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

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INOCULATION OF ECTOMYCORRHIZAL FUNGIIN THE IDFdk2 BIOGEOCLIMATIC ZONE OF BRITISH COLUMBIA:NEW TECHNIQUES, FUNGI AND OUTPLANTING TRIALSByWILLIAM KENNETH CHAPMANB.Sc. Ag., The University of Alberta, 1978A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF SOIL SCIENCEWe accept this thesis as conformingt rerni41 n-ri1 L-’- d / 1(1 -THE UNIVERSITY OF BRITISH COLUMBIADecember 1991Øwiiiiam Kenneth Chapman, 1991In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Departmentof___________________The University of British ColumbiaVancouver, CanadaDate /?DE-6 (2/88)ABSTRACTEctomycorrhizal fungi were used to alter seedling performancein a normal reforestation situation. This work was conducted ina precisely defined, natural ecological situation, so thatfuture work can be compared to this, and an understanding of thebehaviour of ectomycorrhizal fungi, under the conditions definedin this study, can be developed over time. In addition,specific problems in applied mycorrhizal research, such as theculture of fungi, inoculation in nurseries and examination ofectomycorrhizae were addressed, and new techniques developed.Fungi from a variety of sources were screened, using a newnear-pure culture synthesis apparatus. Certain fungi wereselected and grown to volume, using a new fungus culturetechnique. Seedlings in a commercial nursery were successfullyinoculated, using two procedures. One procedure involved theinjection of mycelial slurry into container plugs and the otherinvolved application of the slurry to the surface of root plugs.The performance of the inoculated seedlings and mycorrhizalfungi in the nursery was evaluated. Shoot growth of theseedlings was increased or decreased, depending on the type offungus. Even very low levels of infection by Suillustomentosus (3.55%) increased the growth of lodgepole pineseedlings. Seedlings of lodgepole pine and Enge].mann sprucethat formed mycorrhizae with E—strain (sensu Mikola), Amphinemabyssoides (Fr.) J. Erikss. or Suillus tomentosus (Kauff.) Sing.,Snell & Dick were transplanted to the field into a “normalreforestation” situation. In general, the differences in growthfrom the nursery, persisted after one field season. The11behaviour of the inoculated mycorrhizal fungi on egressed rootsis described, including the encroachment of wild fungi, thegrowth of the inoculated fungus onto new roots and the behaviorof Thelephora terrestris Ehrhart: Fr. In the field. Alow—toxicity dye (FDA Blue No. 1), not previously used forexamining ectomycorrhizae, was evaluated and a procedure for therelatively fast and detailed description of ectomycorrhizae isoutlined.This work suggests a minimum basic protocol for conductingoutplanting trials to insure that meaningful information on thebehaviour and function of ectomycorrhizae can be collected infuture experiments. The major tenets of the protocol are thatsites should be described using a comprehensive ecologicalclassification system, mycorrhizae on natural seedlings at thestudy site should be described, the mycorrhizae on seedlings atoutplantlng should be described and monitored over the shortterm and long term, a wide variety of fungi should be used inoutplanting trials in normal reforestation situations, culturaland inoculation techniques need to be improved and growth andsurvival need to be monitored over the short and long terms. Ifectomycorrhizae evaluation trials do not supply certain basicinformation, it may be a very long time before it is understoodhow ectomycorrhizal fungi respond to the type of site conditionsfound in normal reforestation situations.iiiTABLE OF CONTENTSAbstract iiTable of Contents ivList of Tables viiList of Figures ixAcknowledgements X1.Introduction 12. Objectives 113. Literature Review3.1 Experiment One 133.2 Experiment Two 153.3 A New Low Toxicity Stain Useful for theExamination of Ectomycorrhizal Fungi 173.3 Experiment Three 203.3.1 344. Experiment One: In Vitro Growth of Ectomycorrhizal FungiOn Dilute Agar 375. Experiment Two: Two Inoculation Techniques forVegetative Mycorrhizal Inoculum In a Commercial Nursery. .415.lMaterialsandMethods 425.2 Results 465.3Discussion 506. A Low Toxicity Stain forExaminingEctomycorrhizae 547. Experiment Three: The Selection and Evaluation ofEctomycorrhizal Fungi To Enhance Performance ofSeedlings Planted tinder Normal Reforestation Conditions. .57iv7.1 Materials and Methods .617.1.1 Site Selection and Description 617.1.2 Collection of Isolates 657.1.3 Isolation Techniques 667.1.4 Prescreening of Cultures 697.1.5 Near Pure Culture Synthesis 707.1.6 Trial Inoculations in the Nursery 747.1.7 Preparation of Inoculum for the Nursery 757.1.8 Inoculation of Seedlings in the Nursery 787.1.9 Evaluation of Nursery Trials 797.1.10 Outplanting Trials 837.1.11 Examination of Seedlings Fromthe Outplanting Trials 847.1.12 Statistical Procedures 857.2 Results 877.2.1 Pure Culture Synthesis 877.2.2 Trial Inoculation in the Nursery 887.2.3 Observation of Nursery Seedlings 887.2.4 Observations of Seedlings After OneSeason in the Field 957.2.4.1 Observations on Pine Seedlings 967.2.4.2 Observations on Spruce Seedlings 1027.3 Discussion 1077.3.1 Pure Culture Synthesis Technique 1077.3.2 Nursery Trials 1107.3.3FieldTrials 1148. Overall Conclusions 123VLiterature Cited 132Appendix I: Fungi Used in Pure Culture Synthesis andNursery Screening Trials 146Appendix II: Descriptions of Mycorrhizae 153Appendix III: Nursery Fertilizer Regime, 1989 163Appendix IV: Summary of Statistics 164viLIST OF TABLESTable I. Mycelial Mass of Ectomycorrhizal Fungus AfterOne Month of Growth in Liquid or 0.3% Agar 40Table II. Comparison of Iniection Into the Plug Versus TopApplication of Ectomycorrhizal Mycelial Slurry toTwo Month Old Engelmann Spruce 47Table III. Comparison of Injection Into the Plug Versus TopApplication of, Ectomycorrhizal Mycelial Slurry toTwoMonthOidLodgepolePine .49Table IV. Detailed Description of Outplanting Site 63Table V. Concentrations of Inoculum Used in the NurseryInoculationTrial 77Table VI. Observations on Lodgepole Pine Seedlings Grown Inthe Heffley Reforestation Centre Nursery for 4Months Following Inoculation With EctomycorrhizalFungalMyceliumat2Months 89Table VII. Observations on Engelmann Spruce Seedlings GrownIn the Heffley Reforestation Centre Nursery for 4Months Following Inoculation With EctomycorrhizalFungal Mycelium at 2 Months 92Table VIII. Observations on Lodgepole Pine SeedlingsInoculated With Ectomycorrhlzae in the Nurseryand Grown for 5 Months in the Field (F-ieldMeasurements) 96Table IX. Observations on Lodgepole Pine SeedlingsInoculated With Ectomycorrhizae in the Nurseryand Grown for 5 Months in the Field (LaboratoryMeasurements ) 9 8viiTable X. Volunteer Fungi on Egressed Roots of LodgepolePine Grown in the Field For Five Months WithDifferent Mycorrhizal Treatments 99Table XI. Foliar Nutrient Analyses For Lodgepole PineGrown in the Field For Five Months With DifferentMycorrhizal Treatments 101Table XII. Observations on Engelmann Spruce SeedlingsInoculated With Ectomycorrhlzae in the Nurseryand Grown For 5 Months in the Field (FieldMeasurements) 103Table XIII. Observations on Engelmann Spruce SeedlingsInoculated With Ectomycorrhizae in the Nurseryand Grown For 5 Months in the Field (LaboratoryMeasurements) 104Table XIV. Volunteer Fungi on Egressed Roots of EngeimannSpruce Grown in the Field For Five Months WithDifferent Myeorrhizal Treatments 105Table XV. Foliar Nutrient Analyses for Engelmann SpruceGrown in the Field For Five Months With DifferentMycorrhizal Treatments 106viiiLIST OF FIGURESFIG. 1. Pure Culture Synthesis Apparatus 139FIG. 2. Inoculation in Pure Culture Synthesis 139FIG. 3. Dilute Agar Culture 140FIG. 4. Dilute Agar Liquid Cultures 140FIG. 5. E—strain Hyphae 141FIG. 6. E—strain growing From a Broken Root 141FIG. 7. E—strain Hartig net 141FIG. 8. Type 12 Suilloid Fungus 142FIG. 9. Type 10 Suilloid Rhizomorph 142FIG. 10. Amphlnema byssoldes extramatrical hyphae 143FIG. 11. Amphinema byssoldes in the field 143FIG. 12. Type 3 mycorrhiza stained with FDA Blue No 1....144FIG. 13. Type 3 mycorrhiza longitudinal section 144FIG. 14. Type 8, Lactarlus showing lactifers 145FIG. 15. Pine roots 145ix VACKNOWLEDGEMENTSTo my three dear children, Laticia, Oleh and Roman, who havehad to live without so many of the amenities while their dadstruggled from fellowship to grant. Thank you to Tim Ballardand Shannon Berch, two of the best teachers I have ever known.Tim Ballard loaned me money from his own pocket when times werebad. I will never forget his kindness and his love of learning.He is an inspiration to all teachers, and I hope he is not lostin the swollen maw of administration. Gary Hunt helped in manyways, from all manner of technical advice to arranging fundingfrom Balco Canf or Reforestation Centre Ltd. (now the HeffleyReforestation Centre Ltd.). Thank you, Gary and HeffleyReforestation Centre Ltd. My friends in the department of SoilScience, especially Sharmin Gamiet and Guoping Xiao, helped tomake the department one of the most lively and interestingplaces I have ever been. The Soil Science coffee room is a truecentre of excellence. A special thanks to Ada and Jim Chapman,my mom and dad, who were also there to pull us through whentimes were so bad financially and emotionally. This thesis isdedicated to my collaborator in the experiment of life, my wife,my love Louisa, who has been raising three beautiful childrenunder the most meagre of conditions. Everything I have done isnothing compared to what she has done.x1. INTRODUCTIONThe existence of ectomycorrhizae has been formally recognizedin our culture since the early 19th century. The function ofectomycorrhizae was proposed by Vittadini in 1842 (Trappe andFogel, 1977). Frank named this association in 1885 whilestudying truffles in the Kingdom of Prussia; at that time, healso suggested it was a mutualistic symbiotic relationship(Mosse, Stribley and LeTacon, 1981). Since then, it has becomeabundantly clear that ectomycorrhizae are essential for thenormal growth of most temperate conifers and many angiosperms aswell (Harley and Smith, 1983). The important role thatmycorrhizae play in the development and survival of youngseedlings has been repeatedly demonstrated in circumstanceswhere the fungal symbiont is missing, such as in theintroduction of exotic species (Mikola, 1970; Mikola, 1973) orthe aftermath of fungicide use (Trappe and Strand, 1969).However, the application of mycorrhizal inoculum to more normalreforestation situations has not been nearly as well studied andresults are contradictory.Castellano (in press) has thoroughly reviewed 103 publishedand 10 unpublished studies on mycorrhizal (ecto and VAM)inoculation trials that involved outplanted seedlings. Althoughresults are mixed, there are some useful observations on somefungal species. For example, among seedlings inoculated withPisolithus tinctorlus (Pers.) Coker & Couch, only 46% of thetrials showed improved performance and 48% showed no1difference from uninoculated seedlings. In 17 trials,Thelephora terrestris improved growth three times, decreasedgrowth three times and had no effect 11 times. Hebeloma andLaccaria spp. either did not affect growth or decreased it.About 72 fungal species were examined (of these, only about 34actually produced mycorrhizae, Danielson, personalcommunication) and what is noticeably absent is a fungus thatconsistently improved growth. It is also apparent that almostevery fungus has the potential to decrease growth under somecircumstances. It should be noted that the results summarizedby Castellano included studies in extreme circumstances, definedhere as situations likely devoid of the correct ectomycorrhizalfungal inoculum. Examples of extreme situations are minespoils, reclamation of borrow pits, afforestation and theintroduction of exotic species. These are the conditions wherewe might expect the most dramatic response to inoculation, yetno fungus increased growth in more than 5O of the cases. Therehave been very few reports of results in normal reforestationsituations where one might expect less response to inoculationthan would be found in extreme circumstances.The work of Marx (see Literature Review for a brief summary)and the sometimes spectacular results achieved in extremeconditions led to the hope that manipulation of the fungalsymbiont would be a direct means by which dramatic improvementsin seedling performance could be achieved in normalreforestation situations. To this date, the early promise hasnot been fulfilled. This has resulted in a backlash, where manynow believe that ectomycorrhizal fungi cannot be used to2manipulate seedling growth, except in extreme circumstances.However, there have been some studies, Involving normalreforestation situations, where inoculation with ectomycorrhizaedid improve performance (see literature review). Thisundeniably establishes that ectomycorrhizal fungi have thepotential to influence seedling growth In some normalreforestation situations. What is not understood is the way inwhich ectomycorrhizal fungi, trees and the environment interactto influence growth and survival. Without this fundamentalknowledge, it is very difficult to predict how any given funguswill affect growth as conditions vary. There is a strongpossibility that the mixed results do not reflect the generalfailure of ectomycorrhizal fungi to improve performance so muchas our lack of understanding of the ectomycorrhizalrelationship.Investigation into the effects of ectomycorrhizae on seedlinggrowth has been going on for at least 60 years and one mightexpect that the relationship should be well understood by now.A primary obstacle to this understanding has been and is thetremendous diversity of conditions, species and within-speciescharacteristics found in many temperate forests. For example,the range in microsite characteristics that can be found invirtually any plantation is usually enough to significantlyalter seedling growth. The microsite variability is compoundedby the diversity resulting from normal genetic variabilitywithin a tree species and by the almost incredible diversity inectomycorrhizal fungi. The variability in the habitatpreferences of higher plants is so well documented that it does3not need further discussion. As Trofymow and van den Driessche(1991) have summarized, different fungal species also growoptimally at different pH, moisture levels, temperatures andnutrient levels. Even the within—species differences ofectomycorrhizal fungi are very large in such characteristics asresponse to temperature and moisture, phosphate solubilizationand uptake, nitrogen utilization, production of enzymes,metabolites and antibiotics and resistance of hyphae todecomposition, as summarized by Trappe and Fogel (1977). Thereare other examples of the diversity of ectomycorrhizal fungi,and this has no doubt contributed to the difficulty in selectinga fungus to achieve a particular result. But the enormity ofthe task becomes more apparent when one considers that anyparticular fungal isolate might be optimally adapted to aparticular provenance of tree and a particular set ofenvironmental circumstances. Even if one selects the bestfungus for a particular provenance of tree at a particular site,there is still considerable room for environmental variationresulting from uncontrollable factors such as weather. Thiscreates a high level of uncertainty in any experiment designedto monitor the effects of mycorrhizal fungi.The effort to develop guidelines describing thecircumstances where a particular fungus might give a predictableresult is confounded by the ecotypic variations in fungi whichmake it possible that the same species may not give the sameresult in similar circumstances. There is even the possibilitythat changes occurring within an isolate during storage (e.g.,sectoring) might result in different responses with the same4isolate. This makes it very difficult to even define what oneis holding In one’s hand, let alone prescribe it for a treespecies or set of environmental conditions that may also beundefineable, except at very crude levels.The diversity in fungal characteristics begins to soundimplausibly complex unless one remembers that trees may persistin one place for centuries and while the genetic complement ofthe tree is essentially fixed, the fungal component can changewith the inevitable climatic, soil and other changes that occurover time. Fungi are profuse spore formers and it is verylikely that every tree would be exposed to a myriad of sporesover the life of a forest. The heterokaryotic nature of manyectomycorrhizal fungi further increases the possibility that thefungal thalli at any site might each have a large and uniquepool of genetic material to draw on to assure optimum adaptationto that site. Given the length of time that fungi have topersist on one site, the opportunity for exposure to a widevariety of genetic material, and the malleability of fungi, itis not unrealistic at all to expect site and tree specificadaptation. The possibility of extreme site specificity byfungi is borne out by the fact that there are many thousands ofspecies of ectomycorrhizal fungi and subgroups of those species.It is hard to imagine why this diversity would exist if therewere not sufficient niches to support it.It is becoming more apparent that the ectomycorrhizalsymbiosis cannot simply be defined as one where the hostsupplies carbohydrates and any ectomycorrhizal fungus providessome minerals and a few incidental benefits. If Pirozynski and5Malloch (1975) are correct in their hypothesis that terrestrialplants are a result of the combination of aquatic plants and afungus, then the mycorrhizal relationship is, as old asterrestrial plant life itself and almost certainly very complex.If one accepts this complexity, then it is easy to understandwhy it is so difficult to manipulate fungi in normalreforestation situations. However, this is not to say thatmanipulation of ectomycorrhizal fungi could be expected to havelittle effect on tree growth. It makes little more sense tosuggest that altering the mycorrhizal fungus would not affectthe growth of seedlings in normal reforestation situations thanit does to suggest that altering the tree species or provenancewould have no effect on performance. Manipulation of the fungusto achieve specific results is much more difficult because it isso hard to precisely specify the characteristics of the fungusone is working with, the fungus may confer benefits only in avery specific set of circumstances, the desired fungalcomplement of a seedling may change, in the nursery or thefield, with exposure to any stray inoculum and the exact natureof the ectomycorrhizal relationship is not well understood.Nevertheless, the fungal contribution to tree nutrition andgrowth cannot be written off as the component of tree growththat will be looked after by nature. Trees and how to improvetheir growth will never be adequately understood until the twomajor components that constitute a tree, the fungal and theplant, are both understood.The complexity of the mycorrhizal relationship does hint atthe improbability of selecting a single fungus that will6consistently improve seedling performance. The questions thatneed to be answered in order to select the best fungus are suchthings as, how important are the fungi on the roots of seedlingsat outplanting, how long does it take for a seedling to developa full complement of fungal genetic material to be properlyadapted to its environment, how many species of fungi arenormally found on a particular tree species at a particularsite, can introduction of exotic fungal genetic material resultin large changes in performance and does a diverse fungalpopulation result in better performance as environmentalconditions vary? One possibility is that there are so manyfungal spores and the inoculum potential of normal reforestationsites may be so high, that the mycorrhizal populations ofseedlings at outplanting are not important. While this ispossible, it may not be probable for several reasons. The fungifound in a mature forest, that was present before logging, maybe able to form mycorrhizae with seedlings and even replace thefungi on the seedlings at outplanting, but there is apossibility that they might not be the most suitable fungi foryoung seedlings. There is almost certainly some lag timebetween the outplanting of seedlings and infection by theoptimum complement of fungi, even though infection by some fungimay occur quickly. There is a possibility that the fungi on theroots of seedlings from the nursery could interfere withinfection by more beneficial fungi. Seedlings with inadequatemycorrhizae may be more susceptible to disease or environmentalstress in the period before roots develop optimal mycorrhizae.Answers to these questions might suggest completely different7means of manipulating mycorrhizal fungi than simply trying toselect the optimum fungus.To this time, only 72 fungal species (Castellano, in press)have been tested in outplanting trials and only about 34 specieshave actually formed mycorrhizae (Danielson, personalcommunication). Only a few of the 72 species tested have beenused in a variety of situations. These include Pisolithustinctorius, mixed (a euphemism for soil inoculum) and Rhizopogonvinicolor. There are approximately 5000 species ofectomycorrhizal fungi on 2000 species of woody plants (Marx andShafer, 1989) and if the within-species variation is considered,there are potentially tens of thousands of fungi that could beevaluated in a variety of circumstances. It seems clear that ifmore information is to be gathered about the biology and ecologyof ectomycorrhizal fungi, then many more trials need to be doneand these should not be confined to extreme circumstances thatwill tell us little about the normal biology and ecology ofectomycorrhizal fungi. Furthermore, since only a very smallnumber of fungi has been investigated, it seems premature to bemaking general conclusions about the application of mycorrhizalinoculation.If one starts from the well-supported assumption that incertain normal reforestation situations, ectomycorrhizal fungihave the ability to greatly influence seedling performance,then the problem becomes how to achieve a positive resultconsistently. This is not a trivial exercise, given thepreviously mentioned diversity in fungi and the conditions inwhich they grow. The previous discussion has suggested several8areas that need exploration to solve this problem. Theobjective of this thesis was to examine previous approaches toselecting fungi for outplanting trials and then improve themwith regard not only to getting growth responses but also tocollecting useful information on the biology and ecology ofectomycorrhlzae. Once the fungi were selected, they were usedin an outplanting trial that was conducted where these fungimight normally grow.The performance of this experiment involved virtually allaspects of applied mycorrhizal research. Roots were examined todetermine mycorrhizal status. Because of the large number ofroots examined, new techniques were needed to make observationsquickly and confidently about ectomycorrhizal types and thelevel of colonization. Fungi were isolated from the field, andisolation techniques were streamlined to facilitate thelarge—scale isolation necessary to get a representative fungalpopulation from a given site. The fungi were screened forability to infect seedlings. Given what we know about theadaptation of fungi to specific growing conditions, it isimportant that growing conditions in a screening process mimicthose at the place where growth is to be altered as closely aspossible. Traditional screening procedures have used testingconditions that may include very wet growing conditions, buildupof gases, unusual substrates, anaerobic conditions or othervariations that may select against fungi adapted to the veryconditions in which they must grow.Inoculation on a scale large enough to do a reliableevaluation with highly variable natural growing conditions9requires that a fairly large amount of inoculum be grown. Manyproblems are involved with the culture of ectomycorrhizal fungi,and a new culturing technique was developed that is particularlyapplicable to the production of moderate amounts of inoculum.Moderate amounts of inoculum are defined here as sufficient toinoculate tens of thousands of seedlings. Inoculation in acommercial nursery also presents certain obstacles, sinceinoculation must not be too expensive or cumbersome if it is tobe used routinely. For that reason, this study also included anassessment of two different methods of mycorrhizal fungusinoculation.This thesis can be therefore viewed as a protocol for theselection and evaluation of ectomycorrhizal fungi for use inoutplanting trials. Development of this protocol included thecase study evaluation of certain fungal species previouslyuntested or virtually untested in outplanting trials, adescription of a new technique for culturing ectomycorrhizalfungi, new techniques for the examination of ectomycorrhizalroots and an evaluation of two different methods of applicationof fungal inoculum.Since these topics are distinct, they will be presented in thefollowing autonomous sections:1.) In Vitro Growth of Ectomycorrhizal Fungi on Dilute Agar.2.) A Comparison of Two Inoculation Techniques forEctomycorrhizal Vegetative Inoculum in a CommercialNursery.103.) A New Low Toxicity Stain Useful for the Examination ofMycorrhizae.4.) The Selection and Evaluation of Ectomycorrhizal Fungi toEnhance the Performance of Seedlings Planted Under NormalReforestation Conditions.2. OBJECTIVESThe specific objectives of this thesis research are as follows:1.) To try to improve the performance of Engelmann spruce andlodgepole pine seedlings in the nursery and in the fieldby inoculating seedlings in the nursery withectomycorrhizal fungi isolated from the site where theseeedlings will be outplanted;2.) To compare the effectiveness of local isolates ofectomycorrhizal fungi with fungi from distant anddifferent sources;3.) To observe the behaviour of inoculated fungi on the rootsof seedlings in order to determine how they behave in thenursery and the field and to compare their behaviour tothat of volunteer fungi;4.) To compare two methods of applying fungal inoculum, 1) byinjection into the root plug and 2) by applying theinoculum to the plug surface;5.) To try to improve the culture of the fungal isolates usedin this study;6.) To evaluate a new low—toxicity stain for examining11ectomycorrhizae;7.) To develop a protocol for the design of experimentsinvolving field trials of trees inoculated withectomycorrhizal fungi so that sufficient information iscollected in future studies that experiments may becompared and patterns in the behaviour of inoculated fungimay be determined, if they exist.123. LITERATURE REVIEWIn vitro growth of ectomycorrhizal fungi on dilute agar..Perry, Molina and Aniaranthus (1987) note that different fungi dodifferent jobs for different hosts in different environments andthat inoculation with more than one fungal species is a goodstrategy if the fungal mixture is appropriate. About 34ectomycorrhizal fungi have been tested worldwide to this time, inoutplanting trials. Trappe (1977) estimates that there are over2000 species of fungi mycorrhizal with Douglas—fir alone. Manyreasons exist for the lack of variety in inoculation trials, andsome of them were discussed earlier. However, a major limitingfactor is as Trappe (1977) has stated, “it is the commonexperience of mycorrhiza researchers all over the world that manyfungi grow poorly or not at all in the pure-culture methods triedso far.”At least two major types of variations can be made in culturetechniques; to the nutrient composition of the medium and to thephysical substrate. Considerable emphasis has been placed onnutrient composition. For a good summary, see Molina and Palmer(1982). Much less work has been done on the physical componentof the medium, even though it has been noted that certain fungido much better in one type or another (Molina and Palmer, 1982).Fungi are generally grown in small quantities on agar plates, orin large or small quantities in liquid and particulate substrates(vermiculite with or without peat). The liquid medium may bestill or agitated, or aerated or not. Fungi inoculated intoliquid culture often grow poorly, or not at all. To help13overcome this, a separate “fuzzing out” procedure, to give thefungi a headstart, is often employed (Molina and Palmer, 1982).Still, some commonly found genera of mycorrhizal fungi, includingsuch groups as Russula, Lactarius and Gomphidlus cannot beroutinely grown in culture. Other species like Amphinemabyssoides and Cenococcura geophilum tend to be slow growing andindurate.The culture techniques used today are not new, and haveundergone few adaptations to deal with the specialcharacteristics of ectomycorrhizal fungi (slow—growing, requiringspecific nutrients or other symbiont). The particulate substratetechnique was essentially outlined by Moser in 1958 (Marx andKenney, 1982). Marx and Bryan in Marx (1981) made improvementsto Moser’s technique and since then, this technique has beenwidely used in inoculation trials, particularly with Pisolithustinctorius. Limited forays have been made into using grain as agrowing medium (Takacs as reported in Marx and Kenney, 1982; andPark, 1971), which is essentially an old commercial mushroomspawn production technique developed by James Sinden in 1932(Stamets and Chilton, 1983). In spite of repeated admonitions toinvestigate more of the ectomycorrhizal fungi available, the rateof investigation into new cultural techniques has been slow.Marx and Kenney (1982) took for granted that, “Once pure cultureinoculation techniques have been perfected, the value of a fungusshould be tested over a wide range of environmental conditions.”This recommendation, to select the fungi one wishes to use andthen figure out how to grow them seems to have been overshadowedby emphasis on selecting fungi easily grown in culture. As14researchers start to realize the limitations of the handful ofeasily cultured fungi, It is perhaps time to renew interest Inimproving cultural techniques.Two Techniques For Inoculation of Vegetative EctomycorrhizaeFungal Inoculum.