about 15km  Samples taken  seed 1 ings growing along roads or A14:  lodgepole  A13 to A22 were all  from  small  volunteer  in clear cuts.  from a bright yellow mycorrhiza on spruce at the  same site and same time as A13. A16:  From  an  hyphae  approximately  four-year-old  spruce  with white  and rhizomorphs and pinnately branched mycorrhizae.  Produced  colonies  identical  to  A18.  Same site and same  time as A13. A16A: A16B:  Same as A16 but colony morphology differed. Same  as  A16 but colony morphology differed from A16 and  A16A.  146  A17:  Taken  from  a  small, brown with white spots, tuberculoid  mycorrhiza on lodgepole pine at the same site and same time as A13. A18:  This  was  the  most  at  the  site described in A13.  spruce distinct  mass  of  common  type of mycorrhizae found on  extramatrical  It formed the very  hyphae  associated  with  Amphinema  byssoldes, and was identified as being Amphinema  byssaldes  (  in  KOH,  hyphal and  Fr.) 3. Erikss., on the basis of (1) staining slow-growing  (2) clamps,  mycorrhizae  (3)  (4) occasional fine ornamentation on hyphae  general  (5)  indurate colonies on agar,  in  appearance,.  This isolate did not form  the nursery screening trial.  All isolates  from A18 to A22B were collected October 15, 1987. A18B:  This have  isolate the  came from the same root as A18 but did not  usual Amphlnema byssoldes growth characteristics  in culture.  This culture was used in the nursery screening  trial on spruce and did not form mycorrhizae. A19:  From  a  seedling byssoldes, shaped.  spruce had  seedling  dense but  This  the fungus  at  the same site as A13..  extramatrical mycorrhizae was  used  hyphae were  like Amphinema  very  irregularly  in the nursery screening  trial, but did not produce mycorrhizae.  147  This  A20:  This  fungus  slow—growing isolated  produced indurate  from  mushrooms byssoides.  pine.  was  This  extramatrica).  colonies  young  that  dense  It  to  similar had  a  hyphae  and  A18, but was  distinct odor of  not apparent in cultures of Amphinema  was used in the nursery screening trial,  but did not produce mycorrhizae on spruce. A21: From a much branched, palmate, white mycorrhizae on pine in the  same  used  area  in  the  No obvious rhizomorphs.  as A13.  This was  nursery screening trial, but did not produce  mycorrhizae on spruce. A22:  From the  a tuberculoid type on pine very similar to, and from same  nursery  location screening  as  A13.  trial,  This isolate was used in the  but did not form mycorrhizae on  spruce. A22B:  The  same  as  A22  exhibited  but  a  different  colony  morphology. A29:  Collected from an Arnphinema byssoides infected root from a spruce  at  October  the  15,  screening  Reforestation  Heffley  1987.  trial,  This and  Centre  isolate was used in the nursery  was the only isolate to perceptibly  alter the colonization of the nursery seedlings. dense  masses  nursery on  It formed  of  hyphae  that  same  source  as A29 but the colony morphology  tightly  bound  the  plugs  together. A29B:  From  the  differed.  148  A30:  Isolate  1988 from a Suillus brevlpes  taken June 17,  (Pk.)  Kuntze sporocarp growing under a 15-year—old lodgepole pine Community  near The  site  was  1350m  biogeoclimatic  zone.  numbers A31:  Lakes  about 38km south of the study area. in  elevation  and  fungus  This  was  in the MSdm2  was fruiting in large  in the area.  Same place and same time as A30 from a sporocarp of Suillus pseudobrevipes  Smith  &  Thiers.  Only  one sporocarp was  found. From  A31A:  the  same  sporocarp  as A31,  but colony morphology  same  sporocarp  as A31,  but colony morphology  differed. A31B:  From  the  differed. A33: Found at the Sargent Creek site on June 17, very  a  ESSFwm  young  subalpine fir near a creek,  biogeoclimatic  zone.  1988,  probably in the  culture  This  but under  came  from a  sporocarp tentatively identified as a Hebelorna spp. A34A:  Taken from a mycorrhiza on two or three—year—old spruce at the  Sargent Creek site on June 17,  1988.  This was another  Amphinerna byssoides isolate. A34B:  Same as A34A.  A36:  This short  isolate root  was taken on June 17,  1988 from an E-strain  at the Sargent Creek site,  but the culture was  not E—strain. A45:  Found  on  September  22,  1988 at the Sargent Creek site.  From an unidentified sporocarp growing under pine regen. A46:  Same as A45.  149  A48:  This  culture  collected  was  isolated from a Leccinum spp. that was  under the same circumstances as A45.  many tree species growing in this area,  There were  including lodgepole  pine, aspen, willow and subalpine fir. A48B:  Another  isolate from the same sporocarp as A48, but with  different colony morphology. A49:  From  a  found  Suillu.s  under  tomentosus  the  (Kauff.) Sing., Snell & Dick  same circumstances as A45.  A very large  number of sporocarps were present. A51:  Same the  as pure  A49.  This isolate formed mycorrhizae on pine in  culture  synthesis  trial  and  was  used in the  nursery trial and the outplanting trial. Danielson’s  E—strain  obtained the  most  Mikola)  (R947): This isolate was  from Gary Hunt who got it from R.  University  spruce  (sensu  of  seedlings vigorous  Calgary.  It  was  M.  isolated from white  growing in subalpine soil. mycorrhiza  former  in  Danielson at  the  This was the pure  culture  synthesis trial and was used in the nursery trial. E—strain  (0188):  This isolate was obtained from Lynn Husted who  isolated  it from container-grown Douglasfir at the Harrop  Nursery,  near Nelson,  pure  culture  B. C..  synthesis  It formed mycorrhizae in the  trial, but not very well.  used in the nursery trial.  150  It was  Hebelonia  crustulinlforrne  supplied  this  Bledsoe,  of  (Bull: St Arnans) QUEL.  isolate, the  which  University  was of  Gary Hunt  (5):  collected by Caroline  Washington,  in  a mixed  conifer forest at 550m in Wenatchee National Forest.  It is  kept in the University of Washington Collection. crustulini.forme  Hebeloma  from  the  St.  (Bull.:  University  Amans) QUEL.  (8):  Also  of Washington collection of Bledsoe.  