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Sensitivity in growth responses of tree seedlings to variation in identity and abundance of ectomycorrhizal… Karst, Justine Delaney 2007

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S E N S I T I V I T Y IN G R O W T H R E S P O N S E S O F T R E E S E E D L I N G S T O V A R I A T I O N IN IDENTITY A N D A B U N D A N C E O F E C T O M Y C O R R H I Z A L F U N G I by Just ine Delaney Karst B. S c . , University of Alberta, 1999 M. S c . , McGi l l University, 2001 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F ' D O C T O R O F P H I L O S O P H Y in The Faculty of Graduate Stud ies (Botany) T H E U N I V E R S I T Y O F BRIT ISH C O L U M B I A July 2007 © Just ine Delaney Karst, 2007 II Abstract Interdependent o rgan isms such as trees and ectomycorrhizal fungi are descr ibed as coevo lved. Partner spec ies in coevo lved interactions are expected to be sensi t ive to intraspecif ic variation of each partner due to the intimate and interdependent nature of their interactions. In this thesis, I cons idered specif ic aspec ts of variation in e a c h of the ectomycorrhizal partners and how this variation influenced the other partner. In particular, I used exper imental and meta-analyt ical approaches to evaluate 1. how colonizat ion levels, regardless of ectomycorrhizal fungal taxon, correlated to host growth; 2. how ectomycorrhizal fungi differentially inf luenced growth of different genera of plant hosts, and 3. how variation in growth of a single host spec ies w a s correlated to the composi t ion of ectomycorrhizal fungal communi t ies in var ious soil envi ronments. B e c a u s e controll ing for and manipulat ing ectomycorrhizal fungi on host plants is integral to these quest ions, I a lso tested the eff icacy of two methods to control colonizat ion by ectomycorrhizal fungi on host plants and found that fungicides and mesh can be effective barriers to colonizat ion. Resul ts from the meta-analysis and exper iments indicated that colonizat ion levels did not consistently sca le with host growth response , however, suggest ing that colonizat ion levels may not be an ecological ly useful factor to gauge the growth responses of host plants to ectomycorrhizal fungi. In addit ion, there w a s little sensitivity in growth responses of host plants to variation in the identity of ectomycorrhizal fungi. Seed l ings ac ross multiple host genera increased in b iomass and shoot height when inoculated with ectomycorrhizal fungi regardless of the identity of the fungal assoc ia te . W h e n ectomycorrh izas were cons idered in a multi-specif ic context (i.e. one host spec ies assoc ia ted with a community of ectomycorrhizal fungi), variation in host shoot properties was not correlated with spec ies composi t ion of the communi ty of ectomycorrhizal fungi on their roots but rather appeared to be coupled to edaphic condit ions. T h e s e results indicate that the variation in ectomycorrhizal fungi perce ived and se lected for by the host plant may be of a discrete (p resence/absence of ectomycorrhizal fungi) rather than cont inuous nature (variation in identity or abundance of ectomycorrhizal fungi). Ill Table of Contents Abstract ii Tab le of Contents iii List of Tab les vii List of F igures iix Acknowledgements xii Co-authorsh ip Statement xiii 1 Introduction 1 Context 1 Literature Rev iew 4 C a u s e s of special izat ion versus general izat ion 4 Specia l izat ion versus general izat ion in ectomycorrhizal assoc ia t ions 5 Ecolog ica l material for specia l izat ion: what variation is present among ectomycorrhizal fungi to which plant hosts could respond? 6 Overv iew of Thes is .....6 Re fe rences 8 2 The mutual ism-parasi t ism cont inuum in ectomycorrh izas: A quantitative assessmen t using meta-analys is 13 Introduction 13 Methods 15 Data col lection 15 Data analys is 16 Resul ts 17 Seed l ing response to ectomycorrhizal inoculation 17 Seed l ing response to ectomycorrhizal inoculation and phosphorus addit ion 19 D iscuss ion 20 iv Seed l ing response to ectomycorrhizal inoculation 20 The spectrum is reduced: Publ icat ion b ias inflates measures of effect s i zes 20 The spectrum is distorted: Factors that covary with time may cause spur ious effects 21 The spectrum is distorted: Effects of cross ing hosts and ectomycorrhizal fungi not