- Many techniques have been developed for theinoculation of ectomycorrhizal fungi in greenhouse and nurserysituations. These have been subdivided into categories byRiffle and Maronek (1982). The categories are broadcast,banding, slurry dips, basidiospore inoculations, pelletizingseed, ectomycorrhizal seedlings and roots, and others. Most ofthese techniques (broadcast, banding and pelletizing seeds)require that the inoculum be applied before or during seeding.Application of inoculum at this time has several advantages inthat incorporation of the inoculum is easiest before seedlingshave developed to any great extent. If an extra step is neededin the planting procedure, it is easiest to incorporate into theassembly line nature of planting and it is easiest to utilize amechanized procedure at this time (Riffle and Maronek, 1982).Spore inoculation is most effectively accomplished byincorporating spores into the growing medium or somehow assuringintimate contact with the root system. However, Castellano andTrappe (1985) have had very good success with applying spores inan aqueous slurry from a watering can or commercial irrigationboom. Such a technique has obvious benefits in economy and easeof application.Use of mycelial inoculum is further complicated by problemswith survival of the mycelium until infection occurs. Marx15(1980> failed to achieve infection of ectomycorrhizal fungiusing the grain culture technique of Park and Takacs (Park,1971). The grain became heavily infected with saprophytic fungiand bacteria only three weeks after inoculation. There isoften up to an eight--week lag between germination and short rootdevelopment, and it is probable that the fungal inoculum wouldhave to remain viable at least that long. Marx (1980) foundthat peat moss and vermiculite wetted with modifiedMelin-Norkrans nutrient (MMt4) (Schenck, 1982) would supportexcellent growth of several fungal species. This substrate alsosuffers from attack by saprophytes unless the MMN is washed offbefore inoculation. The amounts of inoculum used in thistechnique are large, ranging from O.27L/m to 2.16L/m. About1L/m- seems to be necessary for good inoculation in a barerootnursery. Rates of 6 to 12% by volume have proven effective incontainer nurseries (Marx as reported by Riffle and Maronek,1982). These are not small volumes of inoculum.The techniques developed for inoculation of mycorrhizal fungiare adequate in many regards. However, given the wide varietyof behavioural characteristics in mycorrhizal fungi, it ISprobable that some types of fungi would perform better withdifferent inoculation styles. In particular, application ofmycelial inoculum at a time when the seedlings are receptive toinfection could be particularly helpful in terms of fungalsurvival and infectivity. Boyle, Robertson and Salonius (1987)and Fortin, Fortin, Gaulin, Jomphe and Lemay (1988> have bothreported some success with top applied mycelial slurries, though16infection rates are generally not as high as with in:iectedinoculum, However, very few fungal species have been testedthis way, and the success achieved so far suggests that thisvery simple technique warrants further investigation. Ifmycelial inoculum could be applied as easily as the Rhizopogonspores of Castellano and Trappe (1985>, without reductions inthe effectiveness of inoculation, it would be a great stepforward in mycelial inoculation technology.A New Low Toxicity Stain Useful for the Examination ofEctomycorrhizae.— There are several reasons for stainingectomycorrhizal roots such as to make it easier to determinepercent colonization, to highlight the fungal component of themycorrhiza to assist description and to help distinguish typesbased on differential staining.The Phillips and Hayman (1970) approach to clearing andstaining roots has been applied to ectomycorrhizal roots toassist in evaluating levels of ectomycorrhizal colonization(Wilcox, 1982). This method has been improved upon(Daughtridge, Boese, Pallardy and Garrett, 1986) with the goalof improving estimates of ectomycorrhizal colonization.However, the vigorous handling and disruptive nature of thestaining process renders identification of the mycorrhizal typesvery difficult and one must resort to hyphal characteristics todistinguish types confidently (Roth, 1990). When examininglarge numbers of roots on container plug-size root systems, itis very impractical to examine hyphal characteristics of allshort roots, and in fact, some of the most useful17characteristics for distinguishing mycorrhizal types areextramatrical hyphal appearance, mycelial strand appearance andgross morphological appearance of mycorrhizae (Danielson andVisser, 1990). For this reason, Danielson recommends that rootsbe examined intact and unwashed.More detailed examination of roots to confirm mycorrhizalinfection can be achieved with whole mounts (Danielson,Griffiths and Parkinson, 1984). For even more precise rootexamination, hand sections stained with trypan blue inlactophenol are recommended. Good hand sections can reveal muchof the detail required by Agerer (1986) in his guidelines forthe description of ectomycorrhizal fungi. The Canadian Centrefor Occupational Health and Safety (MSDS record number 26619)describes trypan blue as a carcinogen and teratogen. It isirritating to the eyes and assumed harmful by skin contact.Furthermore, upon heating, it releases highly toxic fumes ofnitrogen and sulphur compounds. The slide heating techniquerecommended by Danielson (1984) may be quite dangerous if notdone carefully, and if many roots are examined in this manner,the potential for some kind of poisoning from trypan blue isprobably high.Other stains routinely recommended for staining ofectomycorrhizal fungi include cotton blue, chiorazol black E,Pianese 111-B stain (chlorazol, malachite green, acid fuchsinand Martius yellow) and the Conant quadruple stain (safranin,crystal violet, fast green and gold orange (Wilcox, 1982). Acidfuchsin is harmful if swallowed, an irritant to skin and eyesand breathing the dust is dangerous. Malachite green is a skin18and eye irritant and produces toxic fumes. Crystal violet isextremely irritating to eyes and contact with skin and clothingshould be avoided. Any of these dyes can be used safely, butwhen examining a large number of roots, it can become extremelytedious to continually don and doff protective clothing and maketrips to the fume hood. Without examining every dye used instaining fungi, it becomes quite apparent that there could be ause for a low—toxicity dye suitable for staining fungi.19The Selection and Evaluation of Ectomycorrhizal Fungi toEnhance the Performance of Seedlings Planted Under NormalReforestation Conditions.— The accumulated knowledge of thebehaviour of ectomycorrhizae in reforestation can best becharacterized as mixed and often contradictory. Slashburning (and broadcast burning) may reduce or increase themycorrhizal inoculation potential of a site. Organic matter maystimulate or decrease the formation of ectomycorrhizae.Mycorrhizae may be stimulated or reduced by rhizosphereorganisms. Litter extracts may increase or decreasemycorrhizae. Some ectomycorrhizal fungi may be able to breakdown cellulose and others may inhibit the rate of litterdecomposition. Ectomycorrhizal fungi have been found to persistin the soil without hosts for long periods of time or shortperiods. Non—crop species in forested areas may help maintainor reduce mycorrhizal Inoculation potential. The same non-cropspecies such as Alnus spp. may at times act as a reservoir forectomycorrhizal fungi and sometimes not. Removal of the litterlayer may increase or decrease the inoculation potential of asite. Dead wood may serve as an excellent reservoir forectomycorrhizae or as a very poor one. Coal spoils may bedeficient in mycorrhizal inoculum or abundant in it. For anexcellent review of the wide variation in reported effects, seeRoth (1990).The seemingly contradictory results described above are anoutcome of several factors, including the extreme variability Inbehaviour both between and within species of ectomyeorrhizal20fungi. The ectomycorrhizal fungi, as noted earlier, vary widelyin 5uch Important characteristics as pH preference, droughttolerance, ability to reduce nitrate and other ways.compounding the variability within the fungal symbiont is thevariability found within the tree species and specificity ofinteraction between fungus and tree at the within-species level(Castellano and Trappe, 1985). Furthermore, micrositevariability is extremely high in many natural forests andhypothetically, there could exist many situations whereconditions could be sufficiently different over a few metresthat the natural fungal population might change dramatically.This tremendous cumulative and compounded variability almostcertainly accounts for a large portion of the range in responsenoted in outplanting trials summarized by Castellano (in press)and Trofymow and van den Driessche (1991).Early guidelines developed for the selection of fungi thatwould be most useful in pure culture synthesis and inoculation(as opposed to inoculation with soil or spores) already tried totake into account the diverse behaviour of ectomycorrhizalfungi. For example, Molina (1977) suggested the major criteriafor 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.21Molina goes on to stress that, “many species and ecotypes offungi are closely adapted to their particular habitats, and soeach fungal isolate must be tested on its own merits”. Theserecommendations have been repeated so often that they seemalmost obvious today. Certainly one must be able to isolate,grow and inoculate fungi before they may be used in pure culturesynthesis. The criteria designed to deal with the specificityof fungi (5, 6 and 7) are not new either, for as Hatch (1937)noted from Rommell (1930), “mycorrhizal fungi are often moreexacting in their site requirements than are the trees withwhich they are associated”. Even though researchers have knownabout fungal specificity for some time, it has seldom been dealtwith in outplanting trials. Part of the reason is thatresearchers are limited to working with fungi that meet criteria1 to 3. The state of technology related to manipulation ofmycorrhizal fungi is not so advanced that any fungus can becultured and inoculated. This is something that needs to beimproved before mycorrhizal research can advance much further.In addition, it is one thing to say that there is tremendousspecificity in ectomycorrhizae and another to predict whichfungus will do well in a given situation. This obviouslyrequires detailed ecological information for individual fungi.Points 3 and 4 also suggest that the fungi used should formabundant mycorrhizae, yet the need for high colonization has notbeen established (Stenstrom and Ek, 1990). So, while it isuseful to have some guidelines for the selection ofectomycorrhizal fungi, it appears that some of the guidelinesare self—evident, some need additional technology before they22can be applied, and one guideline may not be based on a soundassumption. The handicap which the technological limitationsplace on mycorrhizal research is best illustrated by Castellano(in press) who notes, “most research is concentrated inrelatively small geographic regions (Pacific Northwest andSoutheastern United States), on a few host plant genera (Plnusand Pseudotsuga) and with an extremely limited group ofmycorrhizal fungi (Pisolithus tinctorius and Rhizopogonvinicolor)”. Trofymow and van den Driessche (1991) also note,“very few outplanting studies have been conducted with seedlingsinoculated with other species of fungi” (other than Pisolithustinctorius).To offset our inability to adequately evaluate the huge numberof ectomycorrhizal fungi, researchers have suggested criteriafor the selection of outplanting sites to try to insure that“positive” results are obtained. Of the outplanting trials doneto date, the vast majority were conducted under extremeconditions. These conditions are now described as situationswhere mycorrhizal inoculation might be warranted and have beendefined 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 fromvesicular arbuscular mycorrhizae (yAM) to ectomycorrhizae,or some other such radical change.The contradiction inherent in these suggestions is that in23most cases (except option 3), planting of inoculated stock wouldbe done in circumstances far beyond the normal ecological rangeof the fungus being tested and trials conducted in suchcircumstances only reinforce the already well—documentedobservation that virtually any symbiont is better than nosymbiont. Inoculation trials in such circumstances dorelatively little to expand our knowledge of the relationshipbetween the fungus and its normal environment. Similaranomalies occur if we restrict our observations to fungi that wecan grow easily, or fungi that grow well in the nursery, or anyother artificial limitation designed to result in easilyapplicable commercial technology.This suggests that one approach which would be advantageous tolearning about the behaviour of ectomycorrhizal fungi would beto use as many mycorrhizal fungi in inoculation trials aspossible, regardless of their ease of isolation or growth ratein pure culture (by implication, techniques for isolation andculture need to be improved). The effectiveness of the inoculumis important in terms of application to commercial nurseries,but as Hunt (1989) has demonstrated, the types of fungi thatwill grow in a nursery and the degree to which they will infectroots can be greatly affected by manipulation of growingconditions. Fungi should not be excluded from field trials atthis early stage of understanding because they do not do well inthe artificial and easily manipulated environment in a nursery.Fungi may be evaluated In terms of their effect on growth andvigor, but the response of a seedling in any season or a fewseasons may not give a true indication of the potential of the24fungus (Smith, 1985). The objectives of any fungal evaluationbecome further complicated by our lack of understanding of thecomplete ecophysiological function of the symbiosis (Kropp andLanglois, 1990). Furthermore, the normal situation for trees isthat there are several species of fungi on a tree root(Danielson and Visser, 1989; Roth, 1990), even at a young age.Molina and Trappe (1984) caution that inoculation with only onefungus should be considered as only one approach to applyingmycorrhizal technology.The task of evaluating the effect of mycorrhizal inoculationthen appears to be as follows: to determine if mycorrhizalinoculation is beneficial to trees when the beneficial effectmay not show up in all sets of climatic circumstances, thefungal isolate may not match the tree strain, when the micrositeconditions may not match the fungal strain or tree strain, thepresence of the fungus might manifest itself in someunlooked-for way, the other fungal symbionts necessary to give atree a complete complement may not be present, and new fungithat infect seedlings shortly after outplanting might mask theeffect of the inoculated fungus. The only way to deal with suchdiversity is the large scale evaluation of many fungi under avariety of carefully documented growing conditions. Hatchanticipated this situation in 1937 when he noted, “The scienceof forestation is apparently still in its infancy and it willprobably remain so until our knowledge of tree nutrition, ofmycotrophy, and of the influence of environment upon thesurvival of root symbionts has been widely explored. Theobvious need today is for precise information on the influence25of different species of mycorrhizal fungi upon the growth of ourmost important trees planted in a wide variety of habitats. Theproblem, therefore, is not one which may be solved by one oreven several individuals.” Today, we have accumulated a largeamount of information on the behaviour of a few fungi in arelatively small number of environmental circumstances (extremesituations). This has done little to expand the understandingof the intricacies of the plant-fungus relationship and it seemsthat little has changed since Hatch’s seminal paper.To evaluate how far we have come in collecting the informationthat Hatch suggested was necessary, requires a thoroughexamination of the literature. Fortunately, Castellano (inpress) and Trofymow and van den Driessche (1991) have recentlydone this. The discussion so far has suggested that we must bemost concerned with studies done in normal reforestationsituations if we want to really understand the dynamics ofectomycorrhizae in normal reforestation situations. A normalreforestation situation is defined here as reforestation thatdoes not include afforestation, the introduction of exoticspecies, reclamation of mine spoils or other severely disturbedsites or changes from VAN mycorrhizal species toectomycorrhizae, or other similar radical changes. If thestudies involving extreme situations are discounted, then only afew relevant studies remain. I have reviewed all of the studiesof this nature that I could find. I was concerned withdetermining how much each study advanced the technology ofmycorrhizal fungus inoculation. Several studies are brieflydiscussed below.26Castellano and Trappe (1985). In this experiment, severalspecies of hypogeous fungi were spore—inoculated onto severaltree species, including seven species of hypogeous fungi thatwere inoculated onto two provenances of Douglas-fir. Two fungiformed mycorrhizae with Douglas—fir in the nursery andsubsequently, one of these two fungi improved survival andseveral growth characteristics of seedlings in an outplantingtrial. The site for the outplanting trial is given a minimaldescription in the form of location, elevation, slope andaspect. Site specific descriptions of the sources of inoculumare not given and no attempt is made to relate the environmentof the source of inoculum to that of the outplanting trial. Twoyears after outplanting, the seedlings inoculated with one ofthe fungi had greater root collar diameter, greater height andgreater survival. The inoculated fungus persisted on the rootsof the seedlings, even though indigenous fungi also colonizedroots. It was not indicated if the inoculated fungus wasindigenous to the site. This study included some usefulapproaches. It used fungi that were at least from the samegeneral climatic area and rudimentary descriptions of the studyarea were given, though these were insufficient to use asguidelines for future studies involving similar fungi. Littleinformation is given about the outplanting site in terms ofinoculation potential, indigenous species of plants and fungi,soil and biogeoclimatic factors. Since these things are notknown, it is difficult to assess or begin to define a typicalsite for the successful use of this type of fungus. The sitewas previously covered with red alder, which was cut and burned.27It is not stated if herbicides were used, how intense the burnwas, what species were left on the site (if any) and so on.Yet, the impression created by not noting any extremecircumstances is by default that this was a more or less normalreforestation situation.Stenstrom and Ek (1990). This experiment seems to attempt toaddress ecotypic variability in that it involves several fungithat were isolated from similar latitudes and not too dissimilarlongitudes and from generally the same species of tree as wasused in an outplanting trial. However, details of thecollection sites were not given. The soil of the planting siteis described as being a glacial till deposit with a sandycomposition (sic). Site vegetation and site index are alsodescribed. It was not noted if the types of fungi used in theinoculation trial were found on the site, except that indigenousmycorrhizae appeared to be indistinguishable from some of theinoculated fungi. Some description is given of the behaviour ofthe inoculated and other mycorrhizal fungi on the roots afteroutplanting. The inoculated seedlings were smaller in size atoutplanting, but soon passed the uninoculated seedlings in size.The levels of mycorrhizal infection at outplanting were low: 5to 25 in all cases, yet statistically significant growthimprovements were found.The study described above contrasts nicely with a subsequentpaper by Stenstrom, Ek and Unestam (in press), in which mostfeatures, such as site description and description of thebehaviour of the fungi out in the field, are similar. The majordifference between the studies is that in the more recent one,28the fungi (except one) came from exotic locations. The unifyingcharacteristic of the fungi used is that they were “assertivemycorrhizal formers” which is an appropriate term to describefungi that readily form abundant mycorrhizae under artificialconditions. In this case, many of the inoculated seedlings hada number of roots infected by the applied fungus. Theinoculated seedlings grew more slowly in the nursery, and didnot recover even after three years in the field, andfurthermore, showed little sign of recovering. Even thoughthese fungi were effective at forming mycorrhizae in thenursery, they had little effect in the field, which serves as acaution against choosing fungi based solely on their ability toform abundant mycorrhizae.Richter and Bruhn (1989). Several fungi were isolated from thesame tree species and same general area in which the outplantingtrial would be conducted, but not necessarily from the same orsimilar type of environment. The result was that the isolate ofLaccaria bicolor (R. Mre.) Orton, which significantly improvedsurvival on a “droughty sandy” site, came from a moderately wetsite. This isolate was one of the faster growing ones and wasmost successful at forming mycorrhizae in the greenhouse. Thereare rudimentary descriptions of the sources of the fungi and theoutplanting site, though a slightly more detailed description isincluded for the source of the Laccaria bicolor that improvedgrowth. This was done to highlight the paradoxicalcircumstances. It 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 roots29after outplanting.Amaranthus and Perry, (1989). Improvements in survival on avery harsh site that appeared to be mainly devoid of fungalinoculum were achieved by transferring soil to the plantingholes. Included are very good detailed descriptions of theplantation site and because of its similarity to the source ofinoculum, the authors also describe the inoculum source sitewell. The roots of the seedlings were thoroughly examined,though not much detail is reported in the paper. For instance,both controls and inoculated seedlings had Rhizopogon vinicoloron them after outplanting, but it was not reported what theroots had on them at outplanting. This appeared to be a veryextreme site and almost beyond the realm of normalreforestation. As the authors point out, many factors, bioticand abiotic, could be involved with the transfer of soil andcould also have led to the differences in survival, so studiessuch as these may be of little use in dealing specifically withthe mycorrhizal question.Bledsoe and Tennyson (1982). This is an inoculation trial donein north central Washington State, where fungi from what wasprobably a considerably different climate in western Washingtonwere inoculated onto Douglas—fir seedlings that were thenplanted on a harsh dry site. The fungi were selected becausethey formed mycorrhizae in nurseries and grew well. Theoutplanting sites were quite well described but this is notbalanced by a description of the source of inoculum. Thebiomass of the outplanted seedlings was reduced and no othergrowth effects were reported. The roots became colonized by an30indigenous fungus that was different from the inoculated fungus,but unidentified. The root observations were quite useful Inthat the inoculated fungi (Hebeloma crustullnlforme (Bull.: St.Amans) Quelet and Laccaria laccata (Scop.: Fr.) Berk.& Br.> did not grow onto new roots on the outplanted seedlings.Loopstra, Shaw and Sidle (1988). This study evaluates threefungal isolates, two of which came from the same approximatelocation on the continent as the outplanting site. The studyconfirmed that the trees grown under normal high fertilityregimes in the nursery tended to grow faster than treesinoculated with fungi but with lower fertility regimes. Theauthors conclude, “we caution against sweeping generalizations,unsupported by quantitative, site—specific data, that suggestinoculation with ectomycorrhizal fungi will necessarily improveseedling performance.” This is more or less a restatement ofthe well—documented observation that not all fungi improvegrowth in every situation.Kropp, Castellano and Trappe (1985). Western hemlock wasinoculated with Cenococcurn geophilum and the seedlings wereplanted on separate sites to represent “a variety of aspect,slope, vegetation, and soil types.” However, the sites weredescribed only in terms of elevation and rainfall, which weresimilar for all sites. The different planting sites did notaffect the inoculation effect reported, which was thatinoculated seedlings had greater leader growth than uninoculatedseedlings. There were no differences between seedlings heavilycolonized by Cenococcum geophilurn and those moderatelycolonized. No other growth data is given, in particular, how31the heights of the seedlings compared at outplanting.Non-mycorrhizal seedlings became infected with Cenococcum fromthe site.Heidmann and Cornett (1986). The outplanting site in thistrial presented a quite “normal” reforestation problem. It wasan open area within a young forest that was not properlyrestocked because of poor nursery stock. The site was describedwith brief notes on elevation, slope, vegetation and soil. Thesoil description was “silt barns derived from basalt parentmaterial.” No other details such as humus form, pH or soilseries were given. Two types of inoculum were used: screenedlitter layer (duff) and Pisolithu5 tinctorius spores fromOregon. The descriptions of the inoculum sources were evensparer than those of the outplanting site. The mycorrhizalinfection rates after growth in the nursery were given, but thetypes of fungi were not described. This is a point of concern,since the inoculated seedlings seem to show the same levels ofcolonization as the controls. Nevertheless, the seedlingsinoculated with the duff had a significantly higher survivalrate. Some growth effects occurred that are difficult tosummarize because of interactions with a fertilizer treatment.The rnycorrhizae on the roots were not described after time inthe field, so it is not clear what the fungi were doing.Marx in General. No review of outplanting trials withectomycorrhizal inoculurn would be complete without somediscussion of Don Marx and Pisolithus tinctorius (“Pt”). Marxhas rather consistently reported improvements in seedlingperformance after inoculation with Pt. The normal situation for32bareroot nursery seedlings in the southeastern United Statesseems to be the almost complete domination by Thelephoraterrestrls. When this fungus is replaced by Pt, there are oftengreat improvements in survival and performance of outplantedseedlings. Higher quality planting sites tend to not have asgreat a response to Pt inoculation. Despite many, many trials,the mode of action of Pt is not clear. For example, Marx,Cordell and Clark (1988> reported that when Pt colonization wasgreater than a certain level, then improvements in growthcorrelated with level of colonization. The site used was a goodone with high nutrient levels. Even though many researchershave reported that high fertility reduces ectomycorrhizaldevelopment, Marx (1990) reported that higher N fertilityresulted in more mycorrhizae at three different pH levels, andthe mycorrhizal development was affected more strongly by soilacidity than by nitrogen levels. The plantation site wasformerly an agricultural field and may have been lacking infungal inoculum. However, this point was not discussed indetail. Even though the site reportedly had good moistureholding capacity, the seedlings underwent considerable droughtstress over the period of the experiment, which may haveincreased response to inoculation. Walker, West and McLaughlin((1982), described in Marx, Cordell and Clark, (1988)], reportedlowered internal water tension in seedlings with abundant Pt.It appears that many factors contribute to the effectiveness ofPt inoculation, and these include the extent of rootcolonization at outplanting, the presence or absence ofinoculum, the fertility and moisture holding capacity of the33site, the climatic conditions over the time of the study, soilacidity and inoculum potential of the site. Because of this, itis not possible to accurately predict when Pt inoculation willbe effective. Even when all the most extreme conditions towhich Pt has been applied are included, inoculations haveimproved seedling growth only 46o of the time in outplantingtrials (Castellano, in press).Summary. Considerably more literature on ectomycorrhizalinoculation exists, but most of it deals with the type ofextreme circumstance described previously. The work onoutplanting trials in more or less normal reforestations hasbeen almost entirely published since the inception of thisresearch project. The types of projects undertaken have inreality paid little attention to ecotypic specificity. Often,the only mention made is the general geographic area of theorigin with little description of specific site conditions. Thevocabulary, to describe sites in a way that conveys muchecological significance, seems to be lacking.Hardly ever is there any evaluation of whether the fungusinoculated is actually found naturally at the outplanting site.This may not be a critical factor for selecting the fungi for anoutplanting trial, as in the one case where the environments ofthe fungal inoculum were compared to the outplanting site, itwas found that the fungus from the most disparate site improvedgrowth. Other evidence suggests that fungi from the samegeneral area are better than fungi from further away. However,it is the interaction of the fungi and their behaviour in thefield that will give clues to the mode of action and yield a34procedure for selecting appropriate fungi. For example, it maybe that a fungus found at a site may improve performance if itis given a headstart in the nursery, or it may be that certaintypes of fungi consistently perform better on certain types ofsites, so these fungi should be introduced regardless of theindigenous fungi.Another apparent shortcoming in ectomycorrhizal research ingeneral is the paucity of types of fungi evaluated in normalsituations. When planting in extreme situations, any fungus isbetter than none, but in normal situations, where it isincreasingly evident that inoculation potential is likely to bequite high, the crux of the matter may be to have an appropriatefungus. Yet certain researchers repeatedly test the same fungiin “similar” conditions, and perhaps, somewhat surprisingly,come up with inconsistent results. This may be due to the factthat conditions (e.g. soil, microclimate) are often notdescribed well enough to ascertain if they are in fact similar.Most singularly missing are any attempts to correlate theindigenous ectomycorrhizae at a site with the success of aparticular fungal inoculum.In spite of the little success with the relatively smallnumbers of assertive ectomycorrhizae, almost no work ispublished on techniques to improve inoculation and culturetechniques for ectomycorrhizae. The literature onectomycorrhizae can be summed up as, “a few people working witha few fungi, using a few techniques, working diligently todefine the few situations that might give positive growthresults with the above.” It is not surprising that the35ectomycorrhizal symbiosis has not been fully exploited, even 60years after Hatch remarked on the enormous difficulty involvedwith accurately characterizing mycorrhizal relationships.364. EXPERIMENT ONEIN VITRO GROWTH OF ECTOMYCORRHIZAL FUNGI ON DILUTE AGAR’Ectomycorrhizal fungi often grow very slowly in ordinaryliquid culture, or not at all (Stevens, 1981; Marx and Kenny,1982). Agar-solidified nutrient media may be used to producesmall amounts of mycelium. Other substrates such as peat mosscan be used to produce greater amounts, but this addsconsiderable bulk to the medium and makes inoculation difficultby means other than incorporation of the inoculum into thegrowing medium.We have had good success with culturing fungi in a semi—liquidmedium made thixotropic by including agar at low concentration.The technique has several advantages. Initial growth is fastercompared to standard liquid culture and a separate “fuzzing out”(Molina and Palmer, 1982) procedure is not necessary. Fastergrowth is sustained without frequent shaking or addition ofglass fragments. Several isolates that grow very poorly inliquid will grow reasonably well. The fungi often grow bestnear the surface of the medium (Fig. 3), whereas in liquidculture, the colonies frequently settle near the bottom.We compared two types of media: modified Melin—Norkrans (MMN)formula (Marx, 1969) with no agar added or with 3g/L agar. Themedia were placed in O.25L Erlerimeyer flasks at a rate of O.1Lper flask. All flasks were autoclaved for 20 minutes and* Published in Mycologia, 82(4), 1990, pp. 526—52737allowed to cool. Each inoculation consisted of the addition ofa 4mm agar plug from an agar-plate colony. Twelve differentfungal isolates were used, including Cenococcurn geophilum Fr.,E-strain, Hebeloma crustiliniforrne (Bull.