It was isolated in 1971 from a Douglas—fir forest in Benton County, pure  Oregon.  culture  This  fungus produced mycorrhizae in the  synthesis  trial and was used in the nursery  trial. B122:  This Gary  is  Laccaria  laccata  (Scop.: Fr.) Berk.  & Br.  from  It was isolated in 1985 from a Douglas—fir in  Hunt.  the Heffley Reforestation Centre nursery. B148:  Also Laccaria laccata Gary  Hunt.  This  (Scop. ex Fr.) Berk.  was  isolated  Douglas—fir, Engelmann spruce,  in  1986  & BR.  from Dr.  from  a  mixed  lodgepole pine forest in the  Community Lakes area described under isolate A30. B169:  An  isolate  Hunt,  who  spruce,  of Laccaria glabripes obtained from Dr. Gary  isolated it from a mixed Douglas-fir, Engelmann  lodgepole  pine forest in the Community Lakes area  described at A30. A188—2: The  Cenococcurn  isolate  Oregon, of 305m.  Fr.  geophilum  came  from  11,3km  isolate from Dr. southwest  of  Gary Hunt. Philomath,  U.S.A. at 4428’3O”N, l2329W and at an elevation Aspect was southern and slope variable.  The soil  was Hohnnon gravely loam and is well-drained, moderately  151  deep  and derived from weathered sandstone.  The pH was 5.2  in the litter layer and 5.7 in the Al horizon. mild  and  averages Hunt  All  summers 1905mm.  (1979).  isolates  were  Winters are  warm  and  More  details can be found in Fogel and  dry.  Annual  precipitation  This isolate was used in the nursery trial.  collected  by  indicated.  152  the author,  unless otherwise  APPENDIX II DESCRIPTIONS OF MYCORRHIZAE  Type 1 on lodgepole pine, Type 4 on Engelmann spruce Fungus: Myceliurn radicis atrovirens Melin -like mycorrhizae Abundance:  3.7 on field pine,  O.75 on  Distinguishing Characteristics: basis  a  of  black,  This group was separated on the  sparse  often  field spruce  and  mantle  narrow,  finely  ornamented hyphae.. Macroscopic  Characteristics:  mycorrhizae,  often  secondary  roots.  with  the  without  to  extending up the  were 2.Oum to 2.Sum wide,  Hyphae  clamps and usually with a fine, grainy appearance.  Textura  individual very  simple  No strands were seen.  Characteristics:  varied  mantle  mantle  sparse  were  were usually many, very dark brown to  There  black extramatrical hyphae. Microscopic  these  Usually  in appearance from very loose, intricata.  mantle  up to 8um thick.  hyphae  poorly  The  developed  and  The  individual hyphae  thickness  varied  from  The Hartig net was usually  varied from non-existent to a weak  development around the outer cortical cells. Type 2 on lodgepole pine, Type 1 on Engelmann spruce Fungus:  Thelephora terrestris group  Abundance:  42.2 on field pine,  23.4% on field spruce  Distinguishing Characteristics: This was a very plastic group in which  mantle  Macroscopically,  and the  rhizomorph group  was  colour  varied  distinguished  extremely.  by a generally  smooth and shiny mantle appearance and abundant white to brown  153  strands cystidla more  weak  (to were  rhizomorphs).  visible  Occasional velvety clumps of  under low magnification,  but these were  common on Type 11 on pine. Microscopically,  this group was  distinguished  by  very  abundant  few  to  cystidia  very characteristic cystidia that varied from and large distinct keyhole clamps.  The  were about 2um wide by lOOum to 200um long and clamped  at the base. Macroscopic generally  Characteristics:  with  patterns  on  complete strands white strand thick  dichotomous pine.  white  occasional  The  patches  coverings ranged  highly  mycorrhizae,  simple  sometimes  A  of  from  was  with  spruce,  but  shiny,  typically  with  cystidia, sometimes extending to  of  hyaline,  velvety  cystidia.  Mycelial  Colour varied from  often on the same root in close proximity;  ranged  rhizomorphs.  mantle  on  group  simple pinnate branching  few to very abundant.  to dark brown, thickness  especially very  or  variable  from the thinnest of strands to quite  Extramatrical hyphae uncommonly formed very  dense mats approaching those of Arnphinema byssoic3es. Microscopic  Characteristics:  characteristic  mantle  appearance  This with  type very  had typical  epidermoidia and typical thickness of lOum to l2um. 4um to 5um thick,  a  very Textura  Hyphae were  with abundant keyhole clamps at most septa.  TYPE 3 on lodgepole pine Fungus: Unknown Basidiomycete Abundance:  3.06% on outplanted pine  Distinguishing Characteristic: A smooth, whitish mycorrhiza with a  characteristic plump appearance that could easily be mistaken  154  for  Thelephora mycorrhizae at low magnification.  microscopically  because  of  a  thick  mantle  Very distinct  (7Ourn or greater)  13) with large oil droplets in virtually every cell  (Fig.  (Fig.  12). Macroscopic  Characteristics:  Very  “squiggly’ emanating hyphae  with kinks in them, which at times, gave the mycorrhiza a woolly appearance.  No rhizomorphs present, the mantle was whitish and  the appearance  was  plump, but unlike that of a Suilloid type.  Branching was normally dichotomous and when this type was found, it was usually present in quite large numbers. Microscopic  Characteristics:  The  hyphae  were 2um to 3um wide  with abundant clamps and had a noticeably “squiggly” appearance. The  mantle stained very darkly with FDA Blue No.  was  a  Textura globulosa to T.  1.  The mantle  intricata, with distinction made  difficult by the abundant oil drops in the mantle.  Type 3 on Engelmann spruce Fungus: Araphinerna by.ssoides, the same fungus as isolate A29 Abundance:  2l  of the egressed roots in the outplanted control  and fungus growing medium treatments Distinguishing  Characteristics: This mycorrhizae formed a dense  weft  yellow  of  (Figs.  10  sparser The  pale  began.  11)  cream—coloured  extramatrical hyphae  that was unmistakable.  It also produced a  mass of hyphae, possibly at an earlier stage of growth.  hyphae  made  and  or  also  grew around other types of mycorrhizae, which  it difficult to determine where one type ended and another The hyphae turned dark yellow in KOH.  Macroscopic Characteristics: the mycorrhizae were usually hidden  155  in  mass of hyphae, but when the hyphae were pulled away, the  a  mycorrhizae  were seldom branched, and thin and fragile—looking.  