known to co-occur remain poorly understood 22 The role of variation in fungal properties in host response to ectomycorrhizal inoculat ion 23 Conc lus ions and future directions 26 Re fe rences 42 3 Methods to control ectomycorrhizal colonizat ion: Ef fect iveness of chemica l and physica l barriers 48 Introduction 48 Mater ials and methods 50 Field soi l col lection 50 Plant material 51 Fungic ide exper iment 51 Exper imental des ign and treatments 51 Seed l ing measurements 52 M e s h barrier experiment 53 Exper imental des ign and treatments 53 Seed l ing measurements 54 Molecu lar confirmation of ectomycorrhizal fungal spec ies identification 54 Statist ical analys is 55 Resu l ts 56 Fungic ide treatments 56 M e s h barrier treatments 57 D iscuss ion 58 Fungic ide effects on ectomycorrhizal colonizat ion 58 M e s h barrier effects on hyphal penetration 61 V Conc lus ions 62 Refe rences 78 4 Ectomycorrhizal colonizat ion and intraspecific variation in growth responses of lodgepole pine 83 Introduction 83 Methods : 84 Greenhouse experimental set-up 84 A s s e s s m e n t of ectomycorrhizal fungal colonizat ion 85 Molecular ana lyses 86 Statistical ana lyses 86 Resu l ts 87 D iscuss ion 88 S e e d family effects on the relationship between colonizat ion level and host growth 88 Ectomycorrhizal colonizat ion and host phenotypic variation 89 The prevalence of contaminat ion on seedl ings 91 Conc lus ions 91 References 97 5 Interactions among soil character ist ics, host intraspecific variation and ectomycorrhizal fungal communi t ies 100 Introduction 100 Materials and methods 101 Overv iew 101 Origin of soi ls 102 Soi l col lect ion 102 Plant material 103 Main tenance of soil moisture 103 Final harvest 104 Statistical ana lyses 105 vi Resu l ts 105 References 119 6 Conc lus ions 123 The relationship between colonizat ion level and host growth response is inconsistent 124 There is little sensitivity in growth responses of host plants to variation in the identity of ectomycorrhizal fungi 124 Publ icat ion b ias exists in the ectomycorrhizal literature 125 Future research directions 125 Final conc lus ion 127 Refe rences 128 Append i ces 130 A . Identity of host plant and fungal spec ies pairings and effect s i zes (Ln R) for seedl ing b iomass , shoot height and shoot:root ratio for each study used in meta-analys is 130 Full citation of e a c h study used in meta-analys is (excluding studies involving manipulat ions of nutrients) 152 B. Identity of host plant and fungal spec ies pair ings with assoc ia ted effect s i zes (Ln R) for seedl ing b iomass 160 Full citation of each study used in the meta-analysis examining the effects of phosphorus addit ion on the outcome of ectomycorrhizal assoc ia t ions 169 VII List of Tables Table 2.1 :Means and standard errors (SE) for the influence of location of experiment on the magnitude of contaminat ion 28 Table 3.1: Ana lys is of var iance for effect of fungicide type (F), concentrat ion (C), and applicat ion f requency (A) on square root percent ectomycorrhizal colonizat ion (%) and s ize of Douglas-f ir (Pseudotsuga menziesii var. glauca) seedl ings after five months 64 Tab le 3.2: Descript ion of morphological character ist ics of ectomycorrh izas observed on Douglas-f i r {Pseudotsuga menziesii var. glauca) seedl ings grown in the fungicide (F) and mesh (M) study 65 Table 3.3: Effect of steril ization on growth and ectomycorrhizal (EM) colonizat ion of Douglas-f i r {Pseudotsuga menziesii v rar. glauca) seedl ings. A ser ies of t-tests were used to determine di f ferences among source (S) and recipient (R) seedl ings grown for each mesh barrier treatment 68 Table 3.4: Effect of mesh treatment on growth and ectomycorrhizal (EM) colonizat ion of Douglas-f i r (Pseudotsuga menziesii var. glauca) seedl ings. R e s p o n s e di f ferences between source and recipient seedl ings were calculated for each pot. Th is single number was used in the A N O V A for each response variable. Statistically signif icant mesh treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05) 70 Table 4 .1 : Ana lys is of covar iance for effects of seed family, percent ectomycorrhizal fungal colonizat ion of root tips (% colonization) and their interaction on growth responses of Pinus contorta Dougl . ex Loud. var. latifolia Enge lm. seedl ings 93 Table 4.2: M e a n shoot height of full sib