> Quelet, Laccarialaccata (Scop.:Fr.) Cke., Amphinema byssoides (Fr.) 3. Erikss.and others.The inoculating plug was placed gently in the agar flask sothat it would float on top of the agar. Liquid (no agar)cultures were shaken continuously at 100 reciprocations perminute (Boyle et al., 1987). (Intermittent shaking of theliquid cultures gave similar results.) The agar cultures wereallowed to rest undisturbed until growth had begun on the top ofthe medium, then shaken vigorously once, and allowed to rest atroom temperature for one month.To evaluate growth, cultures were heated to boiling anddrained on filter paper in a Buchner funnel with suction andrinsed with 0.2L of boiling water. Mycelium was air-dried fortwo days and dry mass was determined.Eleven of the 12 isolates grew faster in dilute agar than inthe liquid medium (Table I) (Fig. 4). A paired t-test indicatedthat differences were highly significant (P=0.004). The ratioof growth rates in agar to those in liquid ranged from 0.4x to7x.We have used this technique to grow numerous species ofectomycorrhizal fungi. Resultant cultures have been used toinoculate seedlings in a variety of applications, including acommercial nursery. When dilute agar cultures are fragmented ina blender, they can be injected through a large bore needle or38pipette. We note that it is difficult to separate the agar fromthe fungal mycelium, using this technique. However, results ofour inoculation studies show that separation of the agar mediumfrom the mycelium is not necessary to achieve good formation ofmycorrhizae.Centrifugation can be used to facilitate separation, but thishas proven to be a bit messy. The fungus can be caused tostratify, but it is difficult to remove all traces of the agar.Accurate mycelial concentration can be determined by subsamplingthe mycelium and washing with boiling water as outlined above.39Table IIsolateA14A18Al 8 BA21A2 9 BA31BA46E01C.g.Hecr5Fungal SpeciesunknownunknownunknownunknownAmph I nemabyssol desunknownSuillus sp.E-strainCenococcumgeophi lumHebel amacrustul ml forrneHebel omacrustul ml £ormeLaccaria laccata 0.0713Mean difference=0.028SD difference=0.027Paired—sample t=3.566(Degrees of freedom=ll)P=0.0040.02920.05330.00280.0197Mycelial Mass of Ectomycorrhizal Fungi AfterOne Month of Growth in Liquid or 0.3 AgarMMNDry MycelialMass (g)DiluteAgar Liquid0.0340 0.00530.0789 0.04350.0098 0.02180.1356 0.1280.0553 0.00860.08770.12650.00520.0217Hecr8S2380.0202 0.00400.0188 0.00310.0100405. EXPERIMENT TWOTWO INOCULATION TECHNIQUES FORVEGETATIVE MYCORRHIZAL INOCULUMINACOMMERCIAL NURSERYApplication of ectomycorrhizal fungal inoculation technologyis dependent not only on performance of the inoculum but also oncost effectiveness. The fungal culture technique perfected •byMarx uses peat moss and vermiculite as a substrate, which meansthat it must be incorporated in the growing medium beforeplanting. This does not allow much flexibility in timing ofapplication, if, for example, inoculation is desired over thegrowing season, and it does require a certain amount ofadditional manipulation during planting. The technique ofapplying spores in an aqueous suspension is very flexible withregard to timing, cheap and easy. Work with slurry applicationsof vegetative inoculum suggests that this type of inoculum doesnot need as much technology to apply as was generally believed(Boyle, Robertson and Salonius, 1987). Fortin et al, (1988)have tried surface application of mycelial inoculum and foundthat it produced mycorrhizae, but not as well as injectingmycelial slurry. However, relatively few fungi have beeninoculated this way, and few trials under different conditionshave been done. If slurries of vegetative inoculum could beapplied in a manner similar to to that used for fungal spores,41it would increase the opportunities for using vegetativeinoculum. Vegetative inoculum also has some advantages overspore inoculation in that it can be pure culture or mixed, itcan be grown anytime (so it is not dependent on collection ofsporocarps), quality and consistency can be controlled, somefungi that are difficult or impossible to obtain sporocarps forcan be grown vegetatively and vegetative inoculum may infectmore quickly than spores (Marx and Kenney, 1982).This experiment consisted of applying slurries of vegetativeinoculum to container—grown seedlings by Injection into the plugor by squirting inoculum on top of the plug. An importantdifference from all other trials is that the slurry used in thistrial consisted of the fungus suspended in the dilute agarculture described in Experiment One. The nutrients were notremoved from the growing medium before inoculation, which is adeparture from virtually all other procedures in use today. Thecombination of dilute agar, no washing and top application offungi represents one of the least labour-intensive approaches toinoculation of fungal mycelium yet tried.MATERIALS AND METHODSThe experiment was conducted in the research greenhouse at theHeffley Reforestation Centre, about 20km north of Kamloops, B.C.The research greenhouse was treated the same as the commercialportion of the nursery, except for the experiments conductedthere. The Heffley Reforestation Centre nursery is somewhatdifferent from many nurseries because it does not use42slow-re1ease fertilizer in most stock types; the growing mediumused is mainly coarse peat moss to facilitate aeration of roots;and watering Is reduced, but compensated for by frequent mistingto control temperature. These steps have created an environmentthat is much more conducive to the formation of mycorrhizae thanwas present under the previous and more typical growing regime(Hunt, 1989).Two species of trees were used, Engelmann spruce and lodgepolepine. The seed used was from the appropriate provenance for theplanting site. The fungi were grown on the dilute agar and MMNmedium described in Experiment One. The dilute agar slurry wasfragmented in a blender for 15 to 45 seconds to allow it to beinjected. Both the spruce and pine seedlings were inoculatedwith 5mL of slurry. The slurry was applied with an Oxfordpipetter (Model SA). A length of tubing connected the pipetterto a lmL pipette. The irioculum was applied by (1) injecting themycelium throughout the plug or (2) squirting the inoculum ontop of the plug. In either case, the body of the pipetter waskept below the injector in order to avoid siphoning. The sprucewere grown in used PSB 313a styroblocks (198 cavities perblock, 947 cavities/m, Beaver Plastics, Edmonton, Alta.) andthe pine were grown in used PSB 211 (240 cavities per block,1130 cavities/m2)styroblocks. The growing medium was peat andvermiculite (4.4:1 v:v). Fertilizer was applied according tothe schedule shown in Appendix III. This fertility regime hasbeen shown by Hunt (1989) to increase the level of mycorrhizalcolonization and the number of types of mycorrhizae on the roots43over those using slow release fertilizers. Other details of thegrowing regime can be found in Hunt (1989). Both tree specieswere about eight weeks old at the time of inoculation and hadstarted to develop short roots. The inoculum was applied toapproximately 50 seedlings in each block, excluding the two rowsof cavities around the perimeter of the spruce blocks to reduceedge effects. There were fewer pine seedlings so only the twoend rows and one side row were excluded. The exact number oftrees inoculated was counted and recorded, and the inoculatedarea was marked with felt pen and plastic markers. Eachinoculum was applied to 20 blocks for the spruce (10 blockssurface-applied and 10 injected) and 16 blocks (eight blockssurface—applied and eight blocks injected) for the pine. Eighttreatments were used for each tree species. Two of the fungalspecies used on the pine were an E-strain isolate obtained fromR. M. Danielson (probably the anamorph of Wilcoxlna mikolae:Keith Egger) and an isolate of Suillus tomentosus taken from amushroom collected near the study area. Two of the fungalspecies used on spruce were the same Danielson E-strain isolate,and an isolate of Amphinerna byssoides collected by me from aHeffley Reforestation Centre seedling. The other fungi used,E-strain (0188), Cenococcum geophilum (A188—2), and Hebelomacrustiliniforme (Hecr—8), did not form mycorrhizae and aredescribed in Appendix I. One treatment was the dilute agarmedium used to culture the fungi (referred to hereafter as thefungus growing medium), which was also injected or top applied.There were two separate controls for the injected and topapplied treatments, but they were identical in that they both44had no treatment done to them. The controls had the same numberof trees as the other treatments and they were marked off andcounted in the same manner and treated in the nursery in thesame way. After inoculation, the blocks were placed in arandomized arrangement.The experimental design was nested with blocks nested withintreatments and treatments were crossed with inoculationtechnique. The program that was used for most of thestatistical analyses is called UBC Genlin: A General LeastSquares Analysis of Variance Program by Malcolm Greig and JamesBierring. This program was useful because it could handlemissing data and do multiple comparison tests for randomizedblock and nested designs with more than one independentvariable. Homogeneity of variance was tested using Bartlettsand Layard’s tests. Normality was evaluated using theKolmogorov—Smirnov test. All analyses were checked forhomogeneity of variance and normality, but these data were notused to disregard ANOVA results. In the cases where the datafor the randomized block ANOVA were non-normal, the analysis wasalso done with the Kruskal—Wallis non—parametric ANOVA.Multiple comparisons were done using Tukey and Bonferroni tests.45RES tJL T SSpruce.— Both inoculation techniques produced abundantectomycorrhizae on the seedlings for two types of fungi used.The results of the analyses are shown in Table II. Thedifferences in the levels of colonization between the twoapplication methods were significant (p<0.05) for E—strain. Themean level of colonization by E—strain for the injected E—straintreatment was 71% (SD± 30) and for the top—applied treatment was58% (SD+ 27). The mean level of E—strain in the othertreatments was 0.9%. The mean level of colonization ofAmphlnerna byssoldes for the injected Amphinerna byssoidestreatment was 69% (std. dev. 21) and for the top appliedtreatment was 68% (std. dev. 26). The mean level of infectionby Amphinema byssoides in the control and fungus growing mediumtreatments was 27.5%. Thelephora terrestris—like fungi formedmycorrhizae on an average 47% of the roots that were nottreated with one of the fungi and 16% on the others. The totallevels of colonization (total of all species of fungi present)were not different between injected and top-applied treatments.The inoculated fungi apparently excluded Thelephora and thelevels of Thelephora became significantly different by fungaltreatment, but the levels of Thelephora by application methodwere not significantly different within the Danielson E-strainfungal treatment, even though the levels of E-strain within theE—strain treatment, were significantly different. In spite ofsignificant differences by inoculation technique in colonization46Table IIComparison of In:jection Into the PlugVersus Top Application of Ectomycorrhiza]. MycelialSlurry to Two Month Old Engelmann SpruceE—strainmycorrhizae (96)Thel ephoramycorrhizae (96)Total mycorrhizalcolonization (96)Shoot mass (g)Shoot length (cm)Root CollarDiameter (mm)Root Mass (g)Abundance ofMycorrhizaeRoot to ShootRatioDickson QualityIndex18.334.432.730.382.420.61.560.3915.53.773.330.410.900.272.580.600.600.190 . 390.1215.629 . 030.329. 482.121.51.620.3715.63.513.320.400.950.282.600.600.600 . 190.410.12TreatmentParameter Injected Top Applied pANOVAn 400 3900.06A. byssoidesmycor r hi z a e (96)36.632.735.933.20.070.540.910 . 060.700.860.150.790.960.24The top number is the mean, the number underneath is thestandard deviation. The p value in the table is from ANOVA.47levels by E—strain mycorrhizae, none of the growth parametersmeasured were significantly affected by how the inoculum wasapplied.Pine.— The results of the pine trial are shown in Table III.The colonization levels of E-strain on pine roots were notsignificantly affected by the application method. The meanlevel of infection for the injected treatment was 62% (SD 21)and for the top—applied treatment was 72% (SD 25). However,the levels of A51 (Suillus tornentosus) were marginallysignificantly different (p=0.056) for the two applicationtechniques. The mean level of A51 on the injected treatment was3.55% (SD±. 12) and for the top—applied treatment was 0.76% (SD3.00). The total levels of infection (all species of fungi)were not different between injected or top—applied treatmentsfor any treatment. There were significant growth differencesbetween injected and top-applied treatments. These differencescould not be attributed to one specific group such as the fungaltreatments, growing medium treatment or controls, but applied tothe subset of all trees. The injected treatments had, on theaverage, 10% smaller root weights (p=0.O3), 6% smaller rootcollar diameters (p=0.01) and 10% shorter shoots (p=O.O2) thanthe top-applied treatments. The Dickson Quality Index issignificantly (p=0.Ol) higher in the surface-applied treatment.48Table IIIComparison of Injection Into the PlugVersus Top Application of Ectomycorrhizal MycelialSlurry to Two Month Old Lodgepole PineTreatmentParameter In:Jected Top Applied pANOVAn= 284 318S. tomentosus 0.90 0.23 0.12mycorrhizae (96) 6.23 1.64E—strain 17.4 18.7 0.11mycorrhizae (96) 30.0 33.9Thelephora 77.7 75.2 0.07mycorrhizae (96) 29,6 33.0Total mycorrhizal 98.3 96.3 0.07colonization (96) 7.74 10.9Shoot mass (g) 0.91 0.90 0.610.29 0.29Shoot length (cm) 18.5 17.5 0.023.21 3.19Root Collar 2.96 3.12 0.01Diameter (mm) 0.46 0.51Root Mass (g) 0.50 0.55 0.030.17 0.18Abundance of 2.38 2.30 0.40Mycorrhizae 0.66 0.70Root to Shoot 0.61 0.64 0.46Ratio 0.57 0.23Dickson Quality 0.17 0.20 0.01Index 0.06 0.07The top number is the mean, the number underneath is thestandard deviation. The p value in the table is from ANOVA.49DISCUSSIONContrary to the findings of Boyle, Robertson and Solonius(1987) who worked with Hebeloma longicaudum and Fortin et al,(1988> who worked with Laccaria bicolor, both methods ofapplying the fungal inoculum worked quite successfully forE—strain and Amphinema byssoides. Interestingly, theapplication method did not make any difference for E-strainapplied to pine, but it did for E-strain applied to spruce,though in both cases the infections were quite substantial. Thespruce and pine were inoculated at different times, so theclimatic conditions at the time of application were different.The spruce was inoculated earlier than the pine and the inoculumwas older by the time the pine were inoculated. It is possiblethat the fungus had started to grow after the blenderizationprocess, and so actually increased in vigor by the time the pinewas inoculated. Apparently, some fairly subtle variables affectthe success of these techniques, but in general, mycelialslurries grown in dilute nutrient agar can be used as inoculumfor some fungi without washing away nutrients, whether injectedinto a plug or top—applied.The A51 (Suillus tomentosus) treatment in pine appeared tobehave differently, depending on application method. Thisfungus was very borderline in its ability to form mycorrhizae inthe nursery. The limitation that kept it from vigorouslyforming mycorrhizae in the nursery may or may not be related tothe different response to inoculation technique. One could50speculate that the A51 isolate had a short life expectancy onceit was released Into the environment, so intimate contact withthe roots might have Increased the Inoculation success.However, other explanations are possible, for example, thegrowing conditions near the surface of the plug may not havebeen suitable for Suillus tamentosus. More study is necessaryto answer this question.No significant differences in growth were evident in thespruce, even though there were differences in infection levels.Either application technique could be used, depending on theobjective of the trial. If the desire is to achieve maximumcolonization, then injection into the growing medium hasadvantages for some fungi, or in some as yet undefinedcircumstances. If the object is just to get fungi to grow onthe roots, and ease of application is the primary concern, thenthe inoculum might be top applied. Again, this is species-and/or circumstance—variable, and the limitations of bothtechniques need further exploration.one interesting result is that the injected treatments in pinehad smaller root mass, root collar diameters and shoot lengthsthan the top-applied treatments. These differences could not beattributed to one treatment. The means of the top—applied andinjected controls were grouped closest together for allmeasurements, except root collar diameter. The means of the A51injected and top-applied treatments were contiguously groupedfor shoot weight, but not for shoot length, root weight and rootcollar diameter. The most consistent source of the variationbetween injected and top—applied treatments seems to come from51the E—strain and fungal growing medium treatments, though theA51 treatment, in general, has spreads between the means of theinjected and top-applied treatments. The differences within thegrowing medium treatment seem to indicate that this is not afungus effect. The injected growing medium treatment was biggerthan the control, and the top—applied treatment was smaller thanthe control in shoot length and shoot mass measurements, but.reversed for the root collar diameter and root weightmeasurements. The fungal treatments followed the same patternof having larger shoot length and mass measurements and smallerroot collar diameters for the injected versus top—appliedtreatments. The fact that the inoculum was applied to bothtreatments, just in different manners, seems to suggest that itwas not just a nutrient effect. If it were a nutrient effect,one would have expected the same response in growth, regardlessof the application method. However, some dripping frequentlyoccurred from the bottoms of tubes that had the inoculuminjected and this could be related in some way to the effect.But, if it were an effect that was altered by dripping from thetubes, one might expect it to be more pronounced in thetop—applied treatment, where there was little dripping.However, in three out of four measurements, the top—appliedfungus growing medium treatment is more similar to the means ofthe controls than is the injected fungus growing mediumtreatment.Other possibilities are that the growing medium promoted thegrowth of some antagonistic organism, or that thecarbohydrate-rich growing medium encouraged the growth of52organisms that tied up nutrients, but that this effect somehowdid not occur when the inoculum was surface applied. This couldbe i:’ossible, if for example, the growing medium on the surfacedesiccated and did not promote the growth of organisms. It ispossible that the injected growing medium itself wasantagonistic to seedling roots, and/or that the injectionprocess was disruptive in some way such as the transmission ofdisease or disturbance of roots. The absence of an obviouslygreater similarity between the controls and either the topapplied or injected fungus growing medium treatments makesresolution of this puzzle almost impossible, without furtherstudy.536. A LOW-TOXICITY STAIN FOR EXAMININGECTOMYCORRHI ZAEIt is often useful to stain the fungal portion ofectomycorrhizae to facilitate observation of the mantle orHartig net. Microscopic examinations of fungi in root tissuecommonly use traditional stains, such as Trypan Blue and AnilineBlue, which are carcinogenic and/or teratogenic and/or toxic.When examining large numbers of roots on a daily basis, theexposure to dye can be quite high, even if precautions aretaken. The risk of exposure is particularly high when preparingdyes from powders or when heating slides to drive off bubbles orset stains. The safety procedures that should be used with suchstains can be time—consuming when shifting repeatedly from lowmagnification observation without stain to high powermagnification with stain. Some staining procedures such asclearing and staining (Phillips and Hayman, 1970) requireseveral steps and severely disturb the root, which makesextramatrical hyphae and rhizomorphs virtually useless forseparating types on the plug. The staining technique ofDaughtridge et al. (1986) uses Acid Red 112 as the dye andrequires less manipulation. It could probably be adapted foruse on intact plugs to take advantage of intact extramatricalhyphae and rhizomorphs, but the efficacy of the technique fordistinguishing different types is not evaluated, and it isprobable that the loss of colour would cause more dependence onmicroscopic characteristics. Also, Acid Red 112 is acarcinogen, toxic to breathe, ingest or touch and produces toxic54fumes on heating (Canadian Centre for Occupational Health andSafety, MSDS record no. 244572).As a safe, convenient alternative, we have successfully usedblue food colouring manufactured by Specialty Brands (65International Boulevard, Suite 206, Etobicoke, Ontario, M9W 6L9)to selectively stain the fungal portion of ectomycorrhizae. Thestain is fairly lightfast, and persists at least for severalmonths. It is readily available and consistent in quality,having to meet Food, Drug and Cosmetic Act (FD&C) requirementsand is also presumably quite benign, though Miller and Nicklin(1980) do cite some minor reactions to ingestion of the primarydye contained in this food colourant.The product used in this method is the blue dye in the BlueRibbon Food Colour Preparation. Discussions with themanufacturer indicate that the dye is >85% total colour, with<6% a subsidiary colour. The main colouring ingredient is knownas Brilliant Blue FCF or FD&C Blue No. 1. Its Colour Index is42090 and it is also commonly known to histologists aseriogluacine. Conn (1977) describes it as having similarstaining uses to those of Alphazurine A, C.I. 42080, but no usesfor Aiphazurine A are given. Staining Procedures (1973) alsodoes not list any uses for either dye.The dye is prepared for use in staining ectomycorrhizae bydilution in 70% lactic acid at a rate of about 20 drops of dyeper 5Oml of lactic acid. The whole mycorrhiza or thin sectionis placed in a drop of the staining solution on a slide. Theslide and stain are heated over an alcohol lamp until the lacticacid just about boils. This sets the stain and drives off55bubbles. The stain may be drawn off with absorbant paper. Thisis particularly useful if small amounts of wax are attached toto the thin section. The molten wax can be drawn off withoutoverly disturbing the sections. More stain, clean lactic acid,KOH solution or other mountants, can be put on the specimen.The stain colour is pH—dependent, so putting different pHsolutions on the specimen can change the colour from blue togreen. Slides may be preserved semi-permanently (for severalmonths) by sealing the edge of the coverslip with clearfingernail polish.This stain is useful for most unpigmented or lightly pigmentedfungi. The stain is not visible, or may not be taken up bydarkly pigmented fungi such as Cenococcurn geophilurn. There isalso some variablity in how lightly pigmented hyphae take up thestain. For example, Endogone seems to stain very brightly.This may be a useful characteristic for helping to distinguishfungi.Figures 12 and 13 show a fungal mantle stained with FDA BlueNo. 1. Figure 7 shows an E-strain Hartig net stained the sameway.The simplicity of the procedure and the negligible hazardassociated with the stain make this technique ideal for theresearcher examining large numbers of ectomycorrhizae in detail,for unsupervised student use and for general application wherespecial safety equipment is unavailable or inconvenient to use.567. EXPERIMENT THREETHE SELECTION AND EVALUATION OF ECTOMYCORRHIZAL FUNGITO ENHANCE PERFORMANCE OF SEEDLINGS PLANTED UNDERNORMAL REFORESTATION CONDITIONSIt has been adequately established that, in extremesituations, any mycorrhizae are better than no mycorrhizae(Marx, 1977). Danielson (1988) has countered with “some fungiare better than others” in reforestation situations. Thequestion that needs to be answered before mycorrhizal fungi canbe used for inoculation in normal reforestation situations is“which fungi?” Danielson (1988) points out that before we canask “which”, there are many unsolved mysteries such as: arethere truly early stage and late stage successional fungi andcan nursery fungi be selected on this basis, why can’t we getmany types of putatively late or multi-stage fungi to grow onnursery stock and do nursery fungi continue to offer advantageseven when field fungi colonize the roots. Other unknowns aresuggested in the Introduction. They include such things as: howspecific is ecotypic specificity, is the fungal inoculum leftover from a mature forest suitable for seedlings, do differentfungi become more active as environmental conditions change, howmuch colonization does it take to affect growth, can seedlingperformance be improved by introducing exotic fungi and how longdoes it take for a seedling to acquire the optimum fungalcomplement for a given site.57These unanswered questions suggest some areas that need to bebetter addressed in mycorrhizal research. It has beenestablished that ecotype variability exists (Trappe and Fogel,1977) but what is not clear is how important this variability isto seedling or tree performance. Part of the failure todetermine this may result from inadequate ecologicaldescriptions (see Literature Review). It is easy enough to saythat two ecosystems are different when they are greatlydifferent, but the manipulator of ectomycorrhizal fungi needs toknow how big a difference is relevant to the mycorrhizal fungus.A precise description of ecotype is needed in each outplantingtrial to resolve this question. Problems related to normalreforestation situations include such things as the inoculumpotential at normal sites, the suitability of residual fungalinoculum for seedlings and others as outlined above. It seemsclear that these questions will be answered only by conductingstudies in normal reforestation areas. Recommendations toconsider mycorrhizal inoculation in some extreme circumstancessuch as afforestation should not be mistaken to mean thatfurther studies at more normal sites are 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 typesof fungi normally inhabit a root and so on as outlined above,can be addressed only by careful observation and documentationof the mycorrhizal fungi in defined situations. Finally, thereare problems relating to the inability to isolate, culture andinoculate many fungi. Many questions will remain unanswered aslong we are dealing with only a small fraction of the diverse58population of ectomycorrhizal fungi.For the ectomycorrhizal inoculation component of this thesisresearch, a site was selected that typically had high seedlingmortality, but was within the normal range of conditionsencountered by mycorrhizal fungi in the area. The site ischaracterized in detail using the ecological classificationapproach of V. J. Kraiina as the basis for site description.The details of the approach are included in “A Guide to SiteIdentification and Interpretation for the Kamloops ForestRegion”, Land Management Handbook Number 23, February, 1990.This comprehensive biogeoclimatic classification system coversmany variables but overlooks the critical ectomycorrhizal fungalpopulation. Therefore, this study also includes a descriptionof the fungi present on seedling roots occurring naturally inthe study area. This type of information will serve as a goodguideline for applying the result5 of this study to othersituations.The fungi selected for evaluation in this study have beenderived from a variety of sources, in order to address what hasbeen demonstrated, and is suspected about ecosystem specificity.Fungi have been selected based on their presence on the roots ofseedlings in the study area, but the fungi used are not limitedby this criterion, since exotic (defined here as meaning from adistant location and therefore, most probably, from a situationwhere other site characteristics are different as well) fungiappear as likely to perform well outside their normal range, asin it (at least, at this early stage of investigation). Iwan Ho(1987) demonstrated that there can be significant within—species59variation in important physiological characteristics, betweenisolates from the field or from the nursery, so fungi were alsoisolated from the nursery. In addition, fungi were not selectedbased on their perceived ease of commercial application. Forexample, fungi used in this study vary from very fast to veryslow-growing. Seedlings outplanted in the field had heavy andlight colonization by the inoculated fungus. This study hasintentionally avoided applying previous conceptions of what agood fungal inoculum should be.Finally, included in the observations are detaileddescriptions of the behaviour of the fungi after outplanting.Even if significant seedling growth effects are not found, thistype of observation may be valuable in understanding thebehaviour and action of specific fungi in defined ecologicalsituations. Observations involving some destructive sampling inthis study were made after the possibly critical, first growingseason, but sufficient seedlings were planted so that severalmore years of observation could be made. It is hoped that thisapproach will serve as a model or protocol for futureoutplanting trials, so at least a basic minimum amount ofinformation that will contribute to the understanding of thebehaviour of ectomycorrhizal fungi can be gleaned from eachtrial, and the technology of mycorrhizal fungal inoculation willbe advanced in a systematic and comprehensible way.The issues of culturing and inoculation of fungi have beendealt with in Experiments One and Two of this thesis.60MATERIALS AND METHODSSite Selection.