The extramatrical hyphae often coalesced into loose strands. Type 4 on lodgepole pine Fungus: Suilloid type Abundance:  4.69 on the egressed shortroots of lodgepole pine  Distinguishing  Characteristics:  This  Suilloid type had a very  dark brown mantle with whitish blotches where the outer rind was and  missing  the  hyphae  were  covered  with brownish resinous  exudates. Macroscopic  These  tuberculate  rnycorrhizae  of bunches of dichotomously branched mycorrhizae with  consisted very  Characteristics:  short  branches  The root was covered by a white  (<1mm).  inner  layer and a nearly black outer rind of appressed hyphae.  There  were  abundant brown wire—like rhizomorphs with diameters  of 5Oum and greater. Microscopic  Characteristics:  lacked  clamps.  curved,  with than  diameter  There the that  turn violet in KOH.  hypha  Hyphae  were  were  3um to 4um wide and  sometimes unusual septa that were  on  the  convex side being smaller in  on the concave side.  The exudates did not  The mantle was thick,  usually at least 3Oum  and was Textura intricata to T.  epidermoidea at deeper levels.  Type 5 on lodgepole pine Fungus:  unknown Basidiomycete  Abundance:  0.3% of the egressed shortroots of lodgepole pine  Distinguishing  Characteristics:  A  much  branched  type  with  pinnate initial branching and dichotomous sub-branches. This was  156  a  light  were  brown the  not  myeorrhiza  with occasional white blotches that  same as blotches on Suilloid types.  The overall  appearance was tomentose, often with adhering detritus. Macroscopic  Characteristics:  Abundant  rhizomorphs  lacking.  complex  The  extramatrical branching  hyphae;  pattern  was  distinct. Microscopic Characteristics: which  extended  epidermoidia. unpigmented  from The  and  a  The mantle had a fuzzy outer layer more  hyphae  clamps  compact were  mantle  about  with  2um  in  were common and large.  Textura diameter,  Hyphae did not  have exudates or other surface features. Type 6 on lodgepole pine and Type 2 on Engelmann spruce Fungus: E—strain group Abundance:  3.83% on non-E--strain inoculated field pine and 14.5%  on non—E--strain inoculated field spruce. Distinguishing  Characteristics:  macroscopically  by  reddish  large—diameter  brown,  the  This  presence  of  hyphae  group very  was distinguished distinctive, wiry,  (Figs.  hyphae were not overly abundant but were obvious not  washed.  At  characteristic  higher  Textura  magnification,  intricata  composed  the  5  and 6).  The  if the root was mantle  was  a  of large diameter,  irregularly-shaped hyphae. Macroscopic  Characteristics: The colour ranged from pale brown,  that  easily  could  brown, reddish  almost brown.  be  black. The  confused  with Thelephora,  to very dark  The typical colour was a quite distinct extramatrical  hyphae  were  moderately abundant and had a stiff, wiry appearance.  157  typically The  mycorrhizae  often  had an irregular angular appearance but were  otherwise generally simple in form. Microscopic Characteristics: The hyphae were typically wide, to  7um and usually highly ornamented.  ectendo net  with  arbusculelike  was often quite coarse  The E—strain on pine was  intracellular hyphae.  (Fig.  5um  The Hartig  7), and could be distinguished  from the finer Hartig nets of other fungi. Type 7 on lodgepole pine Fungus: unknown Basidiomycete Abundance:  Found  Found on  of the short roots.  only  in  the control treatment of one block.  Distinguishing Characteristics:  A distinctly yellow mycorrhizae  with a very fuzzy appearance. Macroscopic was  Characteristics:  surrounded  Underneath  the  by  This  yellow mycorrhiza typically  a mass of hyphae,  fuzzy  outer  layer  like a loose cotton ball. was  a smooth mantle.  The  branching was simple dichotomous. Microscopic diameter  Characteristics:  with  large  The  clamps.  hyphae were around 2.5urn in  The compact mantle was a Textura  epidermoidia composed of hyaline hyphal elements. Type 8 on pine and Type 6 on spruce Fungus:  Tonente1la-like  Abundance:  2.O6 average on field pine and O.56 on field spruce.  Distinguishing on  Characteristics:  This group was separated based  the dark brown to blackish mantle that looked very much like  Cenococcurn  hyphae  geophiluin,  often  with  abundant straight emanating  (cystidia?), except that it was a basidiornycete.  158  The  extramatrical  were  Characteristics:  Macroscopic  slightly  present,  roughened.  abundant.  were  usually  The mantle  Often, when this mycorrhiza was  it was present in abundance.  Microscopic Characteristics:  Rhizomorphs were lacking.  The mantle was compact, about lOum  typically a Textura epidermoidia, but on spruce, Textura  angularis in  mycorrhizae  These  but could have one or two dichotomous forks.  appeared  thick,  moderately  normally  there was a brownish—purple cast to the colour.  Frequently,  simple,  hyphae  arrangement  diameter,  with  clamps  and/or  hyphae,  when  was noted.  The hyphae were 2.5um to 4um  a brownish-purple tinge, and sometimes with  fine  The  ornamentation.  present,  straight  emanating  might ha been cystidia, but they were  too long to tell if their length was terminate. Type 8 on spruce Fungus: Lactarius sp., Abundance:  rhizomorphic  (Fig.  deliciosus  two localized infections on outplanted spruce  Distinguishing  Characteristics: mycorrhiza  lactifers  yellow  close to L.  were  This  was  a  pale  with a slight yellowish tinge. present  in  white Golden  the mantle and rhizomorphs  14).  Macroscopic slightly  Characteristics: This mycorrhiza was plain white or  yellow  at  low  magnification  with few extrarnatrical  hyphae and rare rhizomorphs. Microscopic parallel  Characteristics:  hyphae,  golden-yellow  The  mantle  was  made up of neat  Textura epidermoidia and there were beautiful  lactifers  in  the  exuded golden droplets when cut.  159  mantle  and  rhizomorphs that  Type 9 on pine and Type 7 on spruce Fungus: Endogone—like Abundance:  0.19% on egressed roots of outplanted pine and 0.50%  on outplanted spruce Distinguishing distinguish  Characteristics: macroscopically.  