famil ies of Pinus contorta Dougl . ex Loud . var. latifolia Enge lm. Seed l ings grown for 36 weeks (n=8). British Co lumb ia Ministry of Forests seed family identification fol lows in brackets seed family designat ion 94 Table 5.1: Site coordinates and elevation of soil sampl ing locat ions 110 Table 5.2: Fertility character ist ics of soi ls col lected from six sites from the T h o m p s o n -O k a n a g a n region of British Co lumb ia . Va lues are from a composi te of 6 samp les per site 111 Tab le 5.3: Types of statistical ana lyses (canonical cor respondence analys is [CCA] or redundancy analys is [RA]) used and signif icance of explanatory var iables tested to VIII explain measures of ectomycorrhizal fungal community composi t ion or seedl ing growth traits. Only those soil aspec ts of soil fertility found to be significant are reported in table. Numbers in brackets represent percentage of var iance expla ined by each significant explanatory factor 112 ix List of Figures Figure 2 .1 : Cumulat ive mean effect s i zes for total b iomass, shoot height and shoot:root ratio. Error bars are 9 5 % bootstrapped conf idence intervals 29 Figure 2.2: M e a n effect s ize for a) total b iomass and b) shoo t roo t ratio by host genus . M e a n s with 9 5 % bootstrapped conf idence intervals are shown. M e a n s fol lowed by the s a m e letter are not statisticaly different (95% bootstrapped conf idence intervals overlap). For b), positive va lues indicate al location of b iomass to shoots w a s higher than al location to roots 30 Figure 2.3: M e a n effect s ize for shoot roo t ratio by fungal genus . M e a n s with 9 5 % bootstrapped conf idence intervals are shown. M e a n s fol lowed by the s a m e letter are not statistically different (95% bootstrapped conf idence intervals overlap). Posi t ive va lues indicate al location of b iomass to shoots w a s higher than that al located to roots 31 Figure 2.4: Relat ionship between mean effect s i zes and level of ectomycorrhizal fungal colonizat ion of inoculated seedl ings for a) total b iomass, b) shoot height and c) shoot:root ratio. Outl iers (those data points falling above the 9 7 t h percenti le of the distribution) are indicated a s tr iangles; these were retained in the analys is 32 Figure 2.5: Relat ionship between mean effect s i zes and magnitude of contaminat ion for a) total b iomass , b) shoot height and c) shoohroot ratio 34 Figure 2.6: Relat ionship between effect s i zes and duration of assoc iat ion of ectomycorrhizal fungus and host for a) total b iomass, b) shoot height and c) shoo t roo t ratio. Q M / Q T is the amount of total heterogeneity in the data due to variation in effect s i zes expla ined by the model . Statist ics are reported for significant models only 36 Figure 2.7: Relat ionship between magnitude of contamination and duration of associat ion of ectomycorrhizal fungus and host 38 Figure 2.8: Relat ionship between effect s ize residuals and duration of assoc ia t ion of ectomycorrhizal fungus and host for a) total b iomass, b) shoot height and c) shoo t roo t ratio. Stat ist ics are reported for significant models only 39 Figure 2.9: Relat ionship between effect s ize residuals for total b iomass and amount of phosphorus added 41 Figure 3.1: Effect of a) fungicide type and b) application f requency on percent ectomycorrhizal colonizat ion (determined by clearing and staining root tips) of X Douglas-f i r (Pseudotsuga menziesii var. glauca) seedl ings. Fungic ide abbreviat ions: S = Senator® and T = Topas®. Frequency abbreviat ions: A = once upon commencement of the experiment, B = every two months, and C = once a month. Statistically significant fungicide treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05). Error bars are one standard error of the mean 72 Figure 3.2: Abundance of morphotypes {Tomentella-type (Tom) Thelephora terrestris (T); Mycelium radicis afrow'rens-type (MRA) ; Wilcoxina rehmii(\N); Cenococcum geophilum (Cg) ; Rhizopogon/Suillus-type (R/S) ; Piloderma-type (P) and Undifferentiated (Undif) found on morphotyped Douglas-f ir (Pseudotsuga menziesii var. glauca) root sys tems grown in soil treated with different a) fungicide types and b) appl icat ion frequency. Fungic ide abbreviat ions: S = Senator® and T = Topas®. Frequency abbreviat ions: A = once upon commencement of the experiment, B = every two