—The site presents a typical reforestationproblem for the area in that it is hot and relatively dry withseedlings sometimes suffering up to 70% mortality on similarsites (Gary Hunt, personal communication). The site was logged(1988) two years prior to planting but had received no specialtreatment such as burning or scarification.Site Description.— The nursery work was carried out at theHeffley Reforestation Centre and the outplanting site was in theShuswap Highlands, 8km northeast of Barriere (51.16’N_12O.08’W). Heffley Reforestation Centre is about 20km north ofKamloops and Barriere is about 36km north of Heffley Creek.As has been discussed in the Introduction and LiteratureReview, it seems that one of the most critical factors that mayaffect the interpretation of outplanting trials withectomycorrhizae is the description of the site where the funguscame from and where the outplanting trial was done. It may bethat forest sites are so diverse that they defy description on ascale large enough to be of any value in predicting the behviourof inoculated mycorrhizae. However, the British ColumbiaMinistry of Forests uses a hierarchical ecosystem classificationsystem that allows description to a very specific site level. Aswell, the system provides all the information necessary for manytypes of grosser comparisons. Classification is based on a61broad range of characteristics including vegetation, soil andgeographic characteristics. The area where the field study tookplace was recently mapped using a new version of the ecosystemclassification system used by the Ministry of Forests in thearea (Lloyd et al, 1990). In addition, the site was classifiedin detail by the author and Gary Hunt based on existing mappingand procedures as outlined in Lloyd et al, 1990. Theclassification procedures consist of determining such factors aselevation, aspect, slope, soil type, parent material, soiltexture, humus form, soil moisture regime, soil nutrient regime,soil pH, forest cover and types of understory plants. Based onthis information it is possible classify the site to the leveldescribed as the variant level. Once the site has beenclassified, it is possible to infer some other characterisics ofthe site such as general climate and supposed climax forest.The outplanting site was determined to fit into the categorydescribed as IDFdk2. The “IDF” refers to the zonalclassification, which is the coarsest category. IDF meansinterior Douglas—fir, which is the predominant climax foresttype in this zone. The “dk” refers to a subzone rating,specifically “dk” means dry cool. The “2” refers to the variantof the subzone and refers to a very specific mix of vegetation,soil moisture regime, aspect, successional stage, parentmaterial and soil nutrient regime. Thus, this classificationsystem can result in very site specific information about sites.If this information is not sufficient to describe sites for thepurpose of describing the behaviour of ectomycorrhizae, it isprobable that it will not be practical to do so.62Lloyd et al, 1990 indicate that the climate of the IDF iscontinental with warm dry summers and cold winters. Substantialmoisture deficits are common throughout the growing season.Frosts are common in June and late August. The “dk” subzone isabout in the middle of the IDF zone in terms of temperature andmoisture regimes and is very common in the study area. As such,it represents a normal reforestation situation for the area,even though seedling mortalities can be very high in thissubzone. The specific site description is shown in Table IV.TABLE IVDetailed Description of Outplanting SiteElevation— 1150mAspect- SWSlope— 5 to 18%Soil Type- Orthic Humo—Ferric Podzol (Canadian System of SoilClassification)Parent material— glacial tillMineralogy- mixedSoil Texture- sandy loamCoarse Fragment Content— 77% (average for top O.64m)Humus Form- F horizon about 2cm, H horizon about 15cm: Humusat site was disturbed by logging but wasdescribed about 150m from the site and was aHumi—fibrimor (after Bernier, 1968)Annual Precipitation (mm)— 568 (623—843)Growing Season Precipitation (mm)— 221 (185—313)63Annual Snowfall (cm)— 222 (178—264)Mean Annual Temperature (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 (-13.5-—1O.6)Growing Degree Days (>5C)— 1133 (953—1266)Frost Free Period (days)— 95 (62—132)Biogeoclimatic Zone- Cascade Dry Cool Interior Douglas-firVariant, Zonal Site (IDF dk2)Supposed Climax Forest- Douglas-firSoil Moisture Regime— Submesic, borderline subxericSoil Nutrient Regime- MediumSoil pH— Ae 6.0 (5.1 in CaCl,), Bf 5.2 (5.3 in CaCL.)(The Bflmeasurements were repeated four timeson two different pH meters and samepattern occurred each time)Observed Forest Cover— Predominantly lodgepole pine, small(109&) Engelmann spruce componentuphill from clearcut, increasingDouglas-fir downhillObserved Understory- pinegrass (20% of ground cover),kinnikinnick (8%), twinflower (8%),spirea (8%), saskatoon (5%), lupine(3%), Prince’s pine (1%), soopolallie(10%),thimbleberry (5%)64Observed Mycorrhizae On natural lodgepole pine seedlings (1to 5 years old): Suillus—like (60% ofobserved mycorrhizae), E—strain (30%),others (10%)On natural spruce seedlings (1 to 5years old): Arnphinema byssoides (50%),E—strain (30%), others (20%)The common names given here correspond to the scientific namesshown in Appendix 3 of Lloyd et al, 1990.The data in Table 1 were mostly collected using themethodology outlined in Lloyd et al. (1990). The humus formdescription is based on Bernier (1968) not Klinka et al. (1981).The climatic data is from Lloyd et al. (1990). The observationsof mycorrhizae on roots were made by digging up ten naturallyregenerated seedlings in the cutblock and along the edges ofroads in the immediate vicinity (within about lOOm for pine andwithin 2km for spruce) of the planting site. The seedlings wereexamined in the spring when the seedlings were planted (May 23,1990). The percentages shown are crude estimates. The pHanalyses were done as per the Forest Soils Manual (1987-88) byT. M. Ballard.Collection of Isolates.— To increase the odds of getting asuitable isolate, several collection strategies were used. Thefirst strategy was to collect sporocarps from similar sites in65the immediate vicinity (within a few kilometres) of theoutplanting site. Sporocarps were collected from under youngvigorously growing seedlings, both natural and planted and alsofrom mature stands. Unfortunately, the two seasons in whichsporocarps were collected were very dry and there was littlefruiting. This necessitated isolation from mycorrhizae. Again,young vigorously growing seedlings were selected from within theimmediate vicinity of the site to about 38km distant. However,all isolates were taken from the same or very nearly the samebiogeoclimatic zone and from similar slopes, nutrient andmoisture regimes. Isolations were also made from seedlings inthe nursery. The Heffley Reforestation Centre uses a growingregime modified to encourage mycorrhizal growth and this hasresulted in a wide variety of mycorrhizal fungi in the nursery(Hunt, 1989). Finally, isolates of fungi from other sourceswere tested. These included isolates from Dr. G. Hunt (HeffleyReforestation Centre Ltd., Site 10, Comp 10, RR#3, Kamloops, B.C.), Dr. R. M. Danielson (Kananaskis Centre for EnvironmentalResearch, University of Calgary, Calgary, Alberta) and L. D.Husted (Department of Forest Sciences, University of BritishColumbia, B. C.). Certain fungi were selected because they hadreceived a lot of attention from other researchers as goodpotential nursery inoculum so they were evaluated under theconditions in this study. The specific sources of the isolatesare shown in Appendix I.Isolation Techniques.— The isolation technique used forsporocarps was that of Molina and Palmer (1982). Initially,small pieces of sporocarp tissue were placed on MMN (Marx, 1969)66and potato/dextrose agar plates, both with tetracycline. Growthseemed to be consistently better on the MMN so thepotato/dextrose agar was dropped. Trials with and withouttetracycline revealed that bacterial contamination was not aconcern, so the inclusion of the antibiotic was later stopped.Several different techniques were tried for isolating frommycorrhizae. The techniques tried include those of Molina andPalmer (1982), Danielson, Griffiths and Parkinson (1984) andZak and Larsen (1978). These techniques are all quite similarand the procedure finally used is an amalgamation of some ofthese methods. Fairly large pieces of root were rinsed clean intap water. Then, approximately 15 short roots were cut off andplaced in a beaker with about 300ml of water and one drop ofTween or Sunlight dish detergent and stirred with a magneticstirrer for five minutes. The short roots were all removed andplaced in 95% ethanol. They remained in ethanol for a fewseconds up to about five minutes. Then the roots were placed in30% hydrogen peroxide for 5 to 45 seconds. The roots wereremoved at five second intervals. It was felt that treatingeach type of mycorrhiza for varying periods of time would reducethe risk of selecting against any particular fungus. The shortroots were rinsed in cold (3C) sterile distilled water for 15to 30 mm. and placed on MMN or PDA with streptomycin andBenomyl as per Danielson, Griffiths and Parkinson (1984) or onMMN and PDA without Benomyl. Initially, one root was placed perslant, but this was later changed to two roots per plate.Plates were easier to use for cleaning up colonies and observing67colony morphology. After making many isolates, the PDA wasdropped, and later the Benomyl and antibiotic were dropped.There did not seem to be a marked decrease in the success ofisolation without the biocides. Plates were poured as thicklyas possible to delay the effects of drying and wrapped withParafilm to reduce drying and contamination. Many of theectomycorrhizal fungi are extremely slow growing and somecontaminants can ruin the cultures over the long incubationperiod, so it is important to insure that plates are trulysterile. Some isolates grew only after as long as six weeks.Initially, plates had to be watched closely. Any spore-formingcolonies were cut out and discarded. Colonies that looked likepossible ectomycorrhizal fungi were transferred and if the newculture appeared pure, then the isolate was saved on plates,slants and in sterile distilled water under refrigeration, asdescribed by Marx and Daniel (1976). Some cultures remainedviable for at least three years using the sterile distilledwater technique. A slight modification was made to the methodin which cores were taken from the agar plates. A thin-walledhollow glass tube with a firm rubber bulb on one end was used tosuck up cores for transferring. A small stricture was placed inthe glass tube about 5mm above the cutting end to prevent theplugs from being pulled up too far. This is a useful tool fortransferring large numbers of plugs efficiently. Molina andPalmer (1982) note that the cold water storage technique doesnot work well for all fungi so the other two methodssupplemented this one. Plates with thick agar and wrapped withParafilm kept for several months, and served as a good source of68working material for starting cultures or for small scaleinoculation trials.Prescreening of Cultures.— Hutchison and Malloch (1988)suggested four criteria for determining which cultures isolatedfrom mycorrhizae might actually be mycorrhizal fungi. Theseincluded the absence of conidia, slow growth in culture, absenceof cellulase and the absence of pectinase. This guideline isnot foolproof, however, since some ectomycorrhizal fungi, e.g.Wilcoxina mikolae, grow quite rapidly and some ectomyeorrhizalfungi produce cellulase. Furthermore, the complete range incharacteristics of ectomycorrhizal fungi has not beendetermined. To avoid erring on the wrong side, cultures were tobe discarded only if they produced conidia, since this is themost reliable indicator of a non-mycorrhizal fungus. However,it became impossible to use all the isolates in pure culturesynthesis, so some of the faster growing Isolates were not used,and isolates that appeared to be duplicates were also not used.69Near—Pure Culture Synthesis.- After many trials, a new pureculture synthesis apparatus was designed. The base of thesystem was a five—inch clay pot filled with a 4 to 1 by volumepeat-vermiculite mixture. The hole in the bottom of the pot wassealed with silicone rubber sealant. The pots were filled withmoistened growing mixture, placed in Fisher autoclavable plasticbags and autoclaved for one hour. Look oven bags, which arevery thin plastic, were autoclaved separately for one hour.Seedlings were germinated aseptically using the method of Zak(as reported in Molina and Palmer, 1982). Seeds were surfacesterilized in 3096 hydrogen peroxide for times ranging from fiveminutes to one hour at five—minute intervals. Based onnon—quantified evaluation of the plates, it was decided thatabout 30 minutes of surface sterilization was optimum forlodgepole pine and Engelmann spruce. Longer times reducedgermination rates and shorter times resulted in highercontamination.When the pots had cooled, they were each planted in a laminarflow hood with three germinants and the open end of the Lookoven bag was attached to the lip of the pot with tape to form achamber (See Figure 1). The chamber was not sealed in thatmoisture and dissolved nutrient could pass through the porousclay pot and presumably, some gas could diffuse through theplastic bag and the pot. Careful monitoring was neededthroughout this procedure, as the growing medium was a goodsubstrate for molds. After planting, the pots were placed in a4cm deep tray. In later trials the tray depth was changed to70the same depth as the pots (13cm). The tray was made ofpolyethylene supported by a wooden frame. The pots weresubirrigateci by filling the tray with water. The pots wereplaced in a growth chamber, and on an open bench with additionalincandescent and fluorescent lighting. The day lengths were 16hours on the open bench and 21 hours in the growth chamber. Thetemperature in the growth chamber was 18C and the temperatureon the bench was ambient, which was typically around 2OC. Thetrees in the growth chamber grew much faster, and thephotoperiod for the bench seedlings was also increased to 21hours/day.The experiment tested 39 fungal isolates at three differentfertility levels. Only one tree species (lodgepole pine) wasused in the final trial, though spruce and pine were used inpractise runs. However, some isolates from spruce were testedin the nursery for their ability to form mycorrhizae. Threetrees were tested (one pot) at each fertilizer level, thus ninetrees per fungal isolate. The fertility levels were based on Nconcentration in the water used to flood the trays, and werelOOug/L, 5Oug/L and Oug/L of N. The initial fertilizationconsisted of urea two weeks after planting the seedlings,followed by another urea fertilization after three weeks. Afterthis, the plants were fertilized every two weeks with GreenleafShur Gro 20—20—20 water-soluble fertilizer. This is a fairlycomplete fertilizer that also has boron (0.O2), copper (0.O5),chelated iron (0.106), manganese (0.O5), molybdenum (0.0005%)and zinc (0.05%). The application rates were based on the N71rates already described. The fertilization procedure consistedof dissolving the appropriate amount of fertilizer in sufficientwater to fill the tray to the top. The tray was filled, allowedto stand for two days and then siphoned off. If the nutrientmedium was left too long, slime began to form on the pots.Watering was accomplished similarly, except tap water was usedinstead of nutrient solution. The pots were kept moist atfirst, but after two months, an attempt was made to increasemoisture stress by allowing the pots to dry more betweenwaterings. This was a mistake, as the dry peat moss becamehydrophobic and would rewet only very slowly. The plants didnot die, but some were nearly dead and did not recover by theend of the experiment. The growing medium was unsuited to thistype of manipulation. The peat—vermiculite mixture was chosenbecause it is used in the nursery where the next phase of theexperiment was conducted.The germinants were transplanted on October 3, 4 and 5, 1988.Eight weeks later, on December 5,6 and 8, 1988 the trees in thegrowth chamber were inoculated. By this time, the roots werequite—well developed, with a number of short roots. The treeson the open bench were inoculated from January 3 to January 5,1989, at about 12 weeks of age. The inoculum was grown usingthe dilute agar technique described in Experiment One. Theinoculum was prepared by treatment in a blender until it wasfine enough to pass an 18 gauge needle. The different culturesvaried in resistance to disintegration and the blendering timesvaried from 15 to 45 seconds. These were well within the timesdescribed by Boyle, Robertson and Salonius (1987) as having72little effect on inoculum viability. Attempts were made toseparate the growing medium from the fungus, but this provedvery difficult, especially since it was necessary to keep thecultures uncontaminated. The blender cup was sterilized byburning alcohol in the cup while rolling the cup in a laminarflow safety cabinet. The metal blender cup was sealed withpreviously sterilized aluminum foil. The mycelial slurry wassucked into a 5OmL syringe through an 18 gauge needle. Fillingthe syringe this way greatly reduced the likelihood of blockagesduring the inoculation procedure.To inoculate the seedlings, the syringe was simply pokedthrough the bag covering the pot and 2OmL of inoculum weresquirted into the growing medium (See Figure 2). The bag waswiped with 70 ethanol before injecting and the hole was sealedwith a piece of masking tape after inoculation. The medium andlow fertilizer treatments were reinoculated on February 10, 1989and the high fertility treatments were reinoeulated on February14, 1989. The seedlings were reinoculated because preliminaryexaminations revealed few mycorrhizae. The inoculationprocedure was changed slightly for the second inoculation inthat a sterile pipette was poked through a small slit in the bagand the inoculum was placed well down into the growing medium.This was done because most of the roots were located near thebottom of the pots. The pots received 5mL of inoculum thesecond time.The high—fertility treatments were harvested on March 23,1989, the medium—fertility treatments on March 24, 1989 and thelow-fertility treatments on March 28, 1989. The roots were73examined under a dissecting scope after rinsing and submergingthem in a tray of water. Any short roots that appearedmycorrhizal were hand sectioned after allowing the root to dryfor about 30 seconds and then embedding it in paraffin. Thepartial drying of the root was necessary to allow good adhesionto the paraffin. The root was embedded in paraffin by placingit on the end of a drop of paraffin that was stuck to amicroscope slide and then heating the paraffin around the rootwith a hot needle. The paraffin was cooled in cold water andlongitudinal sections were taken with a hand-sharpened scalpel.The sections were placed in a drop of stain (see Section 6) aridheated to melt any clinging paraffin. The melted paraffin wasdrawn off with a piece of absorbant paper and more stain orlactic acid was added before placing a coverslip over theslices. Longitudinal sections allow observation of the wholelength of the root to avoid missing small spots of colonizationas described by Danielson, Griffiths and Parkinson (1984).None of the general procedures of fixation, dehydration andclearing need be used for routine examination of ectomycorrhizaeby this method. Individual short roots are simply embedded inparaffin to immobilize them for sectioning. The whole processof sectioning and staining a root takes a few minutes and it ispossible to see most of the mantle views required by Agerer(1986) for complete description of ectomycorrhizae.Trial Inoculations in the Nursery.— Seven fungal isolates wereinoculated onto eight week old spruce seedlings, in the springof 1988. The isolates used were A18, A18B, A19, A20, A21, A22and A29 (Appendix I). Ten seedlings were inoculated with each74isolate. The inoculation procedure involved fragmenting a largecolony from an agar plate in a blender as per Danielson, Visserand Parkinson (1984), except that the colonies were chopped in aWaring blender and the mycelial suspensions were not centrifugedor washed. Ten millilitres of suspension were inoculated intothe plugs of each tree. The roots were examined on November 29,1988 when the seedlings were eight months old.Preparation of Inoculum for the Nursery.— The fungi selectedfor the nursery trials were grown using the dilute agartechnique described in Experiment One. The inoculum was grownfor different periods. Some of the isolates were started asearly as .January, 1989 because they were slow-growing. Severalof the slow—growing isolates were started well before the pureculture synthesis trials were complete. Cultures that becamecontaminated were discarded and replaced by new cultures, so thefinal inoculum used in all cases consisted of cultures ofvarious ages. It took a minimum of two months to prepareinoculum for these reasonably large scale inoculation trials.The cultures were grown in batches of dilute agar in 75OmL hotpack jars. The contents of several jars were fragmented in ablender and mixed together. The cultures were first fragmentedfor 30 seconds. This seemed more than adequate to break upcolonies in most cases, so time was reduced to 15 seconds,keeping in mind the work of Boyle, Robertson and Salonius (1987)which seemed to suggest that shorter fragmentation timesresulted in better inoculum. Fragmentation was done in a Waringblender (model 31BL92, Waring Products, New Hartford,Connecticut) operated at low speeds. Boyle, Robertson and75Salonius (1987) found good success with inoculations of 2?6 bywet weight of fungus to water. The concentrations of theslurries used were determined by taking a SOmL sample andwashing it with lOOmL of boiling water on a piece of filterpaper in a Buchner funnel with suction. The wet weight of thefilter paper had been previously determined by suctioning thefilter paper on the same apparatus until the free moisture lustdisappeared. The filter paper was weighed five times in thismanner and it was determined that 5OmL of sample would providesufficient mass to mask the moisture fluctuations in the wetpaper. The mycelial slurries were diluted to giveconcentrations in excess of those used by Boyle, Robertson andSalonius (1987).The inoculum was grown in 500mL or 600mL batches. There weredifferent numbers of batches of each fungus and theconcentrations of the fungus within each batch varied. Thebatches were fragmented, combined and then subsampled and washedwith boiling water to determine the concentration of fungus bywet weight. The inoculum was divided in half and the volume wasmade up with distilled water to 5L for the spruce and up to 4Lfor the pine. The dilutions with water were made lust beforeinoculation and were different for pine and spruce because therewere fewer pine seedlings. The final concentrations of theinoculum in percent wet weight of mycelium of total weight ofinoculum are shown in Table V.Most of the spruce seedlings were inlected on May 24 and May25, 1989. The Hecr 8 and A51 treatments were applied to thespruce on June 15, 1989. The inoculum was placed in storage at762C until June 27 and June 28, 1989, when the pine wereInoculated.TABLE VCONCENTRATIONS OF INOCULUM USEDIN THE NURSERY INOCULATION TRIALIsolate 9&mycelial conc. as wet weight/tot, weightA188-2 (Cenococcurn geophilurn): 3.36 spruce, 4.1% pineR947 (Danielson’s E—strain): 4.8% spruce, 6% pine0188 (Husted’s E—strain): 6.15% spruce, 7.89% pineA29 (Arnphinema byssoides): 4.8% spruce, 6% pineA51 (Suillus tornentosus): not measurecl* on spruce or pineHecr-8 (Hebeloma crustuliniforme): not measured on spruce or pine*_ Additional concentrate was added to these two isolates justbefore the trees were inoculated, and the mycelialconcentrations were not calculated. The concentrations wouldhave been the same order of magnitude as the other cultures.77At this time, some additional Hecr 8 and A51 cultures wereadded to freshen the older inoculurn. Little growth of mold orthe ectomycorrhizal fungi were evident in the inoculum afterstorage, even though sterile procedures were not followed inpreparing the dilute agar cultures for injection. There werejust a few small mold colonies in the E0188 culture.Inoculation of Seedlings in the Nursery.- Both the spruce andpine seedlings were inoculated with 5mL of slurry. The slurrywas applied with an Oxford pipetter (Model SA). A length oftubing connected the pipetter to a lmL pipette. The inoculumwas applied by (1) injecting the mycelium throughout the plug or(2) squirting the inoculum on top of the plug. In either case,the body of the pipetter was kept below the injector in order toavoid siphoning. The spruce were grown in used and unsterilizedPSB 313a styroblocks (198 cavities per block, 947 cavities/ma,Beaver Plastics, Edmonton, Alta.) and the pine were grown inused PSB 211 (240 cavities per block, 1130 cavities/m)styroblocks. The growing medium was peat and vermiculite (4.4:1v:v). Fertilizer was applied according to the schedule shown inAppendix III. This fertility regime has been shown by Hunt(1989) to increase the level of mycorrhizal colonization and thenumber of types of mycorrhizae on the roots over those usingslow release fertilizers. Other details of the growing regimecan be found in Hunt (1989). Both tree species were about eightweeks old at the time of inoculation and had started to developshort roots. The inoculum was applied to approximately 5078seedlings in each block, excluding the two rows of cavitiesaround the perimeter of the spruce blocks to reduce edgeeffects. There were fewer pine seedlings so only the two endrows and one side row were excluded. The exact number of treesinoculated was counted and recorded, and the inoculated area wasmarked with felt pen and plastic markers. Each inoculum wasapplied to 20 blocks for the spruce (10 blocks surface—appliedand 10 injected) and 16 blocks (eight blocks surface-applied andeight blocks injected) for the pine. One treatment was thefungus growing medium used to culture the fungi. This mediumwas diluted the same as the media with fungi and was alsoinjected or top applied. This treatment is referred to as thefungus growing medium treatment. There were two separatecontrols for the injected and top applied treatments, but theywere identical in that they both had no treatment done to them.The controls had the same number of trees as the othertreatments and they were marked off and counted in the samemanner and treated in the nursery in the same way. Afterinoculation, the blocks were placed in a randomized arrangement.Evaluation of Nursery Trials.