microscopically but  did  not  examining  It  the  was  usually  found  by  a root that may have been Thelephora  look quite right;  Microscopically,  This was a very hard fungus to  hyphae  for example,  were  it looked too fat.  very irregularly shaped and  stained very dark blue with FDA Blue No. 1. Macroscopic Characteristics: No distinctive characteristics. Microscopic through  Characteristics:  pre—existing  This  mantles.  was  fungus  found  growing  Typically, a Thelephora mantle  would be covered by a loose net of variable width hyphae (2um to Sections  7um).  penetrating but seen  it  that  the  Endogone—like  fungus  was  the existing mantle with haustoria-like structures,  was  to  showed  be  definitely  not a pathogen, as the hyphae could be  forming a Hartig net that was distinctly different  from the pre—existing Hartig net. Type 10 on pine Fungus:  Suilloid  Abundance:  7.44% on outplanted pine  Distinguishing  Characteristics:  Suilloid morphotype. uniform  white  That is,  colour.  This  type  was  a  tomentose  it did not form a rind, and had a  Microscopically,  it produced abundant  exudates (Fig. 9) that did not stain in KOH. Macroscopic Characteristics: Typically, these mycorrhizae were  160  simple,  or with a few irregular branches, and were quite white,  with  woolly  a  appearance.  (strands?)  rhizornorphs  There  and  were  abundant  abundant  fairly  fine white  extramatrical  hyphae. Microscopic Characteristics: Emanating hyphae were 2um to 3um in simple  diameter,  septate  covered with abundant exudates,  and  that did not change colour in KOH.  The mantle about 5Oum thick.  Type 11 on pine Fungus:  Thelephora group  Abundance:  16.26 on outplanted pine  Distinguishing  Characteristics: This Thelephora  type tended to  appear on new roots, sometimes separated from the main plug by a non—mycorrhizal because  it  zone.  formed  It long  was  described to  (3mm  as a different type mycorrhizae  5mm)  and  it  frequently  was covered with, or had prominent blotches of white  cystidia.  The  typically  other  had  cystidia.  only  This  Thelephora a  (Type  few cystidia,  appeared  to  be  2  on lodgepole pine)  or occasional blotches of  different,  possibly a field  strain. Type 12 on pine Fungus:  This  was  a  Suilloid  type that was indistinguishable  from Suillus tornentosus (A51). Abundance: The mean level of this fungus on treatments where A51 was not inoculated was 1.256. Distinguishing but  formed  a  Characteristics:  This type did not form a rind,  woolly  brownish—coloured hyphae.  mass  of  The  hyphae were covered with abundant exudates that turned violet in  161  KOH  (Fig.  8).  Macroscopic clumps  of  tomentose were  many  Characteristics: many  small  mantle.  Mycorrhizae  branches,  The  inner  yellowish-brown  consisted  of  covered with a thick  dense (5Oum),  mantle was more compact.  rhizomorphs  and  fairly  There  abundant  emanating hyphae. Microscopic Characteristics: Emanating hyphae were 4um to 5um in diameter with clamped septa.  The exudates turned violet in KOH.  Mantle type was Textura intricata. Type 13 on lodgepole pine and Type 5 on Engelmann spruce Fungus: Unknown Abundance:  found on one pine seedling and O.38 of spruce short  roots Distinguishing layer it  and black underneath.  looked  except  Characteristics:  for  very the  similar dark  A  translucent  looking  outer  This was an unusual fungus in that  to Amphinerna or sometimes Thelephora,  layer underneath.  The mantle also had a  unique appearance, but it was difficult to determine if this was just because of the darker layer below. Microscopic  Characteristics:  underneath  was  epidermis.  caused  by  It  appeared that the dark layer  heavily  suberized  The Hartig net was well developed.  162  cells  in  the  APPENDIX III NURSERY FERTILIZER REGIME 1989  Engelmann spruce-  sown March 23  Plant Prod Starter 11-41—8 Peter’s Grower 20—7-19 and Plant Prod Grower Plant Prod Finisher 8—20—30  @6weeks after sow @10 weeks after sow @19 weeks after sow  Applications throughout season: 2 applications Plant Prod Starter 11—48—8@600g/1000L 10 applications Peter’s Grower 20—7-19@400g/1000L 1 application Plant Prod Grower 20-8-20@500g/I000L 6 applications Plant Prod Grower 20—8—20@400g/1000L. 12 applications Plant Prod Finisher 8—20-30@400g/I000L Lodgepole pine  sown May 4  Plant Prod Starter 11—41—8 Peter’s Grower 20—7-19 and Plant Prod Grower 20—8—20 Plant Prod Finisher 8—20—30  @4 weeks @7 weeks @ l3weeks  Applications throughout season: 4 applications Plant Prod Starter 11—41—8@600g/1000L 6 applications Peter’s Grower 20—7-19@400g/1000L 1 application Plant Prod Grower 20—8—20@500g/1000L 6 applications Plant Prod Grower 20—8—20@400g/1000L 12 applications Plant Prod Finisher 8-20-30@400g/1000L  Peter’s products are manufactured by W. R. Whittemore Ave.,  Cambridge MA.  62  Plant Prod Products are  manufactured by Plant Products Co. Ontario.  Grace Co.,  Ltd.,  314 Orenda Rd.,  All fertilizers were applied in solution.  163  Brampton,  APPENDIX IV SUMMARY OF STATISTICS Experiment One The different types of inoculum are referred to as treatments, the method of inoculation is referred to as application and the individual styroblocks are blocks. FGM refers to the fungus growing medium treatment. NURSERY PINE ANOVA Root Collar Diameter Sum of d Mean Square f Source Square Treatment Injected Treat*Inj Block Residual Total  1.1926 4.0355 0.4832 32.202 109.08 147.11  3 1 3 53 541 601  0.3975 4.0355 0.1610 0.6076 0.2016  F 0.6543 6.6420 0.2651 3.0134  Means 3.0455 3.0241 2.9875 3.0627 3.1121 2.9575 3.1239  overall A51 ( 1) Dan. E-strain(2) control(3) FGM(4) injected (a) top-applied  P 0.5838 0.0127 0.8502 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 0.4975 0.5069 0.4744 0.5204 0.4738 0. 4655 0.5074  Multiple Comparisons The only significant difference was between application methods. Shoot Length Sum of d Source Square f Treatment Injected Treat*Inj Block Residual Total  103.36 151.86 92.403 1468.5 4498.4 6316.9  3 1 3 53 541 601  Mean Square 34.545 151.86 30.801 27.708 8.3150  F 1.2435 5.4805 1.1116 3.3323  164  P 0.3032 0.0230 0.3526 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  overall A51 ( 1) Dan. E—strain(2) control (3) FGM(4) injected(a) top-applied  Standard Deviations 3.2420 2.9612 3.2289 3.0832 3.5789 3.2110 3.1994  18. 