months, and C = once a month 73 Figure 3.3: Abundance of Wilcoxina reftm/V ectomycorrh izas, as a percentage of all root tips examined on Douglas-f ir (Pseudotsuga menziesii war. glauca) grown in soi l treated with fungicides. Fungic ide abbreviat ions: S = Senator® and T = Topas®. Statistically significant fungicide type treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05). Error bars are one standard error of the mean 75 Figure 3.4: Abundance of morphotypes (Thelephora terrestris (T); Mycelium radicis atrovirens-type (MRA) ; Wilcoxina rehmii{\N); Cenococcum geophilum (Cg) ; Rhizopogon/Suillus-type (R/S) ; and Undifferentiated (Undif), as a percentage of all root tips examined on recipient (R) and source (S) soil seedl ings separated by a mesh barrier 76 Figure 3.5: Ectomycorrhizal community dif ferences, a) Ste inhaus similarity index for ectomycorrhizal communi t ies observed on source and recipient seedl ings separated by a mesh barrier, b) R i chness difference = number of morphotypes observed on source Douglas-f ir (Pseudotsuga menziesii var. glauca) root sys tems minus morphotypes present on recipient Douglas-f ir separated by a mesh barrier. Statistically significant mesh treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05). Error bars are one standard error of the mean 77 XI Figure 4 .1 : The effect of ectomycorrhizal fungal colonizat ion by s e e d family on shoot (top panel) and root m a s s (bottom panel) of Pinus contorta var. latifolia seed l ings. Regress ion l ines are shown for only those s e e d famil ies showing a signif icant relationship between shoot or root mass and level of colonizat ion. S e e Tab le 4.2 for Brit ish Co lumb ia Ministry of Forests seed family identification 95 Figure 4.2: The contribution of ectomycorrhizal fungal colonizat ion to height variation in seedl ings of Pinus contorta Dougl . ex Loud. var. latifolia Enge lm. , independent of s e e d family effects 96 Figure 5.1: Relat ionships between soil moisture (%) and seedl ing height, b iomass and root:shoot ratio J. 113 Figure 5.2: Relat ionships between soil C : N and seedl ing height, b iomass and root:shoot 114 Figure 5.3: Frequency of ectomycorrhizal morphotypes observed ac ross seed l ings of Pseudotsuga menziesii var. glauca 115 Figure 5.4: Canon ica l cor respondence analys is of ectomycorrhizal morphotypes observed on seedl ings of Pseudotsuga menziesii var. glauca ordinated a long gradient of % total soil nitrogen 116 Figure 5.5: Relat ionship between percent ectomycorrhizal colonizat ion and roo tshoot ratio of Pseudotsuga menziesiivar. glauca seedl ings 117 Figure 5.6: Canon ica l cor respondence analys is of ectomycorrhizal morphotypes observed on seedl ings of Pseudotsuga menziesii var. glauca ordinated along gradient of seedl ing height 118 XII Acknowledgements Many people contributed to this thesis. My superv isors, Roy Turkington and Melan ie J o n e s , offered complementary perspect ives on my work. They have been brilliant mentors and I thank them for agreeing to superv ise my endeavors . My family offered stellar support; Dave and C l e a spent count less hours in the g reenhouse just so we could spend "quality" time together as a family. For the thesis chapters I am grateful to J a s o n H o e k s e m a for comments on an earlier draft of Chapter 2. The work in Chapter 3 could not have been f inished if not for the help of Cand i s Staley and Lenka Kudrna, who ass is ted with applying the fungicide treatments, and the molecular analys is , respectively. I thank Aaron Patterson who provided technical ass is tance in the greenhouse to generate the data col lected in Chapter 4 and to Michae l Car l son of British Co lumb ia Ministry of Forests , R e s e a r c h Branch , Ka lama lka Forestry Center , who generously provided seed for that particular experiment. The experimental design of Chapter 4 benefited from thoughtful d iscuss ions with Prof. Sal ly Ai tken. I am grateful to Andrew MacDouga l l , Kate Kirby and Mark Ve l lend for comments on earlier vers ions of the manuscript presented in Chapte r 5. In addit ion, Dan Durall provided fungal D N A sequenc ing results, Frangois Tes te a ided in the morphotyping of ectomycorrh izas and Soren Brothers provided technical ass is tance in the greenhouse. Al though none of my field exper iments were success fu l , it w a s not for