- The seedlings were given apreliminary examination on September 14, 1989, and a sample of10 trees was taken from each block on November 3, 1989. Theobjective was to leave the trees in the nursery as late aspossible before examination, as there is some evidence thatmycorrhizal development proceeds most rapidly in the fall, afterbuds are set (Hunt, personal communication). During theNovember 3 sampling, the surviving trees in the treated areawere counted. Trees for mycorrhizal assessment were randomly79selected, based on their numerical position in the block and atable of 6/49 Lottery numbers. The remaining trees were wrappedin cellophane, put in boxes and placed in cold storage at 2C,according to standard operational storage procedures.The sample trees were placed in plastic bags and put instorage in a refrigerator at OC, from which they were examinedover the next several months. There was not any noticeabledeterioration in the quality of roots over this period. Theroots were initially examined by submerging the intact plug in atray of water and looking at it under the dissecting scope. Theroot was then washed and re-examined. Later, the roots were nolonger submerged, as it was difficult to see extramatricalhyphae when they were wet. The washing stage of observation wasdiscontinued, as this removed extramatrical hyphae andrhizomorphs, which made observations more difficult. Theestimation of the extent of colonization of each plug by thevarious fungi was dependent to a large extent on observations ofextramatrical hyphae. Since several of the fungi in thisexperiment (E-strain, Thelephora terrestris and Ainphinernabyssoides) have- quite indistinct mantle structure at lowmagnification and it was not feasible to examine each short rootunder high magnification, the extramatrical hyphae were used asindicators of the extent of colonization by certain fungi. Forexample, the hyphae of E-strain are thick, reddish—coloured andstiff (Figs. 5, 6 and 7). Even under low magnification (7x),E-strain mycorrhizae can be quite accurately separated fromthose of Thelephora terrestris and other fungi by the presence80of these hyphae. The hyphae of Arnphinerna byssoides are fine,pale yellow and tend to form dense mats. They can be seen quiteeasily when they are dry (Fig. 10) but lose much of theirdistinctness when wet.The fungal types were identified using a synthesis of manyapproaches. The general methodology consisted of identifyingapparent types from intact plugs at low magnification (7x).Minor uncertainties were dealt with by examining whole mounts inlactic acid for mantle and hyphal characteristics. Moredetailed evaluation of the root was accomplished using thelongitudinal section technique described earlier. Hundreds ofshort roots were examined microscopically, and this revealedthat it was not possible to separate the different types ofmycorrhizae with absolute confidence unless each short root wasexamined under high power. Even when examining sections, therewas ambiguity, as it was common for short roots to be infectedby more than one fungus. Roots tentatively identified underlow magnification as being non-mycorrhizal were examined verycarefully, as it was frequently found that rootlets withsloughing epidermal cells and root hairs had Hartig nets. Sinceit was not possible to examine each short root in detail, it wasfelt that the approach of relying on gross characteristics suchas extramatrical hyphal appearance, augmented by frequent highpower microscopic examination was as accurate an approach ascould be achieved on the scale of this experiment. Theparticular features of each fungal type identified are shown inAppendix II. The estimates of percentage colonization were madein increments of 10%, except for a spot colonization of for81example, around 10 mycorrhizae, which is described asOther observations made on seedlings included root collardiameter, shoot length, air dry shoot mass, air dry root massand an estimate of the relative number of short roots. Theobservation of the relative number of short roots is based on ascale of 1 to 3, with 1 being few and 3 being many. Determiningthe root weights presented a problem because some of the hyphae(especially Amphinema byssoides) bound the root so tightly thatit was difficult to get rid of all of the growing medium and thefungal hyphae. Several techniques were tried, including washingin a wide stream of water from a sink with much agitation,washing under a narrow jet, and drying and shaking (very poor).The technique that was finally used consisted of placing theroots on an approximately half-inch mesh and washing with thespray from a Clarke Little Laser power washer. The pressure wasset near the lowest setting and the nozzle was adiusted to aslight fan. This method stripped away all of the fungi andgrowing medium. It would also strip away the root cortex, ifused too vigorously. However, it appeared to be a satisfactorymethod overall. Counts were not made of the number of shortroots lost in this process, but individual short roots weremonitored during spraying, and it appeared that most remainedintact. The amount of root lost generally seemed to beconsiderably less than any technique which required manualmanipulation of the root.Two statistics were derived from the measurements taken.These were the root to shoot ratio, and the Dickson QualityIndex (Dickson, Leaf and Hosner, 1960 as cited in Hunt, 1989).82Both of these measures are used as indicators of seedlingmorphological balance. The Dickson Quality Index is calculatedas: dry weight/height—diameter ratio + shoot-root ratio.Outplanting Trials.- The outplanting trial was done in arandomized block design with five blocks. The three fungaltreatments that produced mycorrhizae in the nursery, the fungusgrowing medium treatment and the control were each representedby 30 seedlings per block. The outplanted seedlings for all ofthe treatments, except A51 (Suillus tomentosus), were selectedat random. The fungal treatments, except A51 (Suillustomentosus), all had very high levels of the inoculated fungus,since almost all the correspondingly treated trees were veryheavily colonized by E—strain and Amphinerna byssoides when theywere placed into cold storage. Trees were selected evenly fromboth the top applied and injected blocks. The A51 (Suillustoraentosus) treatment did not generate heavy infections in mostcases, and seedlings were selected for this group based just onthe presence or absence of the inoculated fungus. Six of theheavily infected A51 (Suillus tomentosus) trees were identifiedbefore outplanting so the behaviour of the fungus on thosespecific trees could be monitored. The trees were all markedwith double—faced write-on aluminum markers from Neville CrosbyInc. of Vancouver, B. C..The trees were planted within each block in a randomizedpattern generated by a double randomizing program written by me.The identity of each tree could therefore be determined by itstag and its position. The blocks were laid out such that onehalf of each block was pine and the other half spruce. The83trees were planted on an approximately lm’ grid with manydeviations for obstacles. The spruce were all planted by onetree planter, and the pine were all planted by another. Iplaced each tree in the approximate planting position, to insurethat the order was correct. The seedlings were planted on May 22and 23, 1990. On July 12, 1990, a mortality check wasconducted. It was assumed that trees dying within the first sixweeks were dead or near dead on planting.Examination of Seedlings From the Outplanting Trial. OnSeptember 13, 1990, the outplanted trees were measured for totalheight from the ground, incremental height, root collar diameterand survival. Miscellaneous forms of damage, such as brokentops and multiple tops, were noted. This work was carried outby Heffley Reforestation Centre employees under the supervisionof Hillary MacMillan. On October 12 and 13, 1990, four treesfrom each treatment in each block were selected at random andexhumed by Dr. T. M. Ballard and me, taking great care to keepthe root systems as intact as possible. Tags from dead treeswere collected and dead trees noted in the earlier fieldevaluation were confirmed by tag and position.The exhumed trees were examined in the laboratory, using muchthe same procedure as described earlier. The roots were notwashed before examination under the dissecting scope. They weregently washed, during examination, with a squirt bottle asrequired. The new roots could be distinguished because the plugshape could still be seen and it was possible to determine whichroots were growing away from the plug. This distinction could84not be made very clearly after the roots were washed. The typesof mycorrhizae and their relative proportion of the total newshort roots was determined. The new short roots consisted ofthose on roots egressing from the plug.The relative difference between the growth and types ofmycorrhizae on roots pointing down versus those growing from theside of the plugs were noted.The same weight of needles was stripped off the leader fromeach tree and combined by treatment for each block. The needleswere air dried and sent for analysis to Pacific Soil TestingInc. of Richmond, B. C. A standard sample of pine needles fromthe U. S. Department of Commerce National Bureau of Standardswas sent with the field material as a reference sample.Statistical Procedures.— The field plots were established in arandomized block design and the nursery trials were arranged ina nested design with styroblocks nested within treatment. Thefield trial experiment had five blocks, with four treatments perblock. There were 30 trees per treatment per block, but onlyfour trees per treatment per block were harvested for fullevaluation. The remaining trees will be examined in futureyears. There were two species of trees, but these were treatedas separate experiments.In the nursery trials, there were eight inoculation treatments(including the control) for each of the two tree species and theinoculum was applied by two methods. Ten blocks were used foreach combination of inoculum and application method in thespruce and approximately 50 trees were treated per block. Thepine was treated similarly, except only eight blocks were85treated per combination of inoculum and application method.Statistical analyses were done after consultation with MalcolmGreig of the Computing Science Department, U. B. C. and Dr. A.Kozak of the Faculty of Forestry, U. B. C. The prograril that wasused for most of the statistical analyses is called UBC Genlin:A General Least Squares Analysis of Variance Program by MalcolmGreig and James Bierring. This program was useful because itcould handle missing data and do multiple comparison tests forrandomized block and nested designs with more than oneindependent variable. Homogeneity of variance was tested usingBartlett’s and Layard’s tests. Normality was evaluated usingthe Kolmogorov-Smirnov test. The ANOVA tests are very robustwith regard to deviations from normality and homogeneity ofvariance. A. Kozak said that ANOVA was still applicable even ifBartlett values are very small. Malcolm Greig confirmed this.Zar (1984)(page 183) states: “Because of the poor performance oftests for variance homogeneity, and the robustness of analysisof variance for multisample testing among means, it is notrecommended that the former be performed as a test of theunderlying assumptions of the latter.” All analyses werechecked for homogeneity of variance and normality, but thesedata were not used to disregard ANOVA results. In the caseswhere the data for the randomized block ANOVA were non-normal,the analysis was also done with the Kruskal—Wallisnon-parametric ANOVA.Multiple comparisons were done using Tukey and Bonferronitests. Mortalities was also analysed using contingency tests onthe SAS statistical package.86RESULTSPure Culture Synthesis.- The pine trees grew reasonably wellat all three of the fertility levels used. All of theunfertilized seedlings developed a distinct purplish tinge. Theplants exhibited unusual growth characteristics in that theneedles were very long (up to 10cm) and the short roots werealmost entirely wye shaped, even when not mycorrhizal. Asmentioned previously, it was not possible to allow the high peatmoss growing medium to dry and stress the trees and then rewetit through the porous pots. There were 30 control seedlings andnone of them became mycorrhizal, so the system was tight enoughto stop contamination by ectomycorrhizal fungal spores in thesurrounding environment.Few isolates formed mycorrhizae, so it was not possible to sayanything about the effects of the fertilizer treatments on theability of the fungi to form mycorrhizae. No patterns wereobvious with the small number of infections that occurred. TheE—strain isolate from R. Danielson formed mycorrhizae mostvigorously. A Hebelorria crustuliniforme isolate obtained fromGary Hunt developed mantles on a few mycorrhizae, but only a fewroots were infected. The A51 isolate (Suillus tomentosus)formed unusual looking roots that were swollen and muchbranched, but the mantle, which is normally a major feature ofSuilloid mycorrhizae, was very weakly developed. This isconsistant with Harley and Smith (1983) where nutrient orphysical stresses are reported to cause poor mantle development,87but Hartig net may be normal.Two E—strain isolates formed mycorrhizae in the pure culturesynthesis technique, but the isolate from Danielson formedmycorrhizae much more vigorously than the E—strain 0188.B-strain 0188 came from the Harrop Nursery near Nelson, B. C.and appeared on agar to be very similar to Danielson’s isolatein colour, rate of growth and hyphal characteristics.Trial Inoculation in the Nursery.— Of the seven fungalisolates used in the nursery inoculation trial, only isolate A29(Amphinerna byssoides) formed mycorrhizae. It caused eachseedling inoculated to become virtually 100 colonized. Thiswas an isolate from the nursery. Other Arnphinema byssoidesisolates from the field did not form levels of colonizationthat were detectable against background levels of Amphinemabyssoides in the nursery.Observations from Inoculation of Nursery Seedlings.— On thelodgepole pine seedlings, the Cenococcum geophilum, Hebeloraacrustuliniforme, Amphinema byssoides and E-strain 0188 fromHarrop did not form any mycorrhizae and no further analyses weredone on these treatments. The results for the other treatmentsare shown in Table VI. The isolate A51 (Suillus tomentosus)which came from a sporocarp under a vigorously growing10—year-old planted tree formed mycorrhizae intermittently inthe injected treatment, and less in the surface appliedtreatment. Even though the level of colonization was low, therewas significantly (p<0.01) more Suillus tornentosus typemycorrhizae on the A51 treated seedlings than on any of theother treatments. The mean percentage of A51 (Suillus88Table VIObservations on Lodgepole Pine Seedlings Grown in theHeffley Reforestation Centre Nursery for 4 Months FollowingInoculation With Ectomycorrhizal Fungal Mycelium at 2 MonthsTreatmentParameter A51 E—strain Fungus Control pR947 Growing ANOVAMediumn= 146 160 149 144b <0.01E-strainmycorrhizae (96)Thelephorarnycorrhizae (96)Shoot mass (g)Shoot length (cm)Root Mass (g)Root to ShootRatioa b b0.030.300.580. 550.310.58The top number is the mean, the number underneath is theS. tomentus—likemycorrhizae (96)0.070.82<0. 01<0. 01<0.012.06 0.00 0.108.75 0.00 0.930.95 b 67.0 a 0.17 b 0.00 b5.98 24.2 1.22 0.0092.8 b 32.3 a 95.1 b 89.0 b13.0 24.4 10.6 14.8Total mycorrhizal 98.2 a 99.7 a 97.5 a 93.5 bcolonization (96) 8.17 3.95 9.58 13.40.98 0.87 0.88 0.870.29 0.27 0.30 0.3018.7 18.1 17.7 17.62.96 3.23 3.58 3.08Root Collar 3.02 2.99 3.11 3.06Diameter (mm) 0.51 0.47 0.47 0.520.52 0.49 0.53 0.540.17 0.16 0.19 0.18Abundance of 2.35 2.39 2.30 2.310.59 0.59 0.65 0.680.62 0.18 0.26 0.49Dickson Quality 0.19 0.18 0.19 0.19Index 0.07 0.06 0.07 0.070.90standard deviation and means with different letters weredetermined to be different based on Tukey’s and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.89tornentosus) type of mycorrhizae in the A51 treatment was 2.06%versus 0.07% for the fungus growing medium treatment. E-strainR947 formed mycorrhizae on virtually every tree inoculated,whether the inoculum was injected into the plug or top applied.The mean level of E—strain on blocks treated with Danielson’sE-strain R947 was 67%, including injected and top appliedtreatments. The mean level for the other treatments was 0.37%.There were significant differences between the injected and topapplied treatments, and these are discussed in Experiment Three.The control and A51 (Suillus tornentosus) treated seedlings wereall heavily infected with Thelephora terrestris (91% meancolonization rate). Thelephora terrestris colonized 32% of theshort roots of the E—strain treatment. The control treatmenthad a significantly (p<0.01) lower level (59o lower) of totalmycorrhizal colonization (sum of all fungi) than the othertreatments. The A51 (Suillus tonientosu,s) treatment (combinedinjected and top applied) had significantly greater shoot mass(13% larger on average) than all other treatments. The highermass was corroborated by the shoot length of the A51 treatment,which was greater on average, (but not significantly), than allother treatments. No other significant differences were foundin growth statistics or indices derived from them. There wasonly one marginally significant difference between the fungusgrowing medium treatment and the control and that was withregard to the total colonization by all fungi. The control was93.5% (std.dev. 13.4%) colonized and the fungus growing mediumtreatment was 97.5% (std.dev. 9.5%) colonized. There wererelatively few other fungi present on the roots, other than90those inoculated and Thelephora terrestris. There were lightinfections (O.36 mean) of Suilloid fungi, mainly with differentmorphology than the type inoculated (A51). The second mostdominant fungus next to Thelephora terrestris was Myceliumradicis atrovirens Melin (MRA—like), at an average colonizationof l.26. Tomentella-like fungi occupied O.28 of the totalshort roots and unknown types constituted O.119o. The MRA-likecolonization was questionably called mycorrhizal. It oftenformed a dense mantle—like layer but rarely had a distinctHartig net. The infections of volunteer fungi (fungi thatcolonize trees without benefit of inoculation) appeared grouped.That is, when a particular type of volunteer fungus was found,it was more likely to appear again in the same styroblock thanin the general population. The styroblocks had been usedpreviously and may have harbored inoculum. The seedlings wereculled during the growing season, so any dead, diseased ordeformed seedlings were removed from the block. There were nosignificant differences between treatments with regard to thenumber of seedlings lost over the growing season.On the spruce seedlings, the Danielson E—strain isolate andthe Amphinema byssoides isolate formed abundant mycorrhizae withvirtually every tree inoculated and the results are shown inTable VII. The other isolates did not form detectable levels ofcolonization. There were also high levels of volunteerArnphinerna byssoides in all of the treatments, but theinoculated trees had a much higher colonization rate, withapproximately 99% of the treated seedlings being infected withArnphinerna byssoldes versus 75% of the control seedlings. Themean91Table VIIObservations on Engelmann Spruce Seedlings Grown in theHeffley Reforestation Centre Nursery for 5 Months FollowingInoculation With Ectomycorrhizal Fungal Mycelium at 2 MonthsTreatmentParameter A51 E—strain Fungus Control pR947 Growing ANOVAMediumn= 200 200 200 190A. byssoides 68.5 a 9.12 b 28.8 c 26.1 c <0.01mycorrhizae () 23.6 19.7 27.4 27.7E—strain 0.75 b 64.7 a 0.55 b 1.35 b <0.01mycorrhizae (%) 4.47 29.6 4.28 7.61Thelephora 10.8 a 21.7 b 42.2 c 52.4 c <0.01mycorrhizae () 18.6 22.5 29.2 28.4Total mycorrhizal 80.1 a 96.3 b 72.0 a 80.5 <0.01colonization (6) 19.5 10.0 24.7 19.3Shoot mass (g) 1.68 a 1.47 b 1.60 a 1.62 a <0.010.38 0.37 0.39 0.36Shoot length (cm) 16.3 a 15.0 b 15.3 15.5 0.023.64 3.54 3.70 3.32Root Collar 3.36 3.27 3.36 3.31 0.38Diameter (mm) 0.40 0.43 0.39 0.39Root Mass (g) 0.93 0.88 0.98 0.90 0.120.29 0.27 0.27 0.27Abundance of 2.68 2.63 2.58 2.47 0.11Mycorrhizae 0.54 0.57 0.66 0.61Root to Shoot 0.58 0.61 0.64 0.57 0.11Ratio 0.22 0.17 0.19 0.17Dickson Quality 0.40 0.39 0.43 0.40 0.16Index 0.12 0.12 0.12 0.12The top number is the mean, the number underneath is thestandard deviation and means with different letters weredetermined to be different based on Tukey’s and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.92percentage of roots infected by Arnphinema byssoides was 68% inthe Arnphinerna bys3oides treated trees and 27 .5% mean for thecontrol and fungus growing medium treatments. The difference inthe percentage of infected roots was significant (p<O.Ol). Themean number of roots infected with E—strain in the E—strain R947treatment was 716 versus O.79& mean level of volunteer E--strainfor the other three treatments. The total level of colonization(sum of all types) was significantly (p<O.Ol) higher (24%) inthe E—strain R947 treatment than for the other treatments.There were significant differences between the injected and topapplied treatments, and these are discussed in Experiment Three.The seedlings treated with Arnphinema byssoides had the largesttops and were significantly taller and heavier than thosetreated with E-strain R947. The Amphinema byssoides treatedseedlings were 8% taller, and 7.5% heavier than the average ofthe other three treatments. There were no other significantdifferences between measured statistics or the indices derivedfrom them.There were high levels of Thelephora terrestris colonizationin most of the treatments, though the levels were not as high asin the pine. The mean level of Thelephora terrestris was 31%.The mean level in the two treatments with introduced fungi was16.2% and the mean level in the control and treatment withfungus growing medium was 76%. The differences were significant(p<O.O1). There were several other types of volunteer fungi onthe spruce. E—strain appeared on about 0.9% of the roots otherthan those inoculated with E-strain. Other types, includingMRA—like, Tomentella—like and unknown, constituted only 0.2% of93the total mycorrhizae. Detailed descriptions of the differenttypes are given in Appendix II. The infections by these fungiwere too sporadic to be able to detect trends in grouping. Ahigher proportion (18) of the spruce roots was non-mycorrhizalthan the pine roots. A typical colonization pattern with thenon—inoculated roots was to have Amphinema byssoides orThelephora terrestris growing on the bottom of the plug with asmaller area of non-mycorrhizal roots near the top, where theremight be a small colonization of some other species of fungus.There were no significant differences between treatments withregard to the number of seedlings lost over the growing season.94Observations on Seedlings After One Season in the Field.— OnJuly 12, 1990 (about seven weeks after planting), the trees weregiven a preliminary examination to try to distinguish whichmight have been dead on planting. The early part of the growingseason was very wet and only one tree, a pine control, was dead.There is an Environment Canada weather station at Barriere,which is eight kilometres southwest of the study area. Therainfall in Barriere was 2.84 times higher (95.9mm) than normalin May, 1990 and 2.5 times higher (101.9mm) than normal in June,1990. In July, August and September, 1990, the mean dailytemperatures were 6.9%, 9.3% and 11.0% higher than normal,respectively. In September the precipitation was 8mm or 25% ofnormal. Mortalities rose considerably by the time the seedlingswere harvested and this may have been related to the hot dryweather during late summer.Most of the parameters were measured on all outplantedseedlings about 15 weeks after outplanting (September 13, 1990)and these results are shown in Table VIII for lodgepole pine andTable XII for Engelmann spruce. Observations of mycorrhizae andmass measurements were gathered from seedlings collected about19 weeks after outplanting (October 12 and 13, 1990) and theseare shown in Table IX for lodgepole pine and Table XIII forEngelmann spruce.The majority of the roots on both pine and spruce seedlingstypically grew straight down from the plug with only a smallproportion (about 10%) of the roots growing from the side of theplug. There were no long lateral roots growing horizontallyfrom any plug. At most, there were individual short roots or95small bunches of short roots growing in cauliflower—like clumpson the sides of the plug. The clumps of roots growing from theside often had one type of fungal colonization and this wasoften different from the predominant colonization on the rootsgrowing down.Observations on Pine Seedlings. The root collar diameter ofthe E-strain R947 treatment was significantly (p<0.0l) smaller(6% difference between means) than the control. The A51(Suillus tomentosus) treatment was significantly (p=.03) taller(6% difference between means) than the control (Table VIII).Table VIIIObservations on Lodgepole Pine Seedlings Inoculated WithEctomycorrhizae in the Nursery and Grown For 5 Monthsin the Field (Field Measurements)TreatmentParameter A51 E-strain Fungus Control pR947 Growing ANOVAMediumn= 120 142 143 145Incremental 10.8 10.14 10.4 9.91 0.14Height (cm) 2.98 3.16 3.31 3.60Total height (cm) 29.1 a 28.5 28.7 27.4 b 0.035.00 5.26 4.85 5.03Root Collar 4.02 3.94 a 4.04 4.18 b <0.01Diameter (mm) 0.66 0.66 0.70 0.67Proportion Living 0.96 0.96 0.94 0.97 0.680.20 0.20 0.24 0.18The top number is the mean, the number underneath is thestandard deviation and means with different letters weredetermined to be different based on Tukey’s and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.96It should be noted that there were significant differencesbetween blocks for every parameter measured. The blocks wereadjacent to one another and quite similar, yet they wereapparently different enough to cause more differences in growththan the fungal treatments. Few lodgepole pine seedlings haddied by the time the seedlings were exhumed (Table VIII). FiveA51 (Suillus tomentosus) treated, six E—strain R947 treated,five fungus growing medium and nine controls died. Trees werecounted as dead only if they had a complete absence of greenneedles.Thirteen different types of ectomycorrhizae were found on thepine roots, with an average of 2.75 types per tree. The numberof types per tree ranged from zero to five. The roots with zerotypes had no discernible root growth from the plug, even thoughthe tree tops appeared live and well in most of these cases.There were significant differences between the treatments in thenumbers of fungi per tree. The fungal growing medium treatmenthad significantly (p=0.04) more types of ectomycorrhizae (3.05types/tree vs. 2.20 types/tree) than the control (Table IX).The A51 treatment was virtually identical to the fungus growingmedium with regard to the number of types per tree, but notsignificantly different from the control. The E—strain R947treatment was halfway between the control and the fungus growingmedium in the number of ectomycorrhiza types on the root.The behaviour of the inoculated fungi was extremely variable.In many cases, mycorrhizae formed with the A51 (Suillustornentosus) treatment appeared to have died after outplanting.There were often large patches of dead roots on the plugs, but97Table IXObservations on Lodgepole Pine Seedlings Inoculated WithEctomycorrhizae in the Nursery and Grown For 5 Monthsin the Field (Laboratory Measurements)TreatmentParameter A51 E-strain Fungus Control pR947 Growing ANOVAMediumn= 20 20 20 20Number of Types of 3.00 2.70 3.05 a 2.20 b 0.04Mycorrhizae/tree 1.17 0.98 1.00 1.06Shoot mass (g) 2.38 a 2.11 b 2.18 b 1.80 b 0.060.67 0.84 0.62 0.55Root Mass (g) 1.15 1.03 1.16 1.14 0.740.32 0.41 0.43 0.43Root to Shoot 0.50 0.52 0.56 0.66 0.31Ratio 0.14 0.19 0.30 0.23The top number is the mean, the number underneath is thestandard deviation and means with different letters weredetermined to be different based on Tukeys and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.these were rarely present on the other treatments. In the caseswhere patches of root were dead, the Suilloid types were oftenmissing, which suggests that the dead patches may have beencolonized by Suilloid types. However, there were also instanceswhere Suilloid types were growing from the roots. ThreeSuilloid types were identified on the egressed roots, and oneof these (type 10) (Fig. 9) was the most common volunteer fungus(other than Thelepl-iora terrestris, which is discussed below)with an average colonization level of 7.44&. Another Suilloidtype (type 4) was the second most common volunteer, with an98colonization level of 4.69%. The third Suilloid type (type 12)(Fig. 8) was on 1.75% of the egressed roots (mean of alltreatments) and was indistinguishable from the inoculatedfungus. It was not possible to determine, in the few caseswhere this fungus was growing on the inoculated seedlings, if itoriginated from the field. However, in at least one case, theSuilloid type fungus on the plug was growing vigorously andappeared to be growing onto egressed roots.The situation was different with E-strain, in that every treethat had been inoculated with E-strain had E-strain colonizingegressed roots. The levels of colonization by E—strain on theegressed roots (side and bottom) varied from 20 to 100%. Thevolunteer level of colonization for E—strain types, on all otherTable XVolunteer Fungi on Egressed Roots ofLodgepole Pine Grown in the Field For Five MonthsWith Different Mycorrhizal TreatmentsFungal Species Percent Infection ofEgressed RootsThelephora terrestris (Types 2 and 11) 69.83(mean excludes E-strairi treatment)Suilloid (Type 10) 7.44Suilloid (Type 4) 4.69Suillous tomentosus (Type 12) 1.75E—strain (mean only includes 3.83uninoculated treatments)MRA (Type 1) 3.69Type 3 3.06Tomentella-like (Type 8) 2.0099treatments, was 3.836. There were a few other types of fungiwith a similar level of colonization, Type 1 (MRA—3.69%), Type 3(oil drop type—3.06%) and Type 8 (Tomentella—like--2%). Thelevels of volunteer fungi on pine are shown in Table X.The fungus that had the highest overall level of colonizationon egressed pine roots was Thelephora terrestris-like. In manycases, the Thelephora terrestris—like fungus from the nurseryappeared to be healthy on the plug and to be growing directlyonto the egressed roots (both side and bottom). In other cases,discrete colonies of Thelephora were separated by zones ofnon—infected roots from the plug and the nursery Thelephora(Fig. 15). In 5 out of 40 cases (the 40 cases consisting of thecontrol and fungus growing medium treatment), the Thelephorafrom the nursery was not growing onto egressed roots. Inaddition, constant differences appeared in the morphology of theThelephora found on the egressed roots, to the point that theywere described as two different types. One type, which appearedon only new roots, formed longer mycorrhizae that werefrequently covered with white blotches of cystidia. The othertype formed shorter mycorrhizae and rarely had the whiteblotches (though cystidia were still present). If the long typewas a separate type of Thelephora from that found in thenursery, then it was by far the most common type of volunteerfungus, with an average colonization level of 16.25. All ofthe fungi found and the levels of colonization are described inAppendix II.No significant differences were found in the nutrient levelsin the tissue samples. The results are shown in Table XI.100Table XIFoliar Nutrient Analyses ForLodgepole Pine Grown in the Field for Five MonthsWith Different Mycorrhizal TreatmentsPhosphorus (6) 0.200.020.250.06Magnesium (6) 0.130.020.640.07Fe (uglg) 87.426.8Manganese (ug/g)Boron (ug/g)1.63 1.74 1.600.21 0.28 0.270.20 0.22 0.20 0.280.02 0.02 0.010.29 0.26 0.26 0.760.04 0.06 0.050.13 0.14 0.12 0.300.01 0.02 0.020.67 0.72 0.66 0.270.05 0.08 0.075.20 5.60 6.20 5.80 0.680.45 1.14 1.79 0.8454.0 46.2 57.6 53.0 0.087.84 4.09 12.7 7.2592.6 92.2 86.6 0.8826.0 22.3 14.0294 327 328 328 0.8119 66 86 6811.8 12.6 13.8 14.4 