011 18.665 18.091 17.598 17.677 18.546 17.532  Multiple Comparisons The only significant difference was between application methods. ****************************************************************  Root Mass Source  Sum of d Square f  Mean Square  Treatment Applic. Treat*Inj Block Residual Total  0.1476 0.3514 0.1230 3.6719 14.048 18.358  0.0492 0.3514 0.0410 0.0692 0.0261  3 1 3 53 539 599  F  0.7094 5.0723 0.5920 2.6582  0.5506 0.0284 0.6230 0.0000  Test Term Block(treat*inj) Block(treat*ini) Block(treat*inj) Residual  Standard Deviations 0.1751 0.1706 0.1600 0.1817 0.1865 0.1664 0.1794  Means 0.5232 0.5215 0.4983 0.5412 0.5340 0.4969 0.5465  overall A51 (1) Dan. E-strain(2) control (3) FGM( 4) injected(a) top-applied(b>  p  Multiple Comparisons The only significant difference was between application methods. ** * ***** * ** * ** *** * * ******* *** *** * *** * ** * * * * * **** * *** ***** *******  Shoot Mass Source  Sum of d Square f  Mean Square  Treatment Applic. Treat*Inj Block Residual Total  1.4936 0.0400 0.5095 8.1858 40.412 50.635  0.4979 0.0400 0.1698 0.1544 0.0751  3 1 3 53 538 598  F 3.2235 0.2570 1.0996 2.0562  165  P 0.0300 0.6143 0.3575 0.0000  Test Term Block(treat*inj) B1ock(treat*in) Block(treat*inj) Residual  Mn  overall A51(1) Dan. E-strain(2) control (3) FGM(4) injected(a) top—applied  StandarL Deviations 0.2910 0 2856 0.2677 0.2981 0.2988 0.2931 0.2894  0.9026 0.9898 0.8712 0.8681 0.8843 0.9106 0.8955  .  Multiple Comparisons There were significant differences between treatments. Neither the Bonferroni nor Tukey tests showed a significant difference at p=O.O5 but the A51 treatment is obviously out of line with the others.  Number of Short Roots Sum of d Mean Source Square f Square Treatment Applic. Treat*Inj Block Residual Total  0.7083 0.8771 0.5140 64.225 213.13 279.51  3 1 3 53 541 601  0.2361 0.8771 0.1713 1.2118 0.3940  F 0.1948 0.7238 0.1414 3 0759 .  0.8995 0.3987 0.9347 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 0.6820 0.7272 0. 6050 0.7103 0.6886 0.6650 0.6957  Means 2.3422 2.3513 2.3937 2.3034 2.3154 2.3837 2.3050  overall A51 (1) Dan. E-strain(2) control (3) FGM(4) injected (a) top-applied(b)  P  Multiple Comparisons There were no significant differences. Root to Shoot Ratio Sum of d Source Square f Treatment Applic. Treat*Inj Block Residual Total  0.8704 0.1303 1.0603 12.729 92.625 107.43  3 1 3 53 538 598  Mean Square  0.2901 0.1303 0.3534 0.2402 0.1722  F  1.2081 0.5426 1.4716 1.3950  166  P  0.3158 0.4646 0.2328 0.0388  Test Term Bloek(treat*inj) Block(treat*inj) Block(treat*inj) Residual  overall A51 (1) Dan. E—strain(2) control (3) FGM(4) injected(a) top-applied (b)  Standard Deviations 0.4238 0.6283 0.1799 0.4888 0.2598 0.5701 0.2266  0.6270 0.5892 0.5936 0.2826 0.6463 0.6104 0.6417  Multiple Comparisons There were no significant differences. Dickson Quality Index Sum of d Mean Source Square f Square  Treatment Applic. Treat*Inj Block Residual Total  0.0243 0.0975 0.0344 0.6516 1.9286 2.7402  3 1 3 53 538 598  0.0081 0.0975 0.0115 0.0123 0.0036  F  0. 6599 7.9320 0.9324 3.4297  Means 0.1884 0.1906 0.1772 0.1930 0.1938 0.1746 0.2006  overall A51 (1) Dan. E-strain(2) control (3) FGM(4 injected(a) top-applied(b)  Test Term  P  0.5804 0.0068 0.4316 0.0000  Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 0.0677 0 0669 0.0604 0.0766 0 0659 0.0643 0.0684 .  .  Multiple Comparisons The injected and top-applied treatments were different. *********** *** **************************************************  Thelepho.ra terrestris Levels Sum of d Mean Source Square f Square Treatment Applic. Treat*Inj Block Residual Total  428E+3 2712.0 2453.7 43005 119E+3 594E+3  3 1 3 53 541 601  0.143E+6 2.712E+3 81.79E+1 81.14E+1 22. 04E+1  F 175.98 3.3423 1.0080 3.6810  167  P 0.0000 0.0732 0.3966 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  overall A51(1) Dan. E-strain(2) control (3) FGM (4) injected(a) top—applied(b)  Standard Deviations 31. 439 13 010 24.391 14.782 10.555 29.575 33.017  76.355 92.824 32.249 89.041 95.013 77.657 75.191  .  Multiple ComDarisons Bonferroni and Tukey homogenous subsets (2..) (3.., 1.., 4..) E-strain Levels Sum of d Square f Source Treatment Applic. Treat*Inj Block Residual Total  523E+3 1559.2 2893.3 31579 62701 620780  3 1 3 53 541 601  Mean Square 174E+3 1559.2 964.45 595.83 115.90  292.87 2.6168 1.6187 5.1410  Means 18.090 0.9459 67.031 0.0000 0.1678 17.450 18.659  overa 11 A51(1) Dan. E—strain(2) control (3) FGM(4) injected(a) top-applied(b)  P  F  0.0000 0.1117 0.1960 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 32.139 5.9860 24.216 0.0000 1.2214 30.048 33.934  Multiple Comparisons Bonferroni and Tukey homogeneous subsets (3.., 4.., 1..,) (2..) (Suillus tomentosus) Levels Sum of d Mean Source Square f Square  A51  Treatment Applic. Treat*Inj Block Residual Total  452.67 68.547 220.48 1456.6 9723.6 11919  3 1 3 53 541 601  150.89 68.547 73.492 27.483 17.973  F  5.4903 2.4942 2.6741 1.5291  168  P 0.0023 0.1202 0.0566 0.0118  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Mn  overall A51 ( 1) Dan. E-strain(2) control (3) FGM(4) injected(a) top-applied(b) al bi a2 b2 a3 h3 a4 b4  nir1  0.5482 2.0608 0.0000 0.1034 0.0671 0.8978 0.2358 3.5507 0.7595 0.0000 0.0000 0.0000 0.1899 0.1449 0.0000  rvitinnq  4.4533 8.7467 0.0000 0.9258 0.8192 6.2333 1.6445 12. 280 3.0052 0.0000 0.0000 0.0000 1 2514 1.2039 0.0000 .  