lack of commitment by summer research assistants. Kristen M a c K a y , Fawn R o s s and J e s s i e Mack ie were remarkable in their t ime, energy and perseverance on all tasks. A spec ia l thanks to B e n Gilbert, my friend and co l league who helped me with all s tages of the thesis. Thanks a lso to S u z a n n e S imard and Gary Bradfield for careful ly reading earlier vers ions of the thesis and being active members on my supervisory committee. XIII Co-authorship Statement Chapter 2 was co-authored with Drs. Laurie Marczak , Melanie J o n e s and Roy Turkington. I identified, des igned and conducted the research, including data col lect ion, analys is and manuscript preparation. Laurie Marczak ass is ted with data col lect ion, analys is and manuscript preparation and revision. Melanie J o n e s and Roy Turkington ass is ted with manuscript revision. Chapter 3 was co-authored with Frangois Teste , Drs. Melan ie J o n e s , S u z a n n e S imard and Dan Dural l . Frangois Teste and I are equal contributors, with authorship ranking determined by a coin toss. He and I identified, des igned and conducted the research, including data col lect ion, analys is and manuscript preparation. Melanie J o n e s , S u z a n n e S imard and Dan Durall ass is ted with manuscript revision. S u z a n n e S imard a lso ass is ted with data analys is and Dan Durall was responsib le for the molecular analys is of fungal samples . Chapter 4 was co-authored by Drs. Melan ie J o n e s and Roy Turkington. I identified, des igned and conducted the research, including data col lect ion, analys is and manuscr ipt preparation. Melan ie J o n e s and Roy Turkington ass is ted with manuscr ipt revision. Chapter 5 was co-authored by Drs. Melan ie J o n e s and Roy Turkington. I identified, des igned and conducted the research, including data col lect ion, analys is and manuscr ipt preparation. Melan ie J o n e s and Roy Turkington ass is ted with exper imental des ign and manuscript revision. 1 1 Introduction Context Interdependent spec ies that adapt to changes in each other are descr ibed as coevo lved. E a c h partner in the relationship exerts select ive pressures on the other, thereby affecting each others' evolution. Coevolut ion is the p rocess resulting from a c lose associat ion between the individuals of two, or more, different spec ies (Thompson 1994). Plants and mycorrhizal fungi appear to have had such an interdependent relationship s ince plants invaded land. The assoc iat ion between plants and mycorrhizal fungi can also be cons idered symbiot ic, def ined by de Bary (1878 as cited in S a p p 1994) as "the living together of unlike named organisms". Ectomycorrh izas are characterist ic of tree spec ies within the famil ies P i n a c e a e , C u p r e s s a c e a e , F a g a c e a e , Myr taceae, Betu laceae, and Sa l i caceae and coevolut ion between phyto- and mycobionts from severa l orders including Agar ica les , Gaut ier ia les, Hymenogast ra les , Phal la les, Lycoperda les, Melanogast ra les , Sc lerodermata les , Aphyl lophora les, Pez i za les and E laphomyceta les has been occurr ing for about 200 million years (Kendrick 2000). A n ectomycorrh iza is the physical assoc iat ion of roots and ectomycorrhizal fungi, with the fungus forming a compact layer of hyphae around the roots (mantle) connected to a network of hyphae growing in between root cel ls (Hartig net). Nutrient transfer (carbon to the mycobiont suppl ied by the host plant and mineral nutrients v ia the fungus to roots of the phytobiont), occurs at the interface between the Hartig net and root cel ls (Smith and R e a d 1997). Historically, ectomycorrh izas have been categor ized as mutualistic because each symbiont w a s deemed to benefit from the exchange of resources (Sapp 1994). Ectomycorrhizal symbionts vary in taxonomic identity, morphology, function and abundance , and symbionts may evolve to these character ist ics in response to each other. In spite of their intimate interactions with their symbiot ic partner, plants and fungi a lso respond to abiotic and biotic factors external to the symbios is . Fungi forming ectomycorrhizal assoc ia t ions with roots of a host plant will interact with the biotic (e.g. bacter ia, microfauna) and abiotic (e.g. soil solution chemistry, water potential) environment of the soi l matrix. For example , soil fauna can consume up to 5 0 % of ectomycorrhizal hyphae (Seta la 1995) and up to 55 isolates of bacteria are reported to 2 occur on ectomycorrh izas formed between a single host-fungus combinat ion (Bending et a l . 