0.412.28 1.95 3.03 5.08The top number is the mean, the number underneath is thestandard deviation and means with different letters weredetermined to be different based on Tukey’s and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.TreatmentNutrient A51 E-strain Fungus Control pR947 Growing ANOVAMediumn= 5 5 5 5Total Nitrogen () 0.631.630.19Calcium ()Potassium (9)Copper (ug/g)Zinc (ug/g)101Observations on Spruce Seedlings. The mortality of the spruceseedlings was much higher than of the pine, averaging 23.5%.The mortalities varied significantly (p<0.O1) by block from 8 to50%. The mortalities varied by treatment from 21 to 27% (TableXII) and were not significantly different. The E—strain R947treatment had significantly (p<0.Ol) smaller (6.4%) root collardiameter (Table XII) than the fungus growing medium treatmentand control.The E-strain R947 and A29 (Amphinema byssoides) treatments hadsignificantly (p<O.O1) smaller (12%) height increments (TableXII) than the fungus growing medium and the control. Seedlingsinoculated with Arnphinerna byssoides were the tallest andsignificantly (p=0.025) taller (6.5%) than the E—strain R947treatment (Table XII). All of the parameters measured weresignificantly different by block. The root mass of the fungusgrowing medium was significantly greater than the control (TableXIII). The E-strain had the greatest root to shoot ratio and itwas nearly significantly different (p=O.O6) (Table XIII). TheA29 treatment (Amphinema byssoide5) had the greatest shoot massthough the difference was not quite significant (p=O.O9). TheA29 treatment also had one of the smaller root to shoot ratios,which was in keeping with its larger top.102Table XIIObservations on Engelmann Spruce Seedlings Inoculated WithEctomycorrhizae in the Nursery and Grown For 5 Monthsin the Field (Field Measurements)TreatmentParameter A29 E-strain Fungus Control pR947 Growing ANOVAMediumn= 121 125 116 122Incremental 11.0 a 10.72 a 12.2 b 12.5 b <0.01Height (cm) 3.68 3.30 2.89 3.00Total Height (cm) 26.2 a 24.6 b 25.7 26.0 0.035.00 5.26 4.85 5.03Root Collar 4.47 4.32 a 4.63 b 4.61 0.06Diameter (mm) 0.80 0.82 0.85 0.81Proportion Alive 0.78 0.77 0.79 0.73 0.570.42 0.42 0.41 0.46The top number is the mean, the number underneath is thestandard deviation and means with different letters weredetermined to be different based on Tukey’s and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.Nine different types of fungi were identified on egressedspruce roots. The mean number of types overall was 1.69/tree.There were significant (p=O.0176) differences in the number oftypes of fungi present by treatment. The E—strain R947 and theAmphinerna byssoides treatments both had 1.4 types/tree versus 2.0 types/tree for control and fungus growing medium treatments(Table XIII). The number of types per plant ranged from zero tofour; in four cases no root growth occurred, even though thetops looked alive.103Table XIIIObservations on Engelmann Spruce Seedlings Inoculated WithEctomycorrhizae in the Nursery and Grown For 5 Monthsin the Field (Laboratory Meaurements)TreatmentParameter A29 E-strain Fungus Control pR947 Growing ANOVAMediumn= 20 20 20 20Number of Types of 1.35 a 1.40 a 2.05 b 1.95 b 0.02Mycorrhizae/tree 0.75 0.67 0.94 1.10Shoot mass (g) 3.85 3.12 3.77 3.57 0.090.92 0.70 1.03 1.15Root Mass (g) 1.89 1.68 2.09 a 1.52 b 0.040.69 0.41 0.90 0.53Root to Shoot 0.49 0.63 0.56 0.44 0.06Ratio 0.14 0.35 0.21 0.14The top number is the mean, the number underneath is thestandard deviation and means with different letters weredetermined to be different based on Tukey’s and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.The mean level of E-strain colonization on new roots of theE-strain R947 treatment was 886. The mean level of E-strain onthe other treatments was 14.5&. The mean level of Amphinemaby,ssoides on the new roots of the Amphinerna byssoides treatedtrees was 84, while the mean for the other treatments was 15.These two fungi were by far the most common volunteer fungi(other than Thelephora). All the other types are present onfewer than 1% of the roots. Only 4.38 of the new short rootswere non—mycorrhizal. The types of fungi and the statistics onthem are summarized in Appendix II. The levels of volunteerfungi are shown in Table XIV.104Table XIVvolunteer Fungi on Egressed Roots otEngelmann Spruce Grown in the Field For Five MonthsWith Different Mycorrhizal TreatmentsFungal Species Percent Infection ofEgressed RootsThelephora terrestris (Type 2) 43.38(mean excludes the A. byssoides andE—strain treatments)Amphirieriia byssoldes (Type 3) 20.89(mean excludes the A. byssoides andE—strain treatments)E—strain (uninoculated treatments) 14.50Other Types <1.0The mean level of Thelephora on the egressed roots of thecontrol and fungus growing medium treatment was 439o and the meanlevel on the E-strain and Amphirserna byssoides treatments was 3.5%. Seven (out of 41) of the control and fungus growing mediumtreatments did not have any Thelephora on the egressed roots.All of the Danielson E-strain treated trees had E—strain growingon the new roots and all (except one, where a large piece of theroot was missing) of the Ainphineina byssoides treated trees hadAmphinema byssoides growing on the new roots. The level ofE—strain on new roots of E—strain treated trees ranged from 40to 100%. The level of Arnphinema byssoides on new roots ofArnphinema byssoides treated trees ranged from 30 to 100%.There was one significant difference in tissue nutrient leveland this is shown in Table XV. The control had a significantly105(p=O.02) lower calcium level than the E—strain treatment.Table XIIFoliar Nutrient AnalysesEngelmann Spruce Grown in the Field For Five MonthsWith Different Mycorrhizal TreatmentsTreatmentNutrient A29 E—strain Fungus Control pR947 Growing ANOVAMediumn= 5 5 5 5Total Nitrogen (p6) 0.441.18 1.31 1.29 1.230.14 0.16 0.19 0.10Phosphorus (6) 0.21 0.24 0.23 0.200.02 0.01 0.04 0.01Calcium (%) 0.32 0.41 a 0.38 0.290.04 0.06 0.09 0.03Magnesium () 0.12 0.13 0.14 0.110.01 0.02 0.02 0.01Potassium (%) 0.80 0.83 0.84 0.760.09 0.05 0.16 0.09Copper (ug/g) 4.60 5.20 5.00 5.000.55 0.45 0.00 1.00Zinc (ug/g) 40.8 43.6 46.2 39.64.97 5.13 7.79 8.26Fe (ug/g) 71.0 105.2 82.2 87.819.3 40.3 12.8 30.8Manganese (ug/g) 262 327 272 26734 48 73 58Boron (ug/g) 11.4 12.8 11.0 10.61.52 1.48 2.45 1.520 . 11b 0.020.090.670.370.500.260.260.22The top number is the mean, the number underneath is thestandard deviation and means with different letters weredetermined to be different based on Tukey’s and Bonferonni’stests with p= 0.05. The p value in the table is from ANOVA.106DISCUSSIONNear-Pure Culture Synthesis Technique.- Many different pureculture synthesis techniques have been developed to studyectomycorrhizae. A fairly rigorous isolation procedure Isneeded to keep any growing system from aerial sporecontamination, which can be a major problem in or near forestedareas.. Melin (as cited in Molina and Palmer, 1982) was thefirst person to use the quite simple approach of growingseedlings in a flask. Hacskaylo (1973), Marx and Zak (1965),and Molina (1979b) all added modifications to this technique.Also, several other, more radical, techniques such as synthesisin solid agar (Pachlewski and Pachlewska as cited in Molina andPalmer, 1982), in growth pouches (Grenville, Peterson andAshford, 1986> and in petri plates (Wong and Fortin, 1988) havebeen developed. Hatch (1937) anticipated the problems with thetechniques mentioned here when he identified the shortcomings ofpure culture synthesis apparatus as: “(l)excessive humidities...• (2)increased partial pressures of carbon dioxide....(3)accumulation of products (in some cases toxic)....(4)saturated substrates.... (5)10w radiation intensitiesThese abnormal growing conditions led Molina and Palmer (1982)to remark that positive synthesis results confirm the ability ofthe fungus to produce mycorrhizae but negative results areinconclusive. Melin made several elaborate apparatus thatovercame many of these problems, but as noted by Molina andPalmer (1982), the more complex the apparatus, the moredifficult it is to keep it intact. Also,107when evaluating large numbers of fungi, it becomes logisticallydifficult to set up many such devices. There is ample evidencethat some fungi which might grow in the nursery might not growin the unusual conditions found in most pure culture synthesisdevices. Therefore, some effort was put into developing a newpure culture synthesis device.The pure culture synthesis technique developed was not veryuseful for predicting which fungi might grow well in thenursery. Of the five fungi that formed mycorrhizae in the pureculture synthesis, only two formed raycorrhizae in the nursery.Two E—strain isolates were used in the pure culture synthesistrials and one formed mycorrhizae vigorously while the other wasmuch less vigorous. This behaviour was paralleled in thenursery study, where the vigorous isolate from the pure culturesynthesis produced many rnycorrhizae and the other produced none.The E-strain cultures appeared very similar with acharacteristic fuzzy appearance and unique large diameter,ornamented hyphae. The fact that one isolate grew well and theother did not may illustrate ecotype (or possibly species, sinceone E—strain isolate was not identified to species level)variation.One weakness of the pure culture synthesis technique forscreening fungi for use in outplanting trials, was that some ofthe fungi which did not form mycorrhizae were almost certainlycapable of doing so under some conditions. The weak developmentof the mycorrhizae suggested that the seedlings were not allowedto grow long enough after inoculation. The seedlings grew foronly about 2.5 months after inoculation. Molina (1979) grew108seedlings for six months under fluorescent lights, beforeevaluating their mycorrhlzae. The lack of colonization by someisolates may also have resulted, in part, from difficulties inregulating moisture levels and poor gas exchange through theplastic bag cover and porous pot. The dichotomous branching ofnori-mycorrhizal roots suggested a build—up of gases likeethylene. Solution of this problem was one of the mainobjectives of developing a new synthesis technique. Thetechnique used in this experiment has been improved by theaddition of glass cover with a membrane filter, without losingsome of its advantages. The advantages include low cost, easymaintenance (watering, fertilizing and injecting), littlerequirement for infrastructure and good protection againstcontamination.Peat moss was a particularly poor growing medium in thissystem because of its hydrophobicity problems. Peat moss wasused in an attempt to mimic conditions in the nursery, but giventhe other huge variances between the systems, using peat mosswas a futile gesture. It seems inescapable that the bestenvironment for screening fungi is the environment in which theywill be grown. The Arnphinerna byssoides isolate selected fromthe nursery inoculation trial formed mycorrhizae very well, sothere is at least some evidence that the nursery might serve asa suitable screening ground for ectomycorrhizal fungi.In spite of its shortcomings, the pure culture synthesis wasused as a major criterion for selecting the fungi for thenursery trial. Four of the five isolates used in the nurserytrial formed mycorrhizae in the pure culture synthesis109(Amphinema byssoides did not). Most of the fungal isolates usedin this experiment were previously untested for theirmyeorrhiza—forming ability. A limited number of trees in thenursery was available for inoculation and some of the fungalisolates were very difficult to grow, so it was felt that at thevery least, the fungi used in the nursery trial should be provenmycorrhizal fungi.Nursery Trials.- The attempts to inoculate fungi in thenursery using a new type of inoculum were successful with threetypes of fungi CE-strain, Amphinerna byssoides and Suillustomentosus), and highly successful for two of those (Amphinernabyssoides and E-strain). The inoculum was not washed free ofthe nutrients in the growing medium, yet successful inoculationswere achieved. This contradicts dogma with regard to vegetativeinoculation, which suggests that the nutrients should beremoved. More research is needed to clarify how many fungalspecies can be handled this way, if the timing of application iscritical and so on. Danielson (1988) has previously identifiedE—strain as a fungus capable of surviving well over the timebetween inoculation and infection. A list of fungi with thischaracteristic would seem to have some use.With Amphinerna byssoides and E-strain, mycorrhizae were formedwhen the medium was injected into the plug or top applied. Twotypes of fungi that have received a lot of attention as goodpotential commercial inoculants, Hebelorna crustuliniforme andCenococcurn geophilurn, failed to form mycorrhizae in the nursery.This confirms the lack of success in attempts by G. Hunt(personal communication) to get these fungi to form mycorrhizae110in the nursery using other inoculation techniques. Boyle,Robertson and Salonius (1987) also failed to get Cenococcwgeophilurn to form mycorrhizae in a container nursery, eventhough mycorrhizae were formed in growth pouch assays.Cenococcum geophilurn is reputed to do well on hot dry sites, butthe container nursery may be a major barrier to using thisfungus on seedlings destined for such sites.On the pine, the A51 treatment (Suillus tomentosus) formedvery few mycorrhizae, yet this treatment had significantlyheavier shoot weights than other treatments. Stenstrom and Ek(1990) reported similar effects with low levels of colonization(but not this low> by Amanita musearia (L.: Fr.) Hooker,Lactarius rufus (Scop.) Fr., and Tricholoma albobrunneum (Pers.Fr.> Kummer. This illustrates the peril of placing too muchemphasis on percent colonization. Significant alterations ingrowth can be brought about by apparently insignificant levelsof mycorrhizal colonization. Danielson (1988) has also reportedlarge fruitings of Suillus sporocarps under trees with fewSuilloid mycorrhizae. This may suggest several things, such asthe possibility that some Suillus species do not need to formlarge infections to fulfill their role. It may also be that notall of the Suillus mycorrhizae, on a single root system, formthe characteristic Suilloid mycorrhiza.Danielson, Visser and Parkinson (1984) and Molina (1981) bothreported failure with attempts to inoculate Suillus tornentosusand other Suillus species into container nurseries. The Heffleynursery did have a modified growing regime, but even with that,colonization was poor. This work suggests that successful111inoculation with Suillus may not be impossible, just difficult,and that it may be worth pursuing further. It may also be thatapparently low levels of colonization with Suillus fungi are allthat are possible and needed.For the spruce seedlings, the Arnphinerna byssoicIes treatmentresulted in seedlings that were tallest and significantly tallerthan the Danielson E—strain treatment, while the DanielsonEstrain trees had the lightest shoots and were significantlysmaller than the other treatments. The Arnphinema byssoidestreated seedlings had the heaviest shoots and these weresignificantly heavier than the E—strain treatment. Thisconfirms the findings of Husted (1990) but contradicts thefindings of Danielson and Visser (1988). Both of these studiesused the same isolate of E-strain, but neither was done in anormal reforestation situation. The Rusted study involved whitespruce grown in a growth cabinet and the Danielson and Visserstudy involved jack pine planted on oilsands tailings. Thomasand Jackson (1983) also reported that E—strain reduced thegrowth of Sitka spruce seedlings in the nursery and the field,but the field was a partially sterilized seedbed. Hunt(personal communication) has reported that seedlings withE—strain from the same nursery used in this study were smallerat outplariting, but grew better in the field. The Amphinernabyssoides treatment had the largest root collar diameters, shootlengths and shoot weights of all the treatments, including thecontrols, while the Danielson E-strain treatment was smallest ineach of those categories, as well as root weight. The onlyother reported study on the effects of inoculation with112Amphinerna byssoides is the white spruce in a growth chamberstudy of Husted (1990). She reported that Amph1nerta byssoldeshad growth similar to controls. Danielson (1988) failed to getArnphinema by.ssoides to form mycorrhizae on jack pine.The growth effects found in this study may be important froman applied point of view, since there are advantages to eitherincreasing or limiting top growth. C. Hunt (personalcommunication) has indicated that one of the problemsencountered at the Heffley Reforestation Centre is limiting topgrowth in certain circumstances. The results are most importantthough, because they illustrate that manipulation of fungus in anursery can change the growth characteristics of seedlings, evenin the high—fertility, high—moisture environment of the nursery.There were, in essence, three fungal treatments in thisexperiment, since the control and fungal growing mediumtreatment were heavily infected with Thelephora spp. Eachtreatment behaved differently, which undeniably illustrates thatthe role of ectomycorrhizal fungi, in the nursery, cannot bereduced to the notion that “any fungus will do”.The modified growing conditions in this nursery were effectivein promoting formation of mycorrhizae because the roots ofuntreated nursery trees were heavily infected with Thelephoraterrestris—like fungi, and there were several other types offungi in the nursery (in low levels that I hesitate to callinsignificant, in light of what happened with the A51treatment). However, inoculation with fungi resulted in evenhigher total levels of colonization. The inoculated fungi grewmainly by excluding Thelephora. The effect of altering the113nursery environment may be cumulative over time, as populationsof fungal inoculum build up in the styroblocks or the nursery,and the effectiveness of funga]. inoculation might be reduced.This may be possible, but the styroblocks used in thisexperiment were not new and the patterns of mycorrhiza formationsuggested that there was already an accumulation of fungalinoculum in this nursery. Furthermore, it is quite apparentthat significant alterations to nursery fungal populations maybe brought about by relatively simple vegetative inoculationtechniques.This technology is not ready for immediate commercialapplication, since there is a possibility that effects may varyfrom year to year, depending on climatic situations or othervariables. Observations by 0. Hunt (personal communication) atthe Heffley Reforestation Centre nursery suggest that this maybe the case. In addition, there are still many more fungi thatcould be tried, including local isolates of B-strain, morevarieties of Suillus, field isolates of Thelephora spp. andothers. Another very important avenue to explore is theapplication of mycorrhizal “cocktails” with more than one funga].species. If Amphinema increases growth and B-strain decreasesit, what would happen with good infections of both? It may wellbe that increasing the biodiversity on the root systems ofnursery seedlings would stabilize the growth of the seedlings inregard to changing environmental conditions, and thus allow moreuniform results for the grower.Field Trials.— The B—strain treatment resulted in smaller(root collar diameter in pine, root collar diameter and height114in spruce) trees both in the pine and spruce. The A51 (Suillustomentosus on pine) and the Amphinema byssoides (on spruce)treated trees carried on from the nursery as being the tallesttreatments for their species. However, for the spruce, theincrement on the Amphinema byssoides treatment was similar tothat of E-strain, which was significantly smaller than othertreatments. On the pine the tops of the A51 treatment weremarginally significantly heavier and the incremental height waslargest, but not significantly. It is not possible to say whatthese differences in growth might mean over the longer term.The differences in morphology and mycorrhizae were notsufficient to cause differences in survival, even though thespruce had relatively high mortalities. The mortalities mighthave been even higher if the early part of the growing seasonhad not been so wet. Some studies have shown that differencesin height after the first year continue and grow larger withtime. For example, Marx, Cordell and Clark III (1988) reportedthat over eight years, the growth of loblolly pine planted on anold agricultural field was positively related to the initialamount of Pisolithus tinctorius ectomycorrhizae. The differencein yearly basal area growth between fungi with a lot ofPisolithus tinctorius and those with none, grew larger for thefirst five years after outplanting.Many fungi grew onto the egressed roots of the seedlings overthe first summer in the field, though the levels of colonizationreached by the volunteer fungi were much smaller than those ofthe inoculated fungi. There is no other published informationon similar sites. Roth (1990), working at a coastal site in115British Columbia, found a similar pattern, where the maiority offungi on new roots came from the nursery fungi, but significantlevels of field fungi moved onto the roots. G. Hunt (personalcommunication) has made considerable observations of roots inthe general vicinity of this study. He has observed that insome circumstances, seedlings remain with the mycorrhizalpopulation that they had in the nursery until the roots ofectomycorrhizal fungus harboring plants such as Arctostaphylosuva—ursi grow into contact with the root plug. In general,reports have varied on the extent of volunteer colonization, byindigenous fungi, shortly after outplanting. Castellano andTrappe (1985) reported that Douglas-fir seedlings had some rootsinfected by indigenous fungi after two years, but a fungusinoculated in the nursery still constituted more than half ofthe mycorrhizae on the seedlings. Kropp (1982) plantednonmycorrhizal western hemlock seedlings, both on mineral soiland on rotten wood, and seedlings became nearly totallycolonized over one growing season. In general, it seems thatmost normal reforestation sites have some inoculation potential.The extent to which indigenous fungi colonize the roots ofoutplanted seedlings in the first season may be related toseveral factors. Contact with the root is an obvious firstrequirement for colonization, but the presence or absence ofmycorrhizae on the outplanted seedlings, and the type ofmycorrhiza all presumably could have some effect. The fact thatcolonization can occur does not eliminate the possibility ofbeneficial effects from inoculation, as indicated by the resultsin this study, those of Castellano and Trappe (1985) and others116discussed in the Literature Review.The fungi used in the inoculation trials seemed well-chosen,because the more common mycorrhizae from the field were usuallysimilar types to those that were inoculated. The pine providedan interesting case, in that the inoculated fungus A51 (SuIllustamentosus) did not grow very well in the nursery, but yieldedsignificant growth results, and fungi similar to A51 were amongthe most common types to grow on the egressed roots over thesummer. The other inoculated fungi and even the Thelephora thatgrew naturally on the nursery seedlings, continued to growvigorously onto the egressed roats. However, this was not sofor every tree, and there were several (17%) cases where theThelephora did not grow at all onto new roots. There may stillbe some debate over whether Thelephora is a suitable fungus forthe field, but this study, in conjunction with observations ofabundant fruitings of Thelephora seen while collectingsporocarps shows that Thelephora can grow well under theconditions of this study. The amount of fungal growth onto newroots was highly variable for all treatments, which may havebeen attributable to microsite differences, genetic differencesbetween seedlings or presence of fungal inoculum.Some significant results are not easy to rationalize. Thedifferences in the numbers of types of ectomycorrhizae on thepine would seem logical if the E-strain treated trees had fewertypes, since their roots were dominated by the inoculatedfungus. However, the control and the E-strain had the fewesttypes, while the fungus growing medium and A51 had the most. Itmay be possible that the fungus growing medium acted as a117substrate which allowed better growth and colonization byindigenous inoculum, while injected fungi (except A51, which hadlow colonization levels> dominated the root and reduced thenumber of types. In the spruce trial, the control and thefungus growing medium had similar and significantly highernumbers of fungi than the fungal treatments. This could beexplained by the high colonization rates of the two inoculatedfungi, which may have resulted in the exclusion of other fungi.Another unusual result was that for spruce, the control had asignificantly lower tissue calcium level than the DanielsonB-strain treatment. The calcium levels were grouped, with thelevels of the Danielson E-strain treatment and the fungusgrowing medium treatment being similar, and the levels of theAmphinema byssoldes treatment and control being similar. Thecontrol treatment was not obviously out of line, nor was theDanielson B—strain treatment. Further study is needed todetermine if this is a real effect.There are several ways these results might be interpreted withregard to the importance of ectomycorrhizal inoculation innormal reforestation situations. One might say that high levelsof a fungus that can grow in the field (Thelephora) withouthuman assisted inoculation on nursery seedlings, and if that isnot enough, many wild types colonize seedlings rather quickly inthe first year of planting, so inoculation with ectomycorrhizalfungi is not important. Alternatively, one might argue that,even though many fungi grow onto the roots, the largestproportion of the colonization on egressed roots in the fieldclearly comes from the nursery fungus, so the nursery fungus is118important. It is also quite clear that the type of fungus onthe seedling in the nursery can affect growth both in thenursery and in the field. Different fungi can increase ordecrease seedling growth under exactly the same conditions.Many factors other than growth might be altered by mycorrhizalinoculation but the changes in growth illustrate thatmycorrhizal manipulation is indeed an important tool in normalreforestation situations, even though significant differences insurvival, which would have had major and immediate importance,were not realized.These specific results are useful, but it is not possible tosay whether the growth effects achieved in this experiment willbe reproducible from year to year with the inevitable changes inenvironmental conditions. Work by G. Hunt (personalcommunication) suggests that they may not be. Therefore, thefindings from this study have been stated in a general way tohighlight what they contribute to the technology of inoculationwith ectomycorrhizal fungi in normal reforestation situations.This study shows that:1. Ectomycorrhizal fungi can be induced to form heavyinfections, in a nursery with a modified cultural regime,with two relatively simple mycelial inoculationtechniques;2. Different fungi do not affect growth in the same way underthe same growing conditions;3. It is normal for seedlings to have more than one type offungus on their roots in normal reforestation situations,even after a very short time in the field;1194. The fungi that infect roots in the first field season donot mask the effects of the fungi that were on theroots of seedlings in the nursery;5. Heavy infections by an inoculated fungus may reduce thefungal diversity found on roots after one growing seasonin the field;6. Even small levels of colonization by certainectomycorrhizal fungi may have significant effects ongrowth;7. Thelephora terrestris has long been cited as an example ofa fungus that grows well in the high fertility, highmoisture environment in the nursery and does not grow wellin the typically lower fertility, lower moistureenvironment in the field, in this study Thelephora oftengrew very well in the field;8. Local fungal isolates (Suillus tomentosus, Arnphlnemabyssoides) improved growth over nursery fungi (apredominant nursery fungus was Thelephora terrestris-like)but an isolate from a distinctly different area(E—strain) reduced growth, even though the same type offungus is common in the study area;9. Inoculation with fungi in the nursery may increasethe level of colonization even in nurseries with heavynatural colonization of the same fungus or a differentfungus.The first point to note about these general statements is thatnone of them is, in fact, general and they only positively applyto the conditions described in this study. However, they do120describe potential behaviour. The simplicity of the techniquesfor inoculating fungi offers the potential to easily applyfungal mycelium in inoculation trials. The fact that differentfungi may potentially affect growth in different ways under thesame conditions is further confirmation that ectomycorrhizalfungi interact with their hosts in very specific ways. Thepresence of more than one type of fungus on the roots ofseedlings confirms other recent work (Roth, 1990; Danielson andVisser, 1989) showing this and suggests that concentrating onfinding one fungus that completely dominates a root may not bethe only approach to inoculation that should be investigated.This is particularly true in light of the suggestion in thisstudy that one dominant fungus can reduce the number of types offungi that colonize a root. There has been much speculationthat the type of fungus on a seedling in the nursery is notimportant in natural reforestation situations because fungi fromthe field will colonize roots so quickly that there will belittle carryover from the nursery. This study confirms that thenursery fungi continue to influence the seedling in the field,even though field fungi move onto roots soon after outplanting.Other recent studies (Stenstrom and Ek, 1990) have shown thatlow levels of colonization with some fungi are enough toinfluence growth, and this study confirms that. Therefore,infectivity should not be the sole criterion for selecting fungito evaluate in outplanting trials (except, of course, that somecolonization has to occur). Thelephora terrestris that growswell in the high nutrient and moisture environment of thenursery can also grow well in the typically, comparably