Multiple Comparisons Bonferroni and Tukey homogeneous subsets (2.., 4.., 3..) (1..) Treatment*application homogeneous subsets Tukey (a3., a2., b4., b2., a4., b3., bi.) (al. Bonferroni (a3., a2., b4., b2., a4., b3., bi.) (bi., al.) Level of Total Infection by Mycorrhizae Sum of Mean Sum of d Mean Source Square f Square F Treatment Applic. Treat*In Block Residual Total  3169.0 560.18 365.24 8626.9 43369 56140  3 1 3 53 549 601  1056.3 560.18 121.75 162.77 78.996  6.4897 3.4415 0.7480 2.0605  169  P  0.0008 0.0691 0.5284 0.0000  Test Term Block(treat*inj) Block(treat*in) Block(treat*inj) Residual  overall A5l(1) Dan. E—strain(2) control(3) FGM(4) injected(a) top—applied(b) al bi a2 b2 a3 b3 a4 b4  Stanard Deviations 9.6013 8.1708 3.9529 13.438 9.5472 7.7445 10.942 0.000 10.992 5.5902 0.000 12.316 14.301 6.9267 11.278  97.277 98.246 99.687 93.499 97.513 98.327 96.324 100.00 96.712 99.375 100.00 94.971 92.212 98.814 96.375  Multiple Comparisons Homogeneous subsets Tukeys (3..) (4.., 1.., 2..) Treatment* inject ion Tukey (b3., a3., b4., bi.) (a3., b4., bi., a4., Bonferroni (b3., a3., b4., bi., (a3., b4., bi., a4.,  Bonferroni (3.., 4..) (4.., 1.., 2..) homogeneous subsets a2., al.,  b2.)  a4.,) a2., al.,  b2.)  170  NURSERY SPRUCE ANOVA  Root Collar Diameter Sum of d Mean Square f Square Source Treatment Applic. Treat*Inj Block Residual Total  1.1214 0.0109 2.0597 25.490 99.428 128.11  3 1 3 71 711 789  0.3738 0.0109 0.6866 0.3590 0.1398  F 1.0412 0.0304 1.9124 2.5673  Means 3.3266 3.3587 3.2705 3.3142 3.3623 3.3301 0.4097  overall A29(1) Dan. E—strain(2) control(3) FGM(4) in:iected(a) top—applied(b)  P  Test Term  0.3798 0.8620 0.1353 0.0000  Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 0.4030 0.3957 0.4326 0.3869 0.3901 0.4097 0.3964  Multiple Comparisons There were no significant differences. Shoot Length Sum of d Square f Source Treatment Applic. Treat*Inj Block Residual Total  209.70 2.9801 5.3978 1451.9 8770.4 10440  3 1 3 71 710 788  overall A29(1) Dan. E—strain(2) control(3) FGM(4) injected(a) top—applied(b)  Mean Square 69.901 2.9801 1. 7993 20.449 12 . 353  P  F 3.4183 0.1458 0.0880 1.6555  Means 15.531 16.348 14.963 15.521 15.288 15.470 15.594  0.0218 0.7038 0.9664 0.0009  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 3.6400 3.8470 3.5392 3.3204 3.6982 3.7651 3.5101  Multiple Comparisons Tukey and Bonferroni homogeneous subsets (.2., .4., .3.) (.4., .3., .1.)  171  Root Mass Source  Sum of d Square f  Mean Square  Treatment Applic. Treat*Inj Block Residual Total  1.0472 0.3630 0.9933 12.262 45.090 59.767  0.3491 0.3630 0.3311 0.1717 0.0636  3 1 3 71 709 787  overall A29 (1> Dan. E-strain(2) control (3) FGM(4) injected(a) top-applied(b)  F 2.0211 2.1018 1.9172 2.7157  p  Test Terra  0.1187 0.1515 0.1345 0.0000  Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 0.2756 0 2856 0.2676 0.2718 0.2691 0.2698 0.2800  0.9243 0.9343 0.8848 0.8975 0.9788 0.9028 0.9464  .  Multiple Comparisons There were no significant differences. ************** **************************************************  Shoot Mass Source  Sum of d Square f  Mean Square  Treatment Applic. Treat*Inj Block Residual Total  4.7914 0.8697 .51413 16.391 91.473 114.02  1.5971 0.8697 0.1714 0.2308 0.1292  overall A29(1) Dan. E-strain(2) control (3) FGM(4) injected(a) top-applied(b) al a2 a3 a4 bi b2 b3 b4  3 1 3 71 708 786  F 6.9183 3.7673 0.7424 1.7868  Means 1.5916 1.6828 1.4688 1. 6166 1.5978 1.5589 1.6248 1.6204 1. 4538 1.6160 1.5446 1.7453 1. 4839 1.6174 1.6505  p 0.0004 0.0562 0.5303 0.0002  Test Term Block(treat*inj) Block(treat*ini) Block(treat*inj) Residual  Standard Deviations 0. 3809 0.3794 0.3664 0 3612 0.3859 0.3890 0.3700 0.3855 0. 3812 0.3666 0.4037 0.3645 0.3423 0.3571 0.3618 .  172  Multiole Comoarisons Homogeneous subsets Tukey Bonferroni (.2., .4.) (.2.) .4., .3., .1.,) (.4., .3., .1.) Treatment*Application homogeneous subsets Tukey and Bonferroni (a2., b2., a4., a3., b3., al., b4.) (a4., a3., b3., al., b4., bi.) ******************************************************** ********  Number of Short Roots Sum of d Mean Source Square f Square  Treatment Applic. Treat*Inj Block Residual Total  4.4771 0.0514 2.6709 50.146 225.22 282.59  3 1 3 71 710 788  1.4924 0.0514 0.8903 0.7063 0 .3172  F  2.1130 0.0727 1.2605 2.2265  Means 2.5919 2.6800 2.6281 2.4736 2.5800 2.5824 2.6015  overall A29(1) Dan. Estrain(2) control(3) FGM(4) injected(a) top—applied(b)  P  0.1062 0.7882 0.1945 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*in) Residual  Standard Deviations 0.5988 0.5376 0.5707 0.6561 0.6128 0.6034 0.5947  Multiple Comparisons The were no significant differences. Root to Shoot Ratio Sum of d Source Square f Treatment Applic. Treat*Inj Block Residual Total  0.0003 0.5987 0.5827 6.6985 20.588 28.468  3 1 3 71 708 786  Mean Square  0.0003 0.1996 0.1942 0.0943 0.0291  F 0.0026 2.1153 2.0587 3.2444  173  P 0.9580 0.1059 0.1134 0.0000  Test Term Block(treat*inj) Block(treat*ini) Block(treat*inj) Residual  Means 0.5980 0.5752 0.6149 0.5670 0.6338 0.5981 0.5979  overall A29(1) Dan. E—strain(2) control (3) FGM(4) injected(a) top-applied (b)  Standard Deviations o .1903 0.2218 0.1666 0.1664 0.1931 0.1883 0.1926  Multiple Comparisons There were no significant differences. ************* ***************************************************  Dickson Quality Index Sum of d Mean Source Square f Square Treatment Applic. Treat*Inj Block Residual Total  0.0471 0.1794 0.1763 2.3763 8.3704 11.151  3 1 3 71 708 786  0.0471 0.0598 0.0588 0.0335 0.0118  F 1.4068 1.7869 1.7563 2.8309  0.2395 0.1574 0.1634 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 0 .1191 0.1170 0.1159 0.1194 0.1212 0.1168 0.1210  Means 0.4020 0.3996 0.3852 0.3964 0.4263 0.3942 0.4099  overall A29 (1) Dan. E-strain(2) control (3) FGM(4) injected (a) top—applied(b)  p  Multiple Comparisons There were no significant differences. ****************************************************************  Thelephora terrestris Levels Sum of d Mean Source Square f Square  Treatment Applic. Treat*1n5 Block Residual Total  210E+3 644.29 20267 1225+3 3485+3 7015+3  3 1 3 71 710 788  70019 644.2 6755 1718 489.6  F 40.744 0.3749 3.9311 3.5096  174  P 0.0000 0.5423 0.0118 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  ntiri1  overall A29 (1) Dan. E-strain(2) control (3) FGM(4) inected(a) top—applied(b) al a2  31.518 10.775 21.728 52.352 42.209 32.656 30.346 13.650 14.800 58.750 43.630 7.900 28.929 45.244 40.790  a4 bi b2 b3 b4  flriiM-innc  29 827 18.601 22.459 28.369 29 .205 30.255 29.373 20.546 19 .716 24.788 27.573 16.020 22.851 30.472 30.825 .  Multiple Comparisons Homogeneous subsets Tukey Bonferroni (.1.) (.1., .2.) (.2.) (.4., .3.) (.4., .3.) Treatment*Application homogeneous subsets Tukey and Bonferroni (bl., al., a2.) (al., a2., b2.) (b2., b4., a4., b3.) (b4., a4., b3., 13.) ****************************************************************  B—strain Levels Sum of d Source Square f Treatment Applic. Treat*Inj Block Residual Total  606E+3 1780.4 6406.9 38188 145E+3 798E+3  3 1 3 71 710 788  Mean Square 202E+3 1780.4 2135.6 537.86 205.52  F 375.58 3.3100 3.9706 2.6171  175  P 0.0000 0.0731 0.0112 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  ncrt1  Mn  overall A29(1) Dan. E—strain(2) eontrol(3) FGM(4) in5ected(a) top—applied(b) al a2 a3 a4 bi b2 b3 b4  16.967 0.7500 64.673 1.3526 0.5500 18.312 15.582 0.6000 71.050 1.2000 0.4000 0.9000 58.232 1.5222 0.7000  flpiitirnc  31.823 4.4695 29.586 7.6121 4.2765 34.365 28.956 3.1205 30.275 8.9081 2.8141 5.8767 27.552 5.8927 5.3664  Multiple ComDarisons Tukey and Bonferroni homogeneous subsets (.4., .1., .3.) .2.) Treatment*Application homegeneous subsets Tukey and Bonferroni (a4., al., b4., bi., a3., b3.) (b2. (a2.  Amphinema byssoides Levels Sum of d Mean Source Square f Square Treatment Applic. Treat*Inj Block Residual Total  378E+3 5038.0 8560.1 95106 373E+3 860E+3  overall A29(1) Dan. E-strain(2) coritrol(3) FGM(4) injected(a) top-applied(b) al a2 a3 a4 bi b2 b3 b4  3 1 3 71 710 788  126E+3 5038.0 2853.4 1339.5 525.26  F 94.132 3.7611 2.1301 2.5502  Means 33.261 68.549 9.1205 26.094 28.799 30.648 35.945 69.250 8.4000 18.450 26.500 67.850 9.8485 34.589 31.100  P 0.0000 0.0564 0.1040 0.0000  Test Term Block(treat*inj) Block(treat*inJ) Block(treat*in) Residual  Standard Deviations 33.044 23.578 19.659 27.667 27.437 32.704 33.217 20.973 19.973 23.897 26.982 26.011 19.410 29.184 27.830 176  Multiple Comparisons Tukey and Bonferroni homogeneous subsets (.2.) (.3., .4.) (.1.) Treatment*Application homogeneous subsets Tukey Bonferroni b2., a3.) (a2., (a2., b2., a3.) (a3., a4., b4., b3.) (b2., a3., a4.) (bi., al.,) a3., a4.., b4., b3.) (bl., al.,) ****************************************************************  Total Level of Infected Roots Sum of d Mean Source Square f Square  Treatment Applic. Treat*Inj Block Residual Total  62035 13.507 3111.1 77819 206E+3 349E+3  3 1 3 71 710 788  overall A29(1) Dan. E-strain(2) control (3) FGM( 4) injected (a) top-applied ( b) al a2 a3 a4 bi b2 b3 b4  20678 13.507 1037.0 1096.0 290.39  F  18.866 0.0123 0.9462 3.7744  Means 82.256 80.149 96.309 80.473 71.999 82.361 82.145 83.650 95.250 79.950 70.600 76.650 97.370 81.055 73.400  p  0.0000 0.9119 0.4231 0.0000  Test Term Block(treat*inj) Block(treat*inj) Block(treat*inj) Residual  Standard Deviations 21.045 19 . 522 10.003 19.273 24 . 704 20. 583 21.534 17.864 12.378 19.790 22.987 20.549 6.7549 18.776 26 . 351  Multiple Comparisons Tukey and Bonferroni homogeneous subsets (.4., .1., .3.) (.2.) Treatment*Application homogeneous subsets Tukey Bonferonni b4., bl., (a4., a3.,b3.,al.) (a4., b4., bi., a3., b3., al.) (b3., al., a2.) (a3., b3., al., a2.) (al., a2., b2.) (b3., al., a2., b2.) ***** ****** * **** ** * * **************************************** ****  177  FIELD PINE ANOVA Root Collar Diameter Sum of d Mean Source Square f Sre 4.4653 46.774 198.74 249.82  3 1.4884 4 11.693 542 0.3667 549  overall A51(l) Dan. E-strain(2) control (3) FGM (4)  Means 4.0444 4.0176 3.9362 4.1771 4.0396  Treatment Block Residual Total  F 4.0592 32.89  P 0.0072 0.0000  Test Terra Residual Residual  Standard Deviations 0.6746 0. 6558 0.6571 0.6651 0.7009  Multiple Comparisons Tukey and Bonferroni homogeneous subsets (2., 1., 4.,) (1., 4., 3.,) ****************************************************** **********  Incremental Height Sum of d Source Square f Treatment Block Residual Total  57. 717 171.93 5682.8 5916.2  Mean Square  3 19.239 4 42.984 540 10.524 547  F 1.8282 4.0844  P 0.1409 0.0029  tndrd  overall A51 ( 1) Dan. E-strain(2) control (3) FGM(4)  10.300 10.841 10.141 9.9139 10.394  Test Terra Residual Residual  flviticns  3. 2887 2.9756 3.1563 3. 5963 3.3122  Multiple Comparisons There were no significant differences. Total Height Sum of d Source Square £ Treatment Block Residual Total  218.06 503 37 13326 14053 .  Mean Square  3 72.687 4 125.84 542 24.586 549  F 2.9564 5.1185  178  P 0.0320 0.0005  Test Term Residual Residual  overall A51(1) Dan. E—strain(2) control(3) FGM(4)  Means 28.368 29.083 28.484 27.359 28.674  Standard Deviations 5.0594 4.9795 5.2561 5.0321 4.8471  Multiple Comparisons Tukey and Bonferroni homogeneous subsets (3., 2., 4.) (2., 4., 1.) ******************************************** ********************  179  Number of Dead Seedlings Sum of d Mean Source Square f Square Treatment Block Residual Total  0.0597 1.5278 22.324 23.909  3 0.0199 4 0. 3819 565 0.0395 572  overall Dan. E—strain(1) A51(2) control(3) FGM(4)  0.9564 0.9600 0.9591 0.9667 0.9404  F  P  0.5035 9.6665  0.6800 0.0000  Test Term Residual Residual  Standard Deviations 0.2044 0.1968 0.1985 0.1801 0.2375  Multiple Comparisons There were no significant differences. ****************************************************************  Shoot Mass Source  Sum of d Square f  Mean Square  Treatment Block Residual Total  3.5366 1.9218 33.078 38.536  1.1789 0.4804 0.4594  3 4 72 79  overall Dan. E—strain(1) A51(2) control(3) FGM(4)  P  F 2.5660 1.0458  Means 2.1192 2.1126 2.3822 1.7977 2.1844  Residual Residual  Standard Deviations 0.6984 0.8383 0.6705 0.5521 0.6205  Multiple Comparisons Homogeneous subsets Tukey (3., 1., 4.) (1., 4., 2.)  0.0612 0.3898  Test Term  Bonferroni (3., 1., 4.,  180  2.)  Root Mass Source  Sum of d Square f  Mean Square  Treatment Block Residual Total  0.2070 0.2730 11.868 12.348  0.0690 0.0682  3 4 72 79  overall Dan. E--strain(1) A51(2) control (3) FGM(4)  F 0.4185 0.4141  P 0.7402 0.7980  Test Term  Residual Residual  Standard Deviations 0.3954 0.4085 0.3256 0.4314 0. 4318  1.1207 1.0339 1.1531 1.1361 1.160  Multiple Comparisons There were no significant differences. ****************************************************************  Root to Shoot Ratio Sum of d Source Square f Treatment Block Residual Total  0.2914 0.1664 3.6456 4.1033  3 4 72 79  overall Dan. E-strain(1) A51 ( 2) control (3) FGM( 4)  Mean Square 0.0971 0.0416 0.0506  F 1.9181 0.8216  p 0.1343 0.5156  Test Term Residual Residual  Standard Deviations 0.2279 0.1943 0.1408 0.2348 0.2965  0.5590 0.5154 0.5023 0 . 6562 0. 5620  Multiple Comparisons There were no significant differences. Number of Types of Mycorrhizae per Root Sum of d Mean Source Square f Square F Treatment Block Residual Total  9.1375 9.6750 74.675 93.487  3 4 72 79  3.0458 2.4187 1.0372  2.9367 2.3321  181  p  0.0390 0.0639  Test Term Residual Residual  Means 2.7375 2.7000 3.0000 2.2000 3.0500  overall Dan. E—strain(1) A51(2) control(3) FGM(4)  Standard Dviatin 1.0878 0.9787 1.1698 1.0563 0.9987  Multiple Comparisons Homogeneous subsets Tukey (3., 1., 2.) (1., 2., 4.)  Bonferronni (3., 1., 2.,  4.)  ****************************************************************  Foliar Nitrogen Sum of d Square f Source Treatment Block Trt*Block Residual Total  0.1578 0.4605 0.1742 0.0104 0.8334  3 4 7 2 16  Mean Square 0.0526 0.1151 0.0249 0.0052  F 10.115 22.139 4.7845  P 0.0914 0.0437 0.1837  Test Term Residual Residual Residual  Multiple Comparisons There were no significant differences. The other nutrient analyses (P, Ca, Mg, K, Cu, Zn, Fe, Mn, B, Al) for field pine were similar and none of them showed any significant differences.  182  FIELD SPRUCE ANOVA Root Collar Diameter Sum of d Mean Source Square I Square Treatment Block Residual Total  6.1647 87.201 239.14 333.71  3 2.0549 4 21.800 481 0.4972 488  overall A51 ( 1) Dan. E-strain(2) control (3) FGM( 4)  Means 4.5042 4.4702 4.3228 4.6063 4.6277  P  F 4.1332 43.849  0.0066 0.0000  Test Term Residual Residual  Standard Deviations 0 .8269 0.8028 0.8169 0.8074 0.8526  Multiple Comparisons Tukey and Bonferroni homogeneous subsets (2., 1.) (1., 3., 4.) Incremental Height Sum of d Square I Source Treatment Block Residual Total  Mean Square  263.91 315.43 4700.8 5293.9  87.969 3 4 78.858 476 9.8757 483  overall A51 ( 1) Dan. E—strain(2) control (3) FGM(4)  Means 11.584 10.992 10.724 12.523 12.160  P  F 8.9077 7.9851  0.0000 0.0000  Test Term Residual Residual  Standard Deviations 3.3107 3.6821 3.3035 3.0040 2.8723  Multiple Comparisons Tukey and Bonferroni homogeneous subsets (2., 1.) (4., 3.) **************************** ************************************  183  Total Height Sum of d Source Square f Treatment Block Residual Total  Mean Square  169.54 295.79 8726.6 9207.9  3 56.514 4 73.948 481 18.143 488  overall A51(1) Dan. E—strain(2) control(3) FGM(4)  25.606 26.205 24.615 25.989 25.652  F  P  3.1150 4.0759  0.0260 0.0029  Means  Test Term Residual Residual  Standard Deviations 4.3438 4.6106 4.4052 3.7469 4.4058  Multiple Comparisons Homogeneous subsets Tukey Bonferroni 4., (2., 3.) (2., 4., 3., (4., 3., 1.)  1.)  Number of Dead Seedlings Sum of d Mean Source Square f Square Treatment Block Residual Total  0.3120 15.944 91.646 107.86  3 0.1040 4 3.9861 593 0.1548 599  overall A51(1) Dan. E—strain(2) control(3) FGM(4)  Means 0.7650 0.7763 0.7667 0.7297 0.7867  F 0.6718 25.748  P 0.5696 0.0000  Test Term Residual Residual  Standard Deviatinnq 0.4244 0.4181 0.4243 0.4456 0.4103  Multiple Comparisons There were no significant differences  184  Shoot Mass Source  Sum of d Square f  Mean Square  Treatment Block Residual Total  6.3473 3.3950 67.154 76.896  2 .1158 0.8487 0.9327  3 4 72 70  F  P  2.2684 0.9100  Means 3.5802 3.1238 3.8463 3.5750 3.7756  overall Dan. E-strain(1) A51(2) control (3) FGM (4)  0.0878 0.4649  Test Term Residual Residual  Standard Deviations 0.9855 0.7035 0.9215 1.1475 1.0258  Multiple Comparisons There were no significant differences. Root Mass Source  Sum of d Square f  Mean Square  Treatment Block Residual Total  3.7181 1.8326 31.258 36.809  1.2394 0.4582 0.4341  3 4 72 79  overall Dan. E-strain(1) A51(2) control (3) FGM(4)  F  P  2.8548 1.0553  Residual Residual  Standard Deviations 0.6826 0.4096 0.6950 0.5326 0.8984  1.7951 1.6822 1.8862 1.5191 2.0929  Multiple Comparisons Homogeneous subsets Tukey (3., 1., 2.) (1., 2., 4.)  0.0431 0.3850  Test Term  Bonferroni (3., 1., 2.,  185  4.)  Root to Shoot Ratio Sum of d Source Square f Treatment Block Residual Total  0.3865 0.3345 3.6236 4.3446  3 4 72 79  Mean Square 0.1288 0.0835 0.0503  F 2.5598 1.6614  Means 0.5291 0.6280 0.4883 0.4443 0.5558  overall Dan. E—strain(1) A51(2) control(3) FGM(4)  P 0.0616 0.1684  Test Term Residual Residual  Standard Deviations 0.2345 0.3536 0.1408 0.1368 0.2115  Multiple Comparisons There were no significant differences. Foliar Calcium Sum of d Source Square f Treatment Block Residual Total  0.0469 0.0237 0.0362 0.1069  3 4 12 19  overall Dan. E-strain(1) A51(2) control(3) FGM(4)  Mean Square  0.0156 0.0059 0.0030  F  5.1805 1.9636  Means 0.3505 0.316 0.412 0.292 0.382  P  0.0159 0.1645  Test Term  Residual Residual  Standard Deviations 0.075 0.039 0.062 0.033 0.092  Multiple Comparisons Homogeneous subsets Tukey Bonferroni (3., 1.., 4.) (3., 1., 4., (1., 4., 2.)  2.)  The other nutrient analyses were N, P, Mg, K, Cu, Zn, Fe, Mn, and Al. No other differences between treatments were significant.  186  B  CONTINGENCY ANALYSIS FOR SEEDLINGS THAT DIED IN THE FIELD SAS was used to do a contingency analysis of how many seedlings had died over the first growing season. There were two independent variables, treatment and block. The analysis was done controlling for block. Summary Statistics For Field Pine, Controlling For Block  Treatment by the Number Dead:  COCHRAN-MANTEL-HAENSZELL STATISTICS SHOWN) STATISTIC ALTERNATIVE HYPOTHESIS 1 2 3  Nonzero Correlation Row Mean Scores Differ General Association  Total Sample Size  =  (BASED ON TABLE SCORES NOT DF  VALUE  1 3 3  0.003 2.025 2.025  PROBABILITY 0.959 0.567 0.567  600  Summary Statistics For Field Spruce, Dead: Controlling For Block  Treatment by the Number  COCHRAN-MANTEL-HAENSZELL STATISTICS SHOWN) STATISTIC ALTERNATIVE HYPOTHESIS Nonzero Correlation 1 2 Row Mean Scores Differ 3 General Association  (BASE ON TABLE SCORES NOT  Frequency Missing  =  19  DF 1 3 3  VALUE 0.523 1.524 1.524  Effective Sample Size  187  PROBABILITY 0.469 0.677 0.677 =  573  


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items