2002). The ecological ampli tude of host plants (measured by height and b iomass performance) is clearly dependent on soil properties such a s nutrient and moisture availability (Burns and Honka la 1990).Thus, ectomycorrh izas exist in a complex biotic and abiotic mil ieu, and heterogeneity in either the biotic or abiotic portions of that mil ieu will be ecological ly significant to the associat ion. Within a forest stand the number of ectomycorrhizal fungal spec ies is an order of magnitude higher than that of host spec ies (Bruns 1995). Dickie (2007) recently showed that total ectomycorrhizal fungal r ichness is a l inear function of the number of ectomycorrhizal plant spec ies ; one hundred fungal spec ies are predicted to assoc ia te with just 2 host plant spec ies . Within a forest s tand, both edaphic condit ions (Farley and Fitter 1999, J a m e s et a l . 2003) and the distribution of ectomycorrhizal fungi are spatial ly heterogeneous (Jonsson et a l . 2000, Li l leskov et a l . 2004, Izzo et a l . 2005). Root sys tems of individual trees normally exper ience temporal and spatial variation both in soi l properties and in the taxonomic identity and abundance of ectomycorrhizal fungi present in the soi l . A s a result, individual trees form mycorrh izas with a diverse community of ectomycorrhizal fungi. In this thesis I consider speci f ic aspec ts of variation in each of the ectomycorrhizal partners and how this variation may inf luence the other partner. Hosts can vary in taxonomic identity, and within a spec ies , hosts vary genetical ly and phenotypical ly. Similarly, ectomycorrhizal fungal communi t ies vary in spec ies composi t ion, individual fungi vary in anatomy and physiology, and populat ions of different fungal spec ies vary in their abundance on root sys tems and in the soil a s inoculum. W e do not yet have a c lear understanding of how finely-tuned phyto- and mycobionts are to each other. Statistically exp ressed , this means that we do not have a s e n s e of the proportion of the total variation in a particular aspect of one partner that is expla ined by variation in the other. In this thesis, I use experimental and meta-analys is approaches to evaluate: i. how colonizat ion levels, regardless of ectomycorrhizal fungal taxon, correlate to host growth ii. how ectomycorrhizal fungi differentially inf luence growth of different genera of plant hosts, and 3 iii. how variation in growth of a single host spec ies correlates to the composi t ion of ectomycorrhizal fungal communi t ies in var ious soi l environments. Integral to these quest ions, and an issue that is central to my thesis, is how to control for and manipulate ectomycorrhizal fungi on host plants. In my exper iments, I used growth of seedl ings as a measure of host response. I did this for two reasons. First, experiment ing with adult t rees is intractable. S e c o n d , while the seedl ing phase is relatively short in compar ison to the entire l i fespan of the tree, select ion pressures are high at this stage (Harper 1977). It has a lso been shown that tree spec ies are more strongly adapted to their regeneration niche than to the adult niche (Poorter 2007), thus the condit ions influencing seedl ings are important for predicting the distribution of adult trees. B e c a u s e seedl ings cannot reproduce, I use growth as my primary measure of performance as is typically done in ectomycorrhizal s tudies (see those in Chapter 2). Considerat ion of variation in both partners of the ectomycorrhizal symbios is to the growth response of either partner has been investigated over the past few d e c a d e s of mycorrhizal research. The novelty of this thesis is the evaluation of this variation from multi-specif ic and r e v o l u t i o n a r y perspect ives. I use the term multi-specific to denote the situation where a host plant interacts with many spec ies of ectomycorrhizal fungi. Most research has focused on evaluat ion of host plants inoculated by a single, target fungal spec ies (but see Baxter and Dighton 2001 , Kranabetter 2004), yet in nature seedl ings are almost a lways co lon ized by severa l ectomycorrhizal fungi concurrently. I cons ider responses both to different individual fungi and to different communi t ies of fungi. I use the term coevolut ion in the broad sense meaning "trait-matching" (Bronstein et a l . 