lower121nutrient and moisture environment found in field situations.This very adaptable fungus warrants considerably more study. Anessential component of every outplanting trial should be theidentification and evaluation of local fungi. Finally,inoculation of fungi should still be considered, even when theinoculated fungus is present in the nursery at high naturallevels, or when some other fungus appears to dominate the root.The inoculation procedure may give enough of a headstart to theinoculated fungus that significant differences in growth can beachieved.1228. OVERALL CONCLUSIONSIn the Introduction, it was suggested that this study mightserve as a protocol for further studies evaluatingectomycorrhizal fungi in normal reforestation situations. Thekey elements of the protocol are:1. The use of a comprehensive ecological classification systemto describe outplanting sites and the sources of fungal isolates(a suitable system would be one similar to the biogeoclimaticsystem developed by Dr. V. J. Krajina (1969) and expanded by theBritish Columbia Ministry of Forests and others>;2. A description of the types of ectomycorrhizae present onthe natural seedlings at a site;3. A description of the types of ectomycorrhizae on seedlingsat outplanting;4. A description of the changes that occur to the populationsof mycorrhizae on planted seedlings, after outplanting, overthe short and long terms;5. The use of a wide variety of fungi in normal reforestationsituations, including local fungal isolates, exotic isolates,assertive rnycorrhiza formers, minimalist (fungi which are ableto affect growth while colonizing only a small portion of theroot) mycorrhiza formers and those in between;6. Development of improved cultural and inoculation techniquesfor ectomycorrhizal fungi;7. The assessment of growth and survival over the shortand long terms.123In addition to the protocol for evaluating ectomyceorhizalfungi, several questions were posed in the Introduction thatrelate to selection of the best fungus for inoculation. Thesequestions and the answers suggested by this study are presentedbelow.Q. How important are the fungi on the roots of seedlings atoutplanting?A. This study established that fungi inoculated in the nurserydo continue to affect growth after outplanting. Growth can beincreased or decreased, but the duration of this study was notenough to determine if those effects persist. The nurseryfungi persist, and continue to dominate the roots after oneseason in the field, even though other fungi infect the roots.A potentially important observation was that more assertiveinoculated fungi CE-strain and Arnphinema byssoides) tended todecrease the diversity of types of mycorrhizae on seedlingsafter one season in the field and a minimalist fungus (Suillustomentosus) did not. All of these fungi affected growth, butE-strain decreased it. The ability to exclude other types ofmycorrhizae could be advantageous if the the fungus iswell-suited to the site, tree and climate or it could also bedisadvantageous if the fungus is not welladapted. A diversefungal population could reduce the risk of having the wrongfungus. It might also be possible to inoculate withminimalist fungi without reducing diversity.Q. How long does it take for a seedling to develop thecomplement of fungal genetic material necessary to be fullyadapted to its site?124A. While some seedlings had four or five fungi after onegrowing season, the mean number for pine trees was 2.75 and 1.69 for spruce. Many trees had only one type of fungus on theroot after one growing season. There is also no guaranteethat four or five types is the maximum number, or that theyare the optimum fungi for the site. At the site where thisstudy was conducted, it seems clear that the nursery fungicontinue to play a major role, even after the first growingseason.Q. How many species of fungi are normally found on aparticular tree species at a particular site?A. There were four or five species of fungi on severalseedlings after only one growing season. Not all seedlingshad so many types of fungi, but it is probable that moreseedlings will approach this number, given the chance ofexposure to inoculum. The upper limit to the number of typeshas probably not been reached, but perhaps this question isnot relevant now. The important answer is that even veryyoung seedlings are capable of sustaining diverseectomycorrhizal populations.Q. Can the introduction of exotic fungal material result inimprovements in performance in normal reforestationsituations?A. In this study, the local fungal isolates (Amphinemabyssoides and Suillus tornentosus) increased growth, while theisolate from the more distant site (E—strain) decreasedgrowth. This occurred, even though E-strain was indigenous tothe study area.125Q. Does a diverse fungal population improve performance, asenvironmental conditions vary?A. The unexpected inverse relationship between successfulinfection by inoculated fungi and diversity of mycorrhizalpopulations makes this question difficult to deal with in thisstudy. The seedlings that had strong growth of inoculatedfungus had less diverse fungal populations. No differences insurvival were found between the treatments but several factorscould have been interacting. The inoculated fungi may havebeen well adapted to the site or not, and they may havedisplaced fungi that were more or less adapted to the site.This question needs to be more specifically addressed infuture work.Q. Are some types of mycorrhizae more susceptible to diseaseor environmental stress?A. This study did not reveal differences in seedling survival,even though mortalities in spruce were quite high. Since ithas been shown, in other studies, that any mycorrhiza isbetter than no mycorrhiza, it may be possible that allmycorrhizal fungi confer some minimal set of advantages, likeroot protection, generally improved nutrient uptake orpossibly others. It may be that unless a tree is exposed tosome specific stresses or competition, any fungus will do. Asthe specific attributes of different fungal isolates areprobed, the more general attributes of mycorrhizal fungi mayalso become apparent.126—(ftCD(ft0ClDiCHC’i-’0rtH,M-ctt1DiZDlCDCDCDZ0H,SctDiI<Ct$fDiDi<i—0HCDDi(ftDc-i-H,5lC)c-tzClzCDJ0CDClH00CDi—’c-I-()c-I-CDi—-0CDCDot‘-<DiHc-I-0ClCDCDIHDii-a-0i-a-Dic-i-HCDH,i•ri!0DiI—’H0Dl(ftDi(ftH0(ftci--CDI.-.i--Qci-cQCDCDCT)c-I-I—aHCT)1-00ci-(ft(ftDiDi(I)çDi(ftHCl‘<c-I-CD00Clc-I-DiCD0H0i-•-01tQi--(ftc-I-c-i-CDHc-iDlc-I-Cl)(ftDiCDCDH0•)ClCDClI--0CDc-I-CDDiDl0HH,CD<HoHDiHi-c-I-(ftDl0Cl(ftCD‘-<CDDiUIci-c-I-Di(C’CDC-h0HDiHc-I-I--Zc-I-(1)0H.0DiH0CDI—’CDO0‘—Nc-I-0CD0c-I-Di0(ftC’-.uI—.’ZCD-<Xct0Dl‘-•0I--HH,DiDlCDctCD(SIH(ftCDCDQHDlI-I,CDc-I-DiCDHCDCli-c-I-Clc-Ic-I-(ftDlç(Cl(ft(ft(pDirHDlClCDCCl-•c-I-H(I)Illc-I-c-c-‘tJF—’CDc-I---Ht0F-’-c-i-(I>ClZ0CDI—’DlF--<0CDF-’-NCDc-I-00H,Cl)<CD(ftCDCDQCDc-I-tItlDi0DlClCDZH,Di<(ftCDCl)Cc—-Hi—QCDc--.DiClCDci-CDCDc-i-c-I-Di0i-a-•0c-I-c-’-HHDic-—’c—’-Zc-I-c-I-Dl0DiClDii(ft0c-I-i-t-1c-I-Ct’CL’(ftC’iDitr0DiHI—hZDic-I-CD‘V0HF-’-Z(ftFc-I-Dic--’Dl‘-<(ft‘Vc-I-0ClZH0Cici-CDCDDi0ctCD(ftCDc-I-HT‘Vc---CD0tCD0CD(ft‘-“c.Q0‘VCDCDDiCDF-hc-I-CZc-I-(ftH,Cl0LO0Dic-I-c-I-I—‘VDlDlClHI—’‘--(ftDl•)•F-HoHHDlDiCD<‘VH(ftF-H0Di0I—’Hc—’-CDCD0I-’tY’(ftc-I-0‘Vc-i-I<0cI-tI-’ClCl•(ft(F<F-(ftc-I-‘HDiF-’-c-I-0(ftCD0I—’-Qcic-I-•F-CD0Dic-I-H0(ft0I-Ic-I-0c-I-0X(CD(I)(ftc-i-c’Zc-i-C0CD(ftc-I-c-I-F-000C)Clc-I--<tic-I-CDZi-’-I—’c-fiCDc—’HDiF-’.Di0c-’-Dl(1)0DiCDci-CD0CT)0CDDic.QClCDCDDlcC)i,QC’DiDi(ftCD(ftCHDic-I-HDiI--CDHI—’c-—•CDF-rDiCD0oCD0c-I-F-c-I-0(ftF-j-,CDHI—’•Di(ftDlc-I-HCI)0CDc-I-cOZC’<H,t3F-c--<1CDc-I-DiCDTI-’-c-I-Cl)0H<CDHCDCI—-Zc-I-c-I-c-I-CD(ftH‘VCDHCDHDic—HHNH,flCD0H(1)c--ClCl)0HHCD(ftCDI-’C(11Cl)‘QDiCDtY’(ftC1‘30c--I—’HC’CDci-CDc-I-DiF“c--0Dlc-I-DiF-HCtUF-<c-I-<HZH,(Y’c--a-(ftF-<rj)c-II-’-c-I-HDiDlZCDc-’CDZDl(ftDiCDCDH,CD()CD0(tJPiDi(1)c-”DlF-0F-•CD‘.0c-i-F-CDIZ00F-CDCDci-0CD(ftI—-F-’-CDDl00Di(1)0(ft(ft‘-<CltyZc-i-p-’-‘.I—’0ZCDCD•0F-’•F-c-I-HDiCD0c-tCD‘-CD(ft(A)Clc--’Clc-I-H(ftc-i-Dl<Cl)HDiDic-I-H3’Di0H,F-c—’F-’Dl.)CD0CDHClCDc-I-CDDi(ft(ftc-I-Di1-’-CDc-I-tYI-’-‘—40ClzZCDCDI—’-(ft(ftCD(ft(ftF-F-c-’-0tYc-I-H,C)Cl•.00QtYCDCDHc-I-Z0F-0c-I-CD‘.0Di‘-3F-(I)HCDH0tY.—HHc--I-’-H,tY—‘.tY’(ftc-I-HCDc-I-CDHCDc-I-c-I-ci-c-I-0CDCDF-(I)DlDitYCl)CD(ftDi00DlCD(p(ftCDc---c-•0cC0•c--I-DiDi(ftCDDl(ftZC-<(0c-I-c-lCD<<DlYc-I-0a>HZc-i-Cl0CDc-I-CDc-’-CDCDCDDi,-‘-I-ha>Dl-<DiClc-i-‘-<DiCDi—’F-hCl0(1)00‘-—c—•i—iF-Di(ftc-’-z0CDCl0o.0DiCr.cH,H,F-hc-i-c-I-c-I--JF-h(<DlDic-tHHtS(11c-i-0(A)tYC(ft(CDc-i-H-c-i-c-i-(ftC)F-h0tIZCDCDc-Dlc--•Hc--<tYZ’00H‘.0‘.0CDCDDi--c---DlDiDlHcC)c-I-CD(ftI—’-0CDDiCDZ0c-I-tSCc-H-c--•ZH(ft‘<c--<CDi-’-CDClCD(12(ftc-i-‘-<CDtYtYCDc-i-consistantly reduce seedling growth. One might speculate thatthese fungi may confer some general mycorrhizal benefits such asincreased root life, disease protection and increased uptake ofnutrients and water. An unavoidable consequence of rapid growthby the fungus is high carbohydrate drain. Over time, the fungalpopulation of the root could become more attuned to the site, asother fungi establish on the root. It is interesting to notethat some of the most common fungi to infect roots in the fieldin this study were E-strain and Thelephora terrestris, both ofwhich might be considered assertive fungi. Yet, the InoculatedE—strain reduced growth. It was not possible to tell from thisstudy if the volunteer E-strain also reduced growth, but it maybe that a reduction in growth is not as critical as the need tobecome mycorrhizal as quickly as possible.There were two somewhat contradictory observations in thisstudy with regard to fungal populations on roots. As alreadystated, heavy infections by inoculated fungi seemed to reducethe fungal population diversity on seedlings after one season inthe field. However, even seedlings with high levels ofinoculated mycorrhizae began to develop more diverse fungalpopulations in the field. The reduction in population diversitymay not be the result of active exclusion, or outcompetition,but simply a reflection of the ability of the inoculated fungusto get there first. This may also apply to early—stage orassertive fungi in general. There seems to be little difficultyfor new fungi to colonize in the field. The affect on growththat Suillus tornentcisus had with low levels of infection mayindicate that a low percentage colonization is all that is128necessary for some types of mycorrhizae to affect growth.The reductions In growth caused by assertive fungi and theincrease in growth caused by Suillus tomnentosus (Danielson, 1988describes Suillus as multi-stage fungi) suggest a possiblestrategy for improving the performance of nursery seedlings. Ifthe vigorous growth of early—stage fungi is an adaptation toachieving mycorrhizal infections quickly, it may be possible tocircumvent the need for this role by assuring that nurseryseedlings are infected with multi—stage or late—stage fungi.The problem with this idea is that many of these multi- andlate—stage fungi do not culture and/or inoculate well. Someeffort might be put into finding such fungi that do inoculate,or into developing techniques that will allow inoculation.Danielson (1988) gives a brief description of some of themanipulations that have been done to try to get successfulinoculation with multi- and late-stage fungi. Attempts havebeen largely unsuccessful. Since late-stage fungi do replaceearly—stage fungi in the field, it is interesting to speculatethat early—stage fungi may play some type of nurse role in theestablishment of later stage fungi. More information is neededabout the mechanics of mycorrhizal—fungus succession on treeroots.While many questions are raised here, this study has,nevertheless, resulted in some concrete advances in techniques.For example, top—applied mycelial slurries of ectomycorrhizalfungi were used successfully to inoculate Engelrnann spruce andlodgepole pine seedlings in a commercial nursery. Thisinoculation technique was extremely simple and could be adapted129to commercial application with very little expense. A newfungal culture technique was developed that simplifies theprocess of fungal culture and speeds up the growth of most ofthe fungal isolates tested. The fungal culture technique workswell in conjunction with the simple inoculation procedurementioned above. These two procedures could constitute twoeasily applicable steps in a commercial protocol for theinoculation of ectomycorrhizal fungi. However, they are alsouseful tools for applied research, since the culture andsynthesis of ectomycorrhizae are two major obstacles toinvestigating the enormously diverse populations ofectomycorrhizae.An important aspect of this work was the observation of rootsand mycorrhizae to describe qualitatively and quantitativelywhat the mycorrhizae were doing. Many researchers have pointedout that growth statistics can convey only a small part of themycorrhizal picture. Future work will have to rely even moreheavily on the detailed examination of roots. A new stainingtechnique, using a low-toxicity dye suitable for the evaluationof certain features of ectomycorrhizae, was developed andintroduced in this study. It is already being used by otherresearchers to examine ericoid mycorrhizae and should have avery high potential application for routine use.These developments are useful, but in the end, the protocolfor outplanting trials must be stressed. Unless information onectomycorrhizae is collected in a systematic way, such asoutlined in this thesis, it may be a very long time before thethe many questions about ectomycorrhizae in normal reforestation130(ft rt a I— 0 a H a a a H aLITERATURE CITEDAgerer, R. 1986. Studies on ectomycorrhizae. II. Introducingremarks on characterization and identification. Mycotaxon 26:473—492.Amaranthus, M. P. and Perry, D. A. 1989. 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Prentice—Hall,Inc., Englewood Cliffs. pp. 718.138FIG. 1. The pure culture synthesis chamber also showing treesand the watering tray. FIG. 2. Inoculation of the pure culturesynthesis chamber.139FIG. 3. A dilute—agar culture showing the concentration ofgrowth near the top. FIG. 4. Cenococcum geophiluni in dilute—agarculture on the left and liquid medium on the right.140FIG. 5. Characteristically coarse E—strain hyphae on the surfaceof a root. Bar = 0.4mm. FIG. 6. E—strain hyphae growing from asevered root. Bar = 0.3mm. FIG. 7. B—strain Hartig net withcharacteristic circular shape near the centre. Bar = l5um.141FIG. 8. Exudates on Type 12 Suilloid fungus in KOH. Note theviolet—coloured exudates. Bar = lOum. FIG. 9. Typicalbrownish—coloured exudates on a Type 10 Suilloid rhizomorph.Bar = 3Ourn.142FFIG. 10. The typical growth of Amphinerna byssoides extramatricalhyphae on a plug. Bar = 4mm. FIG. 11. Arnphinerna byssoides(arrow) showing the same vigorous growth on a seedling from thefield trial.143FIG. 12. Type 3 mycorrhiza on pine showing the characteristicoil droplets in the mantle. Blue stain is from FDA Blue No. 1described in Experiment Three. Bar = lOum. FIG. 13. Longitudinalsection of Type 3 on pine showing mantle stained with FDA BlueNo. 1. Bar 0.25mm.144FIG. 14. The golden yellow exudates and lactifers of Type 8 onspruce. Bar = 2Oum. FIG. 15. Pine roots growing straight downfrom the plug, showing non—mycorrhizal roots near the top andmycorrhlzal further clown. Bar = 4mm.145APPENDIX IFUNGI USED IN PURE CULTURE SYNTHESISandTHE NURSERY SCREENING TRIALA6: This culture was isolated from a Rhizopogon spp. sporocarpcollected under Douglas-fir (Pseudotsuqa menziesii var.glauca) near Haggard Creek at about 210Gm elevation. Thebiogeoclimatic zone was the IDFmw2 and the collection sitewas about 15km east of the study area.approximately four-year-old spruce with whitehyphae and rhizomorphs and pinnately branched mycorrhizae.Produced colonies identical to A18. Same site and sametime as A13.A16A: Same as A16 but colony morphology differed.A16B: Same as A16 but colony morphology differed from A16 andA16A.A13: Cultureon anCollectedthe MSdm2south ofisolatedseed 1 ingsA14: Isolatedsame siteA16: From antaken from a highly tuberculoid, white mycorrhizaapproximately five-year-old lodgepole pine.October 14, 1987, near Sargent Creek at 1300m inbiogeoclimatic zone. This site is about 15kmthe study area. Samples A13 to A22 were allfrom mycorrhizae taken from small volunteergrowing along roads or in clear cuts.from a bright yellow mycorrhiza on spruce at theand same time as A13.146A17: Taken from a small, brown with white spots, tuberculoidmycorrhiza on lodgepole pine at the same site and same timeas A13.A18: This was the most common type of mycorrhizae found onspruce at the site described in A13. It formed the verydistinct mass of extramatrical hyphae associated withAmphinema byssoldes, and was identified as being Amphinemabyssaldes ( Fr.) 3. Erikss., on the basis of (1) stainingin KOH, (2) slow-growing indurate colonies on agar, (3)hyphal clamps, (4) occasional fine ornamentation on hyphaeand (5) general appearance,. This isolate did not formmycorrhizae in the nursery screening trial. All isolatesfrom A18 to A22B were collected October 15, 1987.A18B: This isolate came from the same root as A18 but did nothave the usual Amphlnema byssoldes growth characteristicsin culture. This culture was used in the nursery screeningtrial on spruce and did not form mycorrhizae.A19: From a spruce seedling at the same site as A13.. Thisseedling had dense extramatrical hyphae like Amphinemabyssoldes, but the mycorrhizae were very irregularlyshaped. This fungus was used in the nursery screeningtrial, but did not produce mycorrhizae.147A20: This fungus produced dense extramatrica). hyphae andslow—growing indurate colonies similar to A18, but wasisolated from young pine. It had a distinct odor ofmushrooms that was not apparent in cultures of Amphinemabyssoides. This was used in the nursery screening trial,but did not produce mycorrhizae on spruce.A21: From a much branched, palmate, white mycorrhizae on pine inthe same area as A13. No obvious rhizomorphs. This wasused in the nursery screening trial, but did not producemycorrhizae on spruce.A22: From a tuberculoid type on pine very similar to, and fromthe same location as A13. This isolate was used in thenursery screening trial, but did not form mycorrhizae onspruce.A22B: The same as A22 but exhibited a different colonymorphology.A29: Collected from an Arnphinema byssoides infected root from aspruce at the Heffley Reforestation Centre nursery onOctober 15, 1987. This isolate was used in the nurseryscreening trial, and was the only isolate to perceptiblyalter the colonization of the nursery seedlings. It formeddense masses of hyphae that tightly bound the plugstogether.A29B: From the same source as A29 but the colony morphologydiffered.148A30: Isolate taken June 17, 1988 from a Suillus brevlpes (Pk.)Kuntze sporocarp growing under a 15-year—old lodgepole pinenear Community Lakes about 38km south of the study area.The site was 1350m in elevation and was in the MSdm2biogeoclimatic zone. This fungus was fruiting in largenumbers in the area.A31: Same place and same time as A30 from a sporocarp of Suilluspseudobrevipes Smith & Thiers. Only one sporocarp wasfound.A31A: From the same sporocarp as A31, but colony morphologydiffered.A31B: From the same sporocarp as A31, but colony morphologydiffered.A33: Found at the Sargent Creek site on June 17, 1988, but undera very young subalpine fir near a creek, probably in theESSFwm biogeoclimatic zone. This culture came from asporocarp tentatively identified as a Hebelorna spp.A34A: Taken from a mycorrhiza on two or three—year—old spruce atthe Sargent Creek site on June 17, 1988. This was anotherAmphinerna byssoides isolate.A34B: Same as A34A.A36: This isolate was taken on June 17, 1988 from an E-strainshort root at the Sargent Creek site, but the culture wasnot E—strain.A45: Found on September 22, 1988 at the Sargent Creek site.From an unidentified sporocarp growing under pine regen.A46: Same as A45.149A48: This culture was isolated from a Leccinum spp. that wascollected under the same circumstances as A45. There weremany tree species growing in this area, including lodgepolepine, aspen, willow and subalpine fir.A48B: Another isolate from the same sporocarp as A48, but withdifferent colony morphology.A49: From a Suillu.s tomentosus (Kauff.) Sing., Snell & Dickfound under the same circumstances as A45. A very largenumber of sporocarps were present.A51: Same as A49. This isolate formed mycorrhizae on pine inthe pure culture synthesis trial and was used in thenursery trial and the outplanting trial.Danielson’s E—strain (sensu Mikola) (R947): This isolate wasobtained from Gary Hunt who got it from R. M. Danielson atthe University of Calgary. It was isolated from whitespruce seedlings growing in subalpine soil. This was themost vigorous mycorrhiza former in the pure culturesynthesis trial and was used in the nursery trial.E—strain (0188): This isolate was obtained from Lynn Husted whoisolated it from container-grown Douglasfir at the HarropNursery, near Nelson, B. C.. It formed mycorrhizae in thepure culture synthesis trial, but not very well. It wasused in the nursery trial.150Hebelonia crustulinlforrne (Bull: St Arnans) QUEL. (5): Gary Huntsupplied this isolate, which was collected by CarolineBledsoe, of the University of Washington, in a mixedconifer forest at 550m in Wenatchee National Forest. It iskept in the University of Washington Collection.Hebeloma crustulini.forme (Bull.: St. Amans) QUEL. (8): Alsofrom the University of Washington collection of Bledsoe.It was isolated in 1971 from a Douglas—fir forest in BentonCounty, Oregon. This fungus produced mycorrhizae in thepure culture synthesis trial and was used in the nurserytrial.B122: This is Laccaria laccata (Scop.: Fr.) Berk. & Br. fromGary Hunt. It was isolated in 1985 from a Douglas—fir inthe Heffley Reforestation Centre nursery.B148: Also Laccaria laccata (Scop. ex Fr.) Berk. & BR. from Dr.Gary Hunt. This was isolated in 1986 from a mixedDouglas—fir, Engelmann spruce, lodgepole pine forest in theCommunity Lakes area described under isolate A30.B169: An isolate of Laccaria glabripes obtained from Dr. GaryHunt, who isolated it from a mixed Douglas-fir, Engelmannspruce, lodgepole pine forest in the Community Lakes areadescribed at A30.A188—2: Cenococcurn geophilum Fr. isolate from Dr. Gary Hunt.The isolate came from 11,3km southwest of Philomath,Oregon, U.S.A. at 4428’3O”N, l2329W and at an elevationof 305m. Aspect was southern and slope variable. The soilwas Hohnnon gravely loam and is well-drained, moderately151deep and derived from weathered sandstone. The pH was 5.2in the litter layer and 5.7 in the Al horizon. Winters aremild and summers warm and dry. Annual precipitationaverages 1905mm. More details can be found in Fogel andHunt (1979). This isolate was used in the nursery trial.All isolates were collected by the author, unless otherwiseindicated.152APPENDIX IIDESCRIPTIONS OF MYCORRHIZAEType 1 on lodgepole pine, Type 4 on Engelmann spruceFungus: Myceliurn radicis atrovirens Melin -like mycorrhizaeAbundance: 3.7 on field pine, O.75 on field spruceDistinguishing Characteristics: This group was separated on thebasis of a black, often sparse mantle and narrow, finelyornamented hyphae..Macroscopic Characteristics: Usually these were simplemycorrhizae, often with the sparse mantle extending up thesecondary roots. There were usually many, very dark brown toblack extramatrical hyphae. No strands were seen.Microscopic Characteristics: Hyphae were 2.Oum to 2.Sum wide,without clamps and usually with a fine, grainy appearance. Themantle varied in appearance from very loose, individual hyphaeto Textura intricata. The mantle thickness varied fromindividual hyphae up to 8um thick. The Hartig net was usuallyvery poorly developed and varied from non-existent to a weakdevelopment around the outer cortical cells.Type 2 on lodgepole pine, Type 1 on Engelmann spruceFungus: Thelephora terrestris groupAbundance: 42.2 on field pine, 23.4% on field spruceDistinguishing Characteristics: This was a very plastic group inwhich mantle and rhizomorph colour varied extremely.Macroscopically, the group was distinguished by a generallysmooth and shiny mantle appearance and abundant white to brown153strands (to weak rhizomorphs). Occasional velvety clumps ofcystidla were visible under low magnification, but these weremore common on Type 11 on pine. Microscopically, this group wasdistinguished by very characteristic cystidia that varied fromvery few to abundant and large distinct keyhole clamps. Thecystidia were about 2um wide by lOOum to 200um long and clampedat the base.Macroscopic Characteristics: A highly variable group withgenerally simple mycorrhizae, especially on spruce, butsometimes with dichotomous or very simple pinnate branchingpatterns on pine. The mantle was typically shiny, withoccasional white patches of cystidia, sometimes extending tocomplete coverings of hyaline, velvety cystidia. Mycelialstrands ranged from few to very abundant. Colour varied fromwhite to dark brown, often on the same root in close proximity;strand thickness ranged from the thinnest of strands to quitethick rhizomorphs. Extramatrical hyphae uncommonly formed verydense mats approaching those of Arnphinema byssoic3es.Microscopic Characteristics: This type had a verycharacteristic mantle appearance with very typical Texturaepidermoidia and typical thickness of lOum to l2um. Hyphae were4um to 5um thick, with abundant keyhole clamps at most septa.TYPE 3 on lodgepole pineFungus: Unknown BasidiomyceteAbundance: 3.06% on outplanted pineDistinguishing Characteristic: A smooth, whitish mycorrhiza witha characteristic plump appearance that could easily be mistaken154for Thelephora mycorrhizae at low magnification. Very distinctmicroscopically because of a thick mantle (7Ourn or greater)(Fig. 13) with large oil droplets in virtually every cell (Fig.12).Macroscopic Characteristics: Very “squiggly’ emanating hyphaewith kinks in them, which at times, gave the mycorrhiza a woollyappearance. No rhizomorphs present, the mantle was whitish andthe 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 widewith abundant clamps and had a noticeably “squiggly” appearance.The mantle stained very darkly with FDA Blue No. 1. The mantlewas a Textura globulosa to T. intricata, with distinction madedifficult by the abundant oil drops in the mantle.Type 3 on Engelmann spruceFungus: Araphinerna by.ssoides, the same fungus as isolate A29Abundance: 2l of the egressed roots in the outplanted controland fungus growing medium treatmentsDistinguishing Characteristics: This mycorrhizae formed a denseweft of pale yellow or cream—coloured extramatrical hyphae(Figs. 10 and 11) that was unmistakable. It also produced asparser mass of hyphae, possibly at an earlier stage of growth.The hyphae also grew around other types of mycorrhizae, whichmade it difficult to determine where one type ended and anotherbegan. The hyphae turned dark yellow in KOH.Macroscopic Characteristics: the mycorrhizae were usually hidden155in a mass of hyphae, but when the hyphae were pulled away, themycorrhizae were seldom branched, and thin and fragile—looking.The extramatrical hyphae often coalesced into loose strands.Type 4 on lodgepole pineFungus: Suilloid typeAbundance: 4.69 on the egressed shortroots of lodgepole pineDistinguishing Characteristics: This Suilloid type had a verydark brown mantle with whitish blotches where the outer rind wasmissing and the hyphae were covered with brownish resinousexudates.Macroscopic Characteristics: These tuberculate rnycorrhizaeconsisted of bunches of dichotomously branched mycorrhizae withvery short branches (<1mm). The root was covered by a whiteinner layer and a nearly black outer rind of appressed hyphae.There were abundant brown wire—like rhizomorphs with diametersof 5Oum and greater.Microscopic Characteristics: Hyphae were 3um to 4um wide andlacked clamps. There were sometimes unusual septa that werecurved, with the hypha on the convex side being smaller indiameter than that on the concave side. The exudates did notturn violet in KOH. The mantle was thick, usually at least 3Oumand was Textura intricata to T. epidermoidea at deeper levels.Type 5 on lodgepole pineFungus: unknown BasidiomyceteAbundance: 0.3% of the egressed shortroots of lodgepole pineDistinguishing Characteristics: A much branched type withpinnate initial branching and dichotomous sub-branches. This was156a light brown myeorrhiza with occasional white blotches thatwere not the same as blotches on Suilloid types. The overallappearance was tomentose, often with adhering detritus.Macroscopic Characteristics: Abundant extramatrical hyphae;rhizomorphs lacking. The complex branching pattern wasdistinct.Microscopic Characteristics: The mantle had a fuzzy outer layerwhich extended from a more compact mantle with Texturaepidermoidia. The hyphae were about 2um in diameter,unpigmented and clamps were common and large. Hyphae did nothave exudates or other surface features.Type 6 on lodgepole pine and Type 2 on Engelmann spruceFungus: E—strain groupAbundance: 3.83% on non-E--strain inoculated field pine and 14.5%on non—E--strain inoculated field spruce.Distinguishing Characteristics: This group was distinguishedmacroscopically by the presence of very distinctive, wiry,reddish brown, large—diameter hyphae (Figs. 5 and 6). Thehyphae were not overly abundant but were obvious if the root wasnot washed. At higher magnification, the mantle was acharacteristic Textura intricata composed of large diameter,irregularly-shaped hyphae.Macroscopic Characteristics: The colour ranged from pale brown,that could easily be confused with Thelephora, to very darkbrown, almost black. The typical colour was a quite distinctreddish brown. The extramatrical hyphae were typicallymoderately abundant and had a stiff, wiry appearance. The157mycorrhizae often had an irregular angular appearance but wereotherwise generally simple in form.Microscopic Characteristics: The hyphae were typically wide, 5umto 7um and usually highly ornamented. The E—strain on pine wasectendo with arbusculelike intracellular hyphae. The Hartignet was often quite coarse (Fig. 7), and could be distinguishedfrom the finer Hartig nets of other fungi.Type 7 on lodgepole pineFungus: unknown BasidiomyceteAbundance: Found only in the control treatment of one block.Found on of the short roots.Distinguishing Characteristics: A distinctly yellow mycorrhizaewith a very fuzzy appearance.Macroscopic Characteristics: This yellow mycorrhiza typicallywas surrounded by a mass of hyphae, like a loose cotton ball.Underneath the fuzzy outer layer was a smooth mantle. Thebranching was simple dichotomous.Microscopic Characteristics: The hyphae were around 2.5urn indiameter with large clamps. The compact mantle was a Texturaepidermoidia composed of hyaline hyphal elements.Type 8 on pine and Type 6 on spruceFungus: Tonente1la-likeAbundance: 2.O6 average on field pine and O.56 on field spruce.Distinguishing Characteristics: This group was separated basedon the dark brown to blackish mantle that looked very much likeCenococcurn geophiluin, often with abundant straight emanatinghyphae (cystidia?), except that it was a basidiornycete. The158extramatrical hyphae were normally moderately abundant.Frequently, there was a brownish—purple cast to the colour.Macroscopic Characteristics: These mycorrhizae were usuallysimple, but could have one or two dichotomous forks. The mantleappeared slightly roughened. Often, when this mycorrhiza waspresent, it was present in abundance. Rhizomorphs were lacking.Microscopic Characteristics: The mantle was compact, about lOumthick, typically a Textura epidermoidia, but on spruce, Texturaangularis arrangement was noted. The hyphae were 2.5um to 4umin diameter, with a brownish-purple tinge, and sometimes withclamps and/or fine ornamentation. The straight emanatinghyphae, when present, might ha been cystidia, but they weretoo long to tell if their length was terminate.Type 8 on spruceFungus: Lactarius sp., close to L. deliciosusAbundance: two localized infections on outplanted spruceDistinguishing Characteristics: This was a pale whiterhizomorphic mycorrhiza with a slight yellowish tinge. Goldenyellow lactifers were present in the mantle and rhizomorphs(Fig. 14).Macroscopic Characteristics: This mycorrhiza was plain white orslightly yellow at low magnification with few extrarnatricalhyphae and rare rhizomorphs.Microscopic Characteristics: The mantle was made up of neatparallel hyphae, Textura epidermoidia and there were beautifulgolden-yellow lactifers in the mantle and rhizomorphs thatexuded golden droplets when cut.159Type 9 on pine and Type 7 on spruceFungus: Endogone—likeAbundance: 0.19% on egressed roots of outplanted pine and 0.50%on outplanted spruceDistinguishing Characteristics: This was a very hard fungus todistinguish macroscopically. It was usually found bymicroscopically examining a root that may have been Thelephorabut did not look quite right; for example, it looked too fat.Microscopically, the hyphae were very irregularly shaped andstained very dark blue with FDA Blue No. 1.Macroscopic Characteristics: No distinctive characteristics.Microscopic Characteristics: This fungus was found growingthrough pre—existing mantles. Typically, a Thelephora mantlewould be covered by a loose net of variable width hyphae (2um to7um). Sections showed that the Endogone—like fungus waspenetrating the existing mantle with haustoria-like structures,but it was definitely not a pathogen, as the hyphae could beseen to be forming a Hartig net that was distinctly differentfrom the pre—existing Hartig net.Type 10 on pineFungus: SuilloidAbundance: 7.44% on outplanted pineDistinguishing Characteristics: This type was a tomentoseSuilloid morphotype. That is, it did not form a rind, and had auniform white colour. Microscopically, it produced abundantexudates (Fig. 9) that did not stain in KOH.Macroscopic Characteristics: Typically, these mycorrhizae were160simple, or with a few irregular branches, and were quite white,with a woolly appearance. There were abundant fine whiterhizornorphs (strands?) and fairly abundant extramatricalhyphae.Microscopic Characteristics: Emanating hyphae were 2um to 3um indiameter, simple septate and covered with abundant exudates,that did not change colour in KOH. The mantle about 5Oum thick.Type 11 on pineFungus: Thelephora groupAbundance: 16.26 on outplanted pineDistinguishing Characteristics: This Thelephora type tended toappear on new roots, sometimes separated from the main plug by anon—mycorrhizal zone. It was described as a different typebecause it formed long (3mm to 5mm) mycorrhizae and itfrequently was covered with, or had prominent blotches of whitecystidia. The other Thelephora (Type 2 on lodgepole pine)typically had only a few cystidia, or occasional blotches ofcystidia. This appeared to be different, possibly a fieldstrain.Type 12 on pineFungus: This was a Suilloid type that was indistinguishablefrom Suillus tornentosus (A51).Abundance: The mean level of this fungus on treatments where A51was not inoculated was 1.256.Distinguishing Characteristics: This type did not form a rind,but formed a woolly mass of brownish—coloured hyphae. Thehyphae were covered with abundant exudates that turned violet in161KOH (Fig. 8).Macroscopic Characteristics: Mycorrhizae consisted of denseclumps of many small branches, covered with a thick (5Oum),tomentose mantle. The inner mantle was more compact. Therewere many yellowish-brown rhizomorphs and fairly abundantemanating hyphae.Microscopic Characteristics: Emanating hyphae were 4um to 5um indiameter with clamped septa. The exudates turned violet in KOH.Mantle type was Textura intricata.Type 13 on lodgepole pine and Type 5 on Engelmann spruceFungus: UnknownAbundance: found on one pine seedling and O.38 of spruce shortrootsDistinguishing Characteristics: A translucent looking outerlayer and black underneath. This was an unusual fungus in thatit looked very similar to Amphinerna or sometimes Thelephora,except for the dark layer underneath. The mantle also had aunique appearance, but it was difficult to determine if this wasjust because of the darker layer below.Microscopic Characteristics: It appeared that the dark layerunderneath was caused by heavily suberized cells in theepidermis. The Hartig net was well developed.162APPENDIX IIINURSERY FERTILIZER REGIME1989Engelmann spruce- sown March 23Plant Prod Starter 11-41—8 @6weeks after sowPeter’s Grower 20—7-19 andPlant Prod Grower @10 weeks after sowPlant Prod Finisher 8—20—30 @19 weeks after sowApplications throughout season:2 applications Plant Prod Starter 11—48—8@600g/1000L10 applications Peter’s Grower 20—7-19@400g/1000L1 application Plant Prod Grower 20-8-20@500g/I000L6 applications Plant Prod Grower 20—8—20@400g/1000L.12 applications Plant Prod Finisher 8—20-30@400g/I000LLodgepole pine sown May 4Plant Prod Starter 11—41—8 @4 weeksPeter’s Grower 20—7-19 andPlant Prod Grower 20—8—20 @7 weeksPlant Prod Finisher 8—20—30 @ l3weeksApplications throughout season:4 applications Plant Prod Starter 11—41—8@600g/1000L6 applications Peter’s Grower 20—7-19@400g/1000L1 application Plant Prod Grower 20—8—20@500g/1000L6 applications Plant Prod Grower 20—8—20@400g/1000L12 applications Plant Prod Finisher 8-20-30@400g/1000LPeter’s products are manufactured by W. R. Grace Co., 62Whittemore Ave., Cambridge MA. Plant Prod Products aremanufactured by Plant Products Co. Ltd., 314 Orenda Rd., Brampton,Ontario. All fertilizers were applied in solution.163APPENDIX IVExperiment OneSUMMARY OF STATISTICSThe different types of inoculum are referred to as treatments,the method of inoculation is referred to as application and theindividual styroblocks are blocks. FGM refers to the fungusgrowing medium treatment.NURSERY PINE ANOVARoot Collar DiameterSum of dSource Square fMeanSquare FoverallA51 ( 1)Dan. E-strain(2)control(3)FGM(4)injected (a)top-appliedMultiple ComparisonsMeans3.04553.02412.98753.06273.11212.95753.1239Standard Deviations0.49750.50690.47440.52040.47380. 46550.5074The only significant difference was between application methods.Shoot LengthSource F P Test TermP Test TermTreatment 1.1926 3 0.3975 0.6543 0.5838 Block(treat*inj)Injected 4.0355 1 4.0355 6.6420 0.0127 Block(treat*inj)Treat*Inj 0.4832 3 0.1610 0.2651 0.8502 Block(treat*inj)Block 32.202 53 0.6076 3.0134 0.0000 ResidualResidual 109.08 541 0.2016Total 147.11 601Sum of dSquare fMeanSquareTreatment 103.36 3 34.545 1.2435 0.3032 Block(treat*inj)Injected 151.86 1 151.86 5.4805 0.0230 Block(treat*inj)Treat*Inj 92.403 3 30.801 1.1116 0.3526 Block(treat*inj)Block 1468.5 53 27.708 3.3323 0.0000 ResidualResidual 4498.4 541 8.3150Total 6316.9 601164Standard DeviationsoverallA51 ( 1)Dan. E—strain(2)control (3)FGM(4)injected(a)top-appliedMultiple Comparisons18. 01118.66518.09117.59817.67718.54617.5323.24202.96123.22893.08323.57893.21103.1994overallA51 (1)Dan. E-strain(2)control (3)FGM( 4)injected(a)top-applied(b>Means0.52320.52150.49830.54120.53400.49690.5465Standard Deviations0.17510.17060.16000.18170.18650.16640.1794Multiple ComparisonsThe only significant difference was between application methods.* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *Shoot MassSourceThe only significant difference was between application methods.****************************************************************Root MassSum of dSquare fMeanSquare F p Test TermTreatment 0.1476 3 0.0492 0.7094 0.5506 Block(treat*inj)Applic. 0.3514 1 0.3514 5.0723 0.0284 Block(treat*ini)Treat*Inj 0.1230 3 0.0410 0.5920 0.6230 Block(treat*inj)Block 3.6719 53 0.0692 2.6582 0.0000 ResidualResidual 14.048 539 0.0261Total 18.358 599SourceSum of dSquare fMeanSquare F P Test TermTreatment 1.4936 3 0.4979 3.2235 0.0300 Block(treat*inj)Applic. 0.0400 1 0.0400 0.2570 0.6143 B1ock(treat*in)Treat*Inj 0.5095 3 0.1698 1.0996 0.3575 Block(treat*inj)Block 8.1858 53 0.1544 2.0562 0.0000 ResidualResidual 40.412 538 0.0751Total 50.635 598165Mn StandarL DeviationsoverallA51(1)Dan. E-strain(2)control (3)FGM(4)injected(a)top—appliedMultiple ComparisonsTreatment 0.7083 3Applic. 0.8771 1Treat*Inj 0.5140 3Block 64.225 53Residual 213.13 541Total 279.51 6010.90260.98980.87120.86810.88430.91060.89550.19480.72380.14143 . 07590.29100 . 28560.26770.29810.29880.29310.2894overallA51 (1)Dan. E-strain(2)control (3)FGM(4)injected (a)top-applied(b)Means2.34222.35132.39372.30342.31542.38372.3050Standard Deviations0.68200.72720. 60500.71030.68860.66500.6957Multiple ComparisonsThere were no significant differences.Root to Shoot RatioTreatment 0.8704 3Applic. 0.1303 1Treat*Inj 1.0603 3Block 12.729 53Residual 92.625 538Total 107.43 5980.29010.13030.35340.24020.1722There were significant differences between treatments. Neitherthe Bonferroni nor Tukey tests showed a significant differenceat p=O.O5 but the A51 treatment is obviously out of line withthe others.Number of Short RootsSum of d MeanSource Square f Square F P Test Term0.23610.87710.17131.21180.39400.8995 Block(treat*inj)0.3987 Block(treat*inj)0.9347 Block(treat*inj)0.0000 ResidualSourceSum of dSquare fMeanSquare F P Test Term1.2081 0.3158 Bloek(treat*inj)0.5426 0.4646 Block(treat*inj)1.4716 0.2328 Block(treat*inj)1.3950 0.0388 Residual166Standard DeviationsoverallA51 (1)Dan. E—strain(2)control (3)FGM(4)injected(a)top-applied (b)Multiple Comparisons0.62700.58920.59360.28260.64630.61040.64170.42380.62830.17990.48880.25980.57010.2266Dickson Quality IndexSum of dSource Square fMeanSquare FTreatment 0.0243 3Applic. 0.0975 1Treat*Inj 0.0344 3Block 0.6516 53Residual 1.9286 538Total 2.7402 5980. 65997.93200.93243.4297overallA51 (1)Dan. E-strain(2)control (3)FGM(4injected(a)top-applied(b)Means0.18840.19060.17720.19300.19380.17460.2006Standard Deviations0.06770 . 06690.06040.07660 . 06590.06430.0684Multiple ComparisonsThe injected and top-applied treatments were different.*********** *** **************************************************Thelepho.ra terrestris LevelsTreatment 428E+3 3Applic. 2712.0 1Treat*Inj 2453.7 3Block 43005 53Residual 119E+3 541Total 594E+3 6010.143E+6 175.98 0.0000 Block(treat*inj)2.712E+3 3.3423 0.0732 Block(treat*inj)81.79E+1 1.0080 0.3966 Block(treat*inj)81.14E+1 3.6810 0.0000 Residual22. 04E+1There were no significant differences.P Test Term0.00810.09750.01150.01230.00360.5804 Block(treat*inj)0.0068 Block(treat*inj)0.4316 Block(treat*inj)0.0000 ResidualSourceSum of d MeanSquare f Square F P Test Term167Standard DeviationsoverallA51(1)Dan. E-strain(2)control (3)FGM (4)injected(a)top—applied(b)Multiple ComDarisons76.35592.82432.24989.04195.01377.65775.191Sum of d Mean31. 43913 . 01024.39114.78210.55529.57533.017Source Square f Square F P Test Term0.0000 Block(treat*inj)0.1117 Block(treat*inj)0.1960 Block(treat*inj)0.0000 Residualovera 11A51(1)Dan. E—strain(2)control (3)FGM(4)injected(a)top-applied(b)Means18.0900.945967.0310.00000.167817.45018.659Standard Deviations32.1395.986024.2160.00001.221430.04833.934Multiple ComparisonsBonferroni and Tukey homogeneous subsets(3.., 4.., 1..,)(2..)A51 (Suillus tomentosus) LevelsSum of d MeanSource Square fTreatment 452.67 3Applic. 68.547 1Treat*Inj 220.48 3Block 1456.6 53Residual 9723.6 541Total 11919 601150.8968.54773.49227.48317.973Bonferroni and Tukey homogenous subsets(2..)(3.., 1.., 4..)E-strain LevelsTreatment 523E+3 3 174E+3 292.87Applic. 1559.2 1 1559.2 2.6168Treat*Inj 2893.3 3 964.45 1.6187Block 31579 53 595.83 5.1410Residual 62701 541 115.90Total 620780 601Square F P Test Term5.4903 0.0023 Block(treat*inj)2.4942 0.1202 Block(treat*inj)2.6741 0.0566 Block(treat*inj)1.5291 0.0118 Residual168Mn nir1 rvitinnqoverallA51 ( 1)Dan. E-strain(2)control (3)FGM(4)injected(a)top-applied(b)albia2b2a3h3a4b4Multiple Comparisons0.54822.06080.00000.10340.06710.89780.23583.55070.75950.00000.00000.00000.18990.14490.0000Level of Total Infection by MycorrhizaeSum of MeanSum of d MeanSource Square fTreatment 3169.0 3Applic. 560.18 1Treat*In 365.24 3Block 8626.9 53Residual 43369 549Total 56140 6011056.3560.18121.75162.7778.9964.45338.74670.00000.92580.81926.23331.644512. 2803.00520.00000.00000.00001 . 25141.20390.0000Bonferroni and Tukey homogeneous subsets(2.., 4.., 3..)(1..)Treatment*application homogeneous subsetsTukey(a3., a2., b4., b2., a4., b3., bi.)(al.Bonferroni(a3., a2., b4., b2., a4., b3., bi.)(bi., al.)Square F P Test Term6.4897 0.0008 Block(treat*inj)3.4415 0.0691 Block(treat*in)0.7480 0.5284 Block(treat*inj)2.0605 0.0000 Residual169Stanard Deviationsoverall 97.277 9.6013A5l(1) 98.246 8.1708Dan. E—strain(2) 99.687 3.9529control(3) 93.499 13.438FGM(4) 97.513 9.5472injected(a) 98.327 7.7445top—applied(b) 96.324 10.942al 100.00 0.000bi 96.712 10.992a2 99.375 5.5902b2 100.00 0.000a3 94.971 12.316b3 92.212 14.301a4 98.814 6.9267b4 96.375 11.278Multiple ComparisonsHomogeneous subsetsTukeys(3..)(4.., 1.., 2..)Treatment* inject ionTukey(b3., a3., b4., bi.)(a3., b4., bi., a4., a2., al., b2.)Bonferroni(b3., a3., b4., bi., a4.,)(a3., b4., bi., a4., a2., al., b2.)Bonferroni(3.., 4..)(4.., 1.., 2..)homogeneous subsets170NURSERY SPRUCE ANOVARoot Collar DiameterSum of d MeanSource Square f Square FTreatment 1.1214 3 1.0412Applic. 0.0109 1 0.0304Treat*Inj 2.0597 3 1.9124Block 25.490 71 2.5673Residual 99.428 711Total 128.11 789Means Standard Deviationsoverall 3.3266 0.4030A29(1) 3.3587 0.3957Dan. E—strain(2) 3.2705 0.4326control(3) 3.3142 0.3869FGM(4) 3.3623 0.3901in:iected(a) 3.3301 0.4097top—applied(b) 0.4097 0.3964Multiple ComparisonsThere were no significant differences.Shoot LengthTreatment 209.70 3 3.4183Applic. 2.9801 1 0.1458Treat*Inj 5.3978 3 0.0880Block 1451.9 71 1.6555Residual 8770.4 710Total 10440 788Means Standard Deviationsoverall 15.531 3.6400A29(1) 16.348 3.8470Dan. E—strain(2) 14.963 3.5392control(3) 15.521 3.3204FGM(4) 15.288 3.6982injected(a) 15.470 3.7651top—applied(b) 15.594 3.5101Multiple ComparisonsTukey and Bonferroni homogeneous subsets(.2., .4., .3.)(.4., .3., .1.)P Test Term0.37380.01090.68660.35900.13980.3798 Block(treat*inj)0.8620 Block(treat*inj)0.1353 Block(treat*inj)0.0000 ResidualSourceSum of dSquare fMeanSquare F P Test Term69.9012.98011. 799320.44912 . 3530.0218 Block(treat*inj)0.7038 Block(treat*inj)0.9664 Block(treat*inj)0.0009 Residual171Root MassTreatment 1.0472 3Applic. 0.3630 1Treat*Inj 0.9933 3Block 12.262 71Residual 45.090 709Total 59.767 787Standard DeviationsoverallA29 (1>Dan. E-strain(2)control (3)FGM(4)injected(a)top-applied(b)Multiple Comparisons0.92430.93430.88480.89750.97880.90280.94640.27560 . 28560.26760.27180.26910.26980.2800There were no significant differences.************** **************************************************Shoot MassoverallA29(1)Dan. E-strain(2)control (3)FGM(4)injected(a)top-applied(b)ala2a3a4bib2b3b4Means1.59161.68281.46881. 61661.59781.55891.62481.62041. 45381.61601.54461.74531. 48391.61741.6505Standard Deviations0. 38090.37940.36640 . 36120.38590.38900.37000.38550. 38120.36660.40370.36450.34230.35710.3618SourceSum of dSquare fMeanSquare F p Test Terra0.3491 2.0211 0.1187 Block(treat*inj)0.3630 2.1018 0.1515 Block(treat*inj)0.3311 1.9172 0.1345 Block(treat*inj)0.1717 2.7157 0.0000 Residual0.0636SourceSum of dSquare fMeanSquare F p Test TermTreatment 4.7914 3 1.5971 6.9183 0.0004 Block(treat*inj)Applic. 0.8697 1 0.8697 3.7673 0.0562 Block(treat*ini)Treat*Inj.51413 3 0.1714 0.7424 0.5303 Block(treat*inj)Block 16.391 71 0.2308 1.7868 0.0002 ResidualResidual 91.473 708 0.1292Total 114.02 786172Multiole ComoarisonsHomogeneous subsetsTukey Bonferroni(.2.) (.2., .4.).4., .3., .1.,) (.4., .3., .1.)Treatment*Application homogeneous subsetsTukey and Bonferroni(a2., b2., a4., a3., b3., al., b4.)(a4., a3., b3., al., b4., bi.)******************************************************** ********Number of Short RootsTreatment 4.4771 3 2.1130Applic. 0.0514 1 0.0727Treat*Inj 2.6709 3 1.2605Block 50.146 71 2.2265Residual 225.22 710Total 282.59 788Means Standard Deviationsoverall 2.5919 0.5988A29(1) 2.6800 0.5376Dan. Estrain(2) 2.6281 0.5707control(3) 2.4736 0.6561FGM(4) 2.5800 0.6128injected(a) 2.5824 0.6034top—applied(b) 2.6015 0.5947Multiple ComparisonsThe were no significant differences.Root to Shoot RatioSum of d MeanSource Square f Square F P Test TermTreatment 0.0003 3 0.0026 0.9580 Block(treat*inj)Applic. 0.5987 1 2.1153 0.1059 Block(treat*ini)Treat*Inj 0.5827 3 2.0587 0.1134 Block(treat*inj)Block 6.6985 71 3.2444 0.0000 ResidualResidual 20.588 708Total 28.468 786SourceSum of dSquare fMeanSquare F P Test Term1.49240.05140.89030.70630 .31720.1062 Block(treat*inj)0.7882 Block(treat*inj)0.1945 Block(treat*in)0.0000 Residual0.00030.19960.19420.09430.0291173Means Standard DeviationsoverallA29(1)Dan. E—strain(2)control (3)FGM(4)injected(a)top-applied (b)Multiple ComparisonsThere were no significant differences.************* ***************************************************Dickson Quality IndexSum of d MeanSource Square f0.2395 Block(treat*inj)0.1574 Block(treat*inj)0.1634 Block(treat*inj)0.0000 ResidualoverallA29 (1)Dan. E-strain(2)control (3)FGM(4)injected (a)top—applied(b)Multiple ComparisonsMeans0.40200.39960.38520.39640.42630.39420.4099Standard Deviations0 .11910.11700.11590.11940.12120.11680.1210There were no significant differences.****************************************************************Thelephora terrestris LevelsSum of d MeanSource Square f SquareTreatment 210E+3 3 70019Applic. 644.29 1 644.2Treat*1n5 20267 3 6755Block 1225+3 71 1718Residual 3485+3 710 489.6Total 7015+3 78840.744 0.0000 Block(treat*inj)0.3749 0.5423 Block(treat*inj)3.9311 0.0118 Block(treat*inj)3.5096 0.0000 Residual0.59800.57520.61490.56700.63380.59810.5979o .19030.22180.16660.16640.19310.18830.1926Square F pTreatment 0.0471 3 0.0471 1.4068Applic. 0.1794 1 0.0598 1.7869Treat*Inj 0.1763 3 0.0588 1.7563Block 2.3763 71 0.0335 2.8309Residual 8.3704 708 0.0118Total 11.151 786Test TermF P Test Term174ntiri1 flriiM-inncoverallA29 (1)Dan. E-strain(2)control (3)FGM(4)inected(a)top—applied(b)ala2a4bib2b3b4Multiple ComparisonsHomogeneous subsetsTukey(.1.)31.51810.77521.72852.35242.20932.65630.34613.65014.80058.75043.6307.90028.92945.24440.79029 . 82718.60122.45928.36929 .20530.25529.37320.54619 .71624.78827.57316.02022.85130.47230.825Treatment 606E+3 3 202E+3Applic. 1780.4 1 1780.4Treat*Inj 6406.9 3 2135.6Block 38188 71 537.86Residual 145E+3 710 205.52Total 798E+3 788375.58 0.0000 Block(treat*inj)3.3100 0.0731 Block(treat*inj)3.9706 0.0112 Block(treat*inj)2.6171 0.0000 Residual(.1., .2.)(.4., .3.)Bonferroni(.2.)(.4., .3.)Treatment*Application homogeneous subsetsTukey and Bonferroni(bl., al., a2.)(al., a2., b2.)(b2., b4., a4., b3.)(b4., a4., b3., 13.)****************************************************************B—strain LevelsSourceSum of dSquare fMeanSquare F P Test Term175Mn ncrt1 flpiitirncoverall 16.967 31.823A29(1) 0.7500 4.4695Dan. E—strain(2) 64.673 29.586eontrol(3) 1.3526 7.6121FGM(4) 0.5500 4.2765in5ected(a) 18.312 34.365top—applied(b) 15.582 28.956al 0.6000 3.1205a2 71.050 30.275a3 1.2000 8.9081a4 0.4000 2.8141bi 0.9000 5.8767b2 58.232 27.552b3 1.5222 5.8927b4 0.7000 5.3664Multiple ComDarisonsTukey and Bonferroni homogeneous subsets(.4., .1., .3.).2.)Treatment*Application homegeneous subsetsTukey and Bonferroni(a4., al., b4., bi., a3., b3.)(b2.(a2.Amphinema byssoides LevelsSum of d MeanSource Square f Square F P Test TermTreatment 378E+3 3 126E+3 94.132 0.0000 Block(treat*inj)Applic. 5038.0 1 5038.0 3.7611 0.0564 Block(treat*inJ)Treat*Inj 8560.1 3 2853.4 2.1301 0.1040 Block(treat*in)Block 95106 71 1339.5 2.5502 0.0000 ResidualResidual 373E+3 710 525.26Total 860E+3 788Means Standard Deviationsoverall 33.261 33.044A29(1) 68.549 23.578Dan. E-strain(2) 9.1205 19.659coritrol(3) 26.094 27.667FGM(4) 28.799 27.437injected(a) 30.648 32.704top-applied(b) 35.945 33.217al 69.250 20.973a2 8.4000 19.973a3 18.450 23.897a4 26.500 26.982bi 67.850 26.011b2 9.8485 19.410b3 34.589 29.184b4 31.100 27.830176Multiple ComparisonsTukey and Bonferroni homogeneous subsets(.2.)(.3., .4.)(.1.)Treatment*Application homogeneous subsetsTukey Bonferroni(a2., b2., a3.) (a2., b2., a3.)(a3., a4., b4., b3.) (b2., a3., a4.)(bi., al.,) a3., a4.., b4., b3.)(bl., al.,)****************************************************************Total Level of Infected RootsSum of d MeanSource Square f Square FoverallA29(1)Dan. E-strain(2)control (3)FGM( 4)injected (a)top-applied ( b)ala2a3a4bib2b3b4Means82.25680.14996.30980.47371.99982.36182.14583.65095.25079.95070.60076.65097.37081.05573.400Standard Deviations21.04519 . 52210.00319.27324 . 70420. 58321.53417.86412.37819.79022.98720.5496.754918.77626 . 351Multiple ComparisonsTukey and Bonferroni(.4., .1., .3.)(.2.)homogeneous subsetsTreatment*Application homogeneous subsetsTukey Bonferonni(a4., b4., bl., a3.,b3.,al.) (a4., b4., bi., a3., b3., al.)(b3., al., a2.) (a3., b3., al., a2.)(al., a2., b2.) (b3., al., a2., b2.)***** ****** * **** ** * * **************************************** ****p Test TermTreatment 62035 3 20678 18.866 0.0000 Block(treat*inj)Applic. 13.507 1 13.507 0.0123 0.9119 Block(treat*inj)Treat*Inj 3111.1 3 1037.0 0.9462 0.4231 Block(treat*inj)Block 77819 71 1096.0 3.7744 0.0000 ResidualResidual 206E+3 710 290.39Total 349E+3 788177FIELD PINE ANOVARoot Collar DiameterTreatmentBlockResidualTotal4.465346.774198.74249.821.488411.6930.3667overallA51(l)Dan. E-strain(2)control (3)FGM (4)Means4.04444.01763.93624.17714.0396Standard Deviations0.67460. 65580.65710.66510.7009Multiple ComparisonsTukey and Bonferroni homogeneous subsets(2., 1., 4.,)(1., 4., 3.,)****************************************************** **********Incremental HeightSum of dSource Square fMeanSquare FTreatmentBlockResidualTotal57. 717171.935682.85916.219.23942.98410.524tndrd flviticnsoverallA51 ( 1)Dan. E-strain(2)control (3)FGM(4)Multiple Comparisons10.30010.84110.1419.913910.3943. 28872.97563.15633. 59633.3122There were no significant differences.TreatmentBlockResidualTotal218.06503 . 371332614053SourceSum of dSquare fMeanSre F P34542549Test Terra4.0592 0.0072 Residual32.89 0.0000 ResidualP34540547Test Terra1.8282 0.1409 Residual4.0844 0.0029 ResidualTotal HeightSourceSum of dSquare £MeanSquare F P3454254972.687125.8424.586Test Term2.9564 0.0320 Residual5.1185 0.0005 Residual178Means Standard Deviationsoverall 28.368 5.0594A51(1) 29.083 4.9795Dan. E—strain(2) 28.484 5.2561control(3) 27.359 5.0321FGM(4) 28.674 4.8471Multiple ComparisonsTukey and Bonferroni homogeneous subsets(3., 2., 4.)(2., 4., 1.)******************************************** ********************179Number of Dead SeedlingsSum of d MeanSource Square f P Test TermTreatment 0.0597 3Block 1.5278 4Residual 22.324 565Total 23.909 572Standard Deviationsoverall 0.9564 0.2044Dan. E—strain(1) 0.9600 0.1968A51(2) 0.9591 0.1985control(3) 0.9667 0.1801FGM(4) 0.9404 0.2375Multiple ComparisonsThere were no significant differences.****************************************************************Shoot MassTreatment 3.5366Block 1.9218Residual 33.078Total 38.536Means Standard Deviationsoverall 2.1192 0.6984Dan. E—strain(1) 2.1126 0.8383A51(2) 2.3822 0.6705control(3) 1.7977 0.5521FGM(4) 2.1844 0.6205Multiple ComparisonsHomogeneous subsetsTukey Bonferroni(3., 1., 4.) (3., 1., 4., 2.)(1., 4., 2.)Square F0.01990. 38190.03950.5035 0.6800 Residual9.6665 0.0000 ResidualSourceSum of dSquare fMeanSquare F P3472791.17890.48040.4594Test Term2.5660 0.0612 Residual1.0458 0.3898 Residual180Root MassTreatment 0.2070 3Block 0.2730 4Residual 11.868 72Total 12.348 790.0690 0.4185 0.74020.0682 0.4141 0.7980ResidualResidualoverallDan. E--strain(1)A51(2)control (3)FGM(4)1.12071.03391.15311.13611.160Standard Deviations0.39540.40850.32560.43140. 4318Multiple ComparisonsThere were no significant differences.****************************************************************Root to Shoot RatioTreatment 0.2914 3Block 0.1664 4Residual 3.6456 72Total 4.1033 790.09710.04160.0506Standard DeviationsoverallDan. E-strain(1)A51 ( 2)control (3)FGM( 4)Multiple Comparisons0.55900.51540.50230 . 65620. 56200.22790.19430.14080.23480.2965There were no significant differences.Number of Types of Mycorrhizae per RootSum of d MeanSource Square f Square F p Test TermTreatmentBlockResidualTotal9.1375 39.6750 474.675 7293.487 793.04582.41871.0372SourceSum of dSquare fMeanSquare F P Test TermSourceSum of dSquare fMeanSquare F p Test Term1.9181 0.1343 Residual0.8216 0.5156 Residual2.9367 0.0390 Residual2.3321 0.0639 Residual181Means Standard Dviatinoverall 2.7375 1.0878Dan. E—strain(1) 2.7000 0.9787A51(2) 3.0000 1.1698control(3) 2.2000 1.0563FGM(4) 3.0500 0.9987Multiple ComparisonsHomogeneous subsetsTukey Bonferronni(3., 1., 2.) (3., 1., 2., 4.)(1., 2., 4.)****************************************************************Foliar NitrogenSum of d MeanSource Square f Square F P Test TermTreatment 0.1578 3 0.0526 10.115 0.0914 ResidualBlock 0.4605 4 0.1151 22.139 0.0437 ResidualTrt*Block 0.1742 7 0.0249 4.7845 0.1837 ResidualResidual 0.0104 2 0.0052Total 0.8334 16Multiple ComparisonsThere 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 anysignificant differences.182FIELD SPRUCE ANOVARoot Collar DiameterSum of dSource Square IMeanSquare FTreatment 6.1647 3Block 87.201 4Residual 239.14 481Total 333.71 4882.054921.8000.4972overallA51 ( 1)Dan. E-strain(2)control (3)FGM( 4)Means4.50424.47024.32284.60634.6277Standard Deviations0 .82690.80280.81690.80740.8526Multiple ComparisonsTukey and Bonferroni homogeneous subsets(2., 1.)(1., 3., 4.)Incremental HeightSum of dSource Square IMeanSquare FTreatment 263.91 3Block 315.43 4Residual 4700.8 476Total 5293.9 48387.96978.8589.8757overallA51 ( 1)Dan. E—strain(2)control (3)FGM(4)Means11.58410.99210.72412.52312.160Standard Deviations3.31073.68213.30353.00402.8723Multiple ComparisonsTukey and Bonferroni(2., 1.)(4., 3.)homogeneous subsets**************************** ************************************P Test Term4.1332 0.0066 Residual43.849 0.0000 ResidualP Test Term8.9077 0.0000 Residual7.9851 0.0000 Residual183Total HeightSum of d MeanSource Square f Square FTreatment 169.54 3Block 295.79 4Residual 8726.6 481Total 9207.9 488Means Standard Deviationsoverall 25.606 4.3438A51(1) 26.205 4.6106Dan. E—strain(2) 24.615 4.4052control(3) 25.989 3.7469FGM(4) 25.652 4.4058Multiple ComparisonsHomogeneous subsetsTukey Bonferroni(2., 4., 3.) (2., 4., 3., 1.)(4., 3., 1.)Number of Dead SeedlingsSum of d MeanSource Square fTreatment 0.3120 3 0.1040Block 15.944 4 3.9861Residual 91.646 593 0.1548Total 107.86 599Means Standard Deviatinnqoverall 0.7650 0.4244A51(1) 0.7763 0.4181Dan. E—strain(2) 0.7667 0.4243control(3) 0.7297 0.4456FGM(4) 0.7867 0.4103Multiple ComparisonsThere were no significant differencesP Test Term56.514 3.1150 0.0260 Residual73.948 4.0759 0.0029 Residual18.143Square F P Test Term0.6718 0.5696 Residual25.748 0.0000 Residual184Shoot MassTreatment 6.3473 3Block 3.3950 4Residual 67.154 72Total 76.896 702 .11580.84870.9327overallDan. E-strain(1)A51(2)control (3)FGM (4)Means3.58023.12383.84633.57503.7756Standard Deviations0.98550.70350.92151.14751.0258Multiple ComparisonsThere were no significant differences.Root MassSum of d MeanSource Square f Square F P Test TermTreatment 3.7181 3Block 1.8326 4Residual 31.258 72Total 36.809 791.2394 2.8548 0.0431 Residual0.4582 1.0553 0.3850 Residual0.4341overallDan. E-strain(1)A51(2)control (3)FGM(4)1.79511.68221.88621.51912.0929Standard Deviations0.68260.40960.69500.53260.8984Multiple ComparisonsHomogeneous subsetsTukey(3., 1., 2.)(1., 2., 4.)Bonferroni(3., 1., 2., 4.)SourceSum of d MeanSquare f Square F P Test Term2.2684 0.0878 Residual0.9100 0.4649 Residual185Root to Shoot RatioSum of d MeanSource Square f Square F P Test TermTreatment 0.3865 3 0.1288 2.5598 0.0616 ResidualBlock 0.3345 4 0.0835 1.6614 0.1684 ResidualResidual 3.6236 72 0.0503Total 4.3446 79Means Standard Deviationsoverall 0.5291 0.2345Dan. E—strain(1) 0.6280 0.3536A51(2) 0.4883 0.1408control(3) 0.4443 0.1368FGM(4) 0.5558 0.2115Multiple ComparisonsThere were no significant differences.Foliar CalciumSum of d MeanSource Square f Square F P Test TermTreatment 0.0469 3 0.0156 5.1805 0.0159 ResidualBlock 0.0237 4 0.0059 1.9636 0.1645 ResidualResidual 0.0362 12 0.0030Total 0.1069 19Means Standard Deviationsoverall 0.3505 0.075Dan. E-strain(1) 0.316 0.039A51(2) 0.412 0.062control(3) 0.292 0.033FGM(4) 0.382 0.092Multiple ComparisonsHomogeneous subsetsTukey Bonferroni(3., 1.., 4.) (3., 1., 4., 2.)(1., 4., 2.)The other nutrient analyses were N, P, Mg, K, Cu, Zn, Fe, Mn, Band Al. No other differences between treatments weresignificant.186CONTINGENCY ANALYSIS FOR SEEDLINGS THAT DIED IN THE FIELDSAS was used to do a contingency analysis of how many seedlingshad died over the first growing season. There were twoindependent variables, treatment and block. The analysis wasdone controlling for block.Summary Statistics For Field Pine, Treatment by the Number Dead:Controlling For BlockCOCHRAN-MANTEL-HAENSZELL STATISTICS (BASED ON TABLE SCORES NOTSHOWN)STATISTIC ALTERNATIVE HYPOTHESIS DF VALUE PROBABILITY1 Nonzero Correlation 1 0.003 0.9592 Row Mean Scores Differ 3 2.025 0.5673 General Association 3 2.025 0.567Total Sample Size = 600Summary Statistics For Field Spruce, Treatment by the NumberDead: Controlling For BlockCOCHRAN-MANTEL-HAENSZELL STATISTICS (BASE ON TABLE SCORES NOTSHOWN)STATISTIC ALTERNATIVE HYPOTHESIS DF VALUE PROBABILITY1 Nonzero Correlation 1 0.523 0.4692 Row Mean Scores Differ 3 1.524 0.6773 General Association 3 1.524 0.677Frequency Missing = 19 Effective Sample Size = 573187

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