2006), in contrast to the more restrictive definition meaning reciprocal evolut ionary change in interacting spec ies (Thompson 1994). No formal definition of trait-matching exists; however, Gomulk iew icz et a l . (2007) illustrate the concept with the example of plant f lowering time synchron ized to time of pollinator emergence. Implicit in my thesis is the understanding that host plants and ectomycorrhizal fungi are coevo lved. Understanding precisely how variation in either partner of the ectomycorrhizal symbios is is matched by the other partner al lows us to make conc lus ions about the sensitivity of the growth responses between symbionts. Host plants showing the s a m e growth response to variation in mycobionts, regardless of fungal taxon or extent of colonizat ion, suggests a response of low sensitivity. Synchron ized responses between variation in host plant growth and that present in mycobionts is suggest ive of a more sensit ive response. Addit ionally, because each symbiont can a lso respond independently to its abiotic environment, variation in the abiotic component may alter the associat ion or even supercede the importance of changes in fungi or host plants involved in the symbios is . Literature Review The level of sensitivity between symbiont responses can be v iewed a s a measure of the general izat ion or special izat ion that has occurred as a result of coevolut ion. For example , if growth responses of a host plant spec ies are independent of variation in taxonomic identity of its ectomycorrhizal fungi, and in nature host plants were found to assoc ia te with a very large number different spec ies of ectomycorrhizal fungi, these f indings would indicate this particular host plant is a generalist. Converse ly , if it were shown that host growth responses were highly sensit ive to the identity of the ectomycorrhizal fungus, and that in nature the host plant w a s found to assoc ia te with a narrower range of fungi, this would suggest that host plants are spec ia l ized. Causes of specialization versus generalization Specia l izat ion is a somewhat arbitrary and relative term used to represent the range of resources a spec ies uses . In the context of coevolut ion of mycorrh izas, this could refer to the number of spec ies with which a particular spec ies interacts. General izat ion and special izat ion are not static categor ies (Holmes 1977) and ev idence has rejected the hypothesis that special izat ion is a "dead-end" . It is c lear that swi tches between each mode over evolutionary time have been frequent (Thompson 1994, J a n z et a l . 2001 , Nosi l and Mooers 2005). Addit ionally, accumulat ing research suggests that coevo lved partners are highly asymmetr ic in their degree of special izat ion (Bronstein et a l . 2006). For example , in plant-pollinator sys tems pollinators tend to spec ia l ize on a plant spec ies , but a given plant spec ies may be visited by many different spec ies of poll inators (Vazquez and A i zen 2004). Most theories on the c a u s e s of special izat ion invoke the role of variation in the environment or in some attribute of the organ isms involved. For example , within trophic groups, special izat ion is thought to be a response to environmental constancy and the presence of interspecific interactions, most notably competit ion (Futuyma and Moreno 1988). The degree of special izat ion ac ross trophic 5 groups, such as that observed between coevolved organisms is hypothesized to be a result of variation in availability of partners. For example , Stebbins (1970) posi ted that general izat ion in plant-pollinator sys tems is favored when the availability of the most effective pollinator is unpredictable and conversely, special izat ion is favored when pollinator availability is reliable. Within symbiotic sys tems, Douglas (1998) reviewed c a u s e s of general izat ion between hosts and symbionts. S h e suggested that when ef fect iveness of symbionts var ies differentially with environmental condit ions, and these condit ions are unpredictable relative to host generat ion time, host specia l izat ion should not be favoured. Specia l izat ion is a lso d isadvantageous when the abundance of symbionts in free-living condit ion is low or their spatial distribution is unpredictable. Specialization versus generalization in ectomycorrhizal associations The degree of special izat ion within ectomycorrhizal assoc ia t ions is typically asymmetr ic between myco- and phytobionts. Host plant spec ies tend to assoc ia te with a higher number of fungal spec ies compared to the number of host spec ies with which an ectomycorrhizal fungal spec ies forms assoc ia t ions (Mal loch et a l . 1980; Borowicz and Jul iano 1991). General ly , most ectomycorrhizal fungi form assoc ia t ions with multiple host spec ies (Horton and Bruns 1998, S imard et a l . 1997, Massicot te et a l . 1999, Kennedy et a l . 2003, Nara 2006), with some except ions: the genera Rhizopogon and Suillus assoc ia te primarily only with Pseudotsuga menziesii and Pinus spp. There are a lso severa l fungal spec ies that are found only in associat ion with Alnus spp . (Mol ina et a l . 1992). Hosts appear broadly receptive to different spec ies of ectomycorrhizal fungi, with Alnus having a somewhat restricted receptivity. O n e except ion to this pattern is the high specif icity observed between plants in the Monotropoideae and their ectomycorrhizal fungi (Bruns et a l . 2002, Bidartondo and Bruns 2005). W h e n cons idered in a multi-specif ic context, plant host attributes often structure the composi t ion of ectomycorrhizal fungal communit ies. For example, the composi t ion of ectomycorrhizal fungal communi t ies var ies among c lones of Picea abies differing in relative growth rate but grown in the s a m e soil (Korkama et a l . 2006). In addit ion, there is a negative relationship between similarity among ectomycorrhizal fungal communi t ies and taxonomic distance among hosts - similarity among ectomycorrhizal fungal communi t ies is higher on hosts of the s a m e genus or family (Ishida et a l . 2007) . Thus , 6 variation in host character ist ics may be an ecological ly important gradient which is partitioned by ectomycorrhizal fungal spec ies . Ecological material for specialization: what variation is present among ectomycorrhizal fungi to which plant hosts could respond? Ectomycorrhizal fungi vary in their abundance and spatial and temporal distribution within the soil ( Jonsson et a l . 2000, Li l leskov et a l . 2004, Izzo et a l . 2005 , Ko ide et a l . 2007). Hence , as roots of an individual host forage through soi l , they will encounter different spec ies and genotypes of ectomycorrhizal fungi. Funga l portions of the mycorrhiza, such as mantle and extramatrical hyphae, vary morphological ly depending on the fungal spec ies involved (Agerer 1987-1998, G o o d m a n et a l . 1996). Funct ional variation among spec ies of ectomycorrhizal fungi has been reported for carbon demand (Bidartondo et a l . 2001), nutrient uptake (Read and Pe rez -Moreno 2003), and pH (Wal lander 2002, Y a m a n a k a 2003, Dunabei t ia et a l . 2004) and drought tolerance (Parke et a l . 1983, Boyle and Hel lenbrand 1991, Dixon and Hiol-Hiol 1992). Variat ion among fungal isolates of the same spec ies has also been reported for nutrient uptake (Cairney 1999, Sawyer et a l . 2003, Guidot et a l . 2005). Overal l colonizat ion levels and hyphal b iomass of ectomycorrhizal fungal usually dec reases in soi ls having high nitrogen or phosphorus levels (Treseder 2004), but different spec ies of ectomycorrhizal fungi differ in their sensitivity to changes in nitrogen and phosphorus (Jones et a l . 1990, Brandrud and T immermann 1998, Wa l lenda and Kottke 1998, N i lsson and Wal lander 2003 , but see C l e m m e n s e n et a l . 2006). Overview of Thesis In Chapter 2 using meta-analys is, I quantitatively a s s e s s which c a u s e s more variation in host growth responses to ectomycorrh izas: changes in host or fungal taxonomic identity? In addit ion, I examine whether colonizat ion levels, regardless of fungal identity, correlate to plant host response. In the meta-analys is, the effect of ectomycorrh izas is based on compar isons of non-inoculated to inoculated seedl ings. I highlight limitations to this approach in Chapter 2, but I a lso review and test the major techniques currently avai lable to create ectomycorrhizal controls in Chapter 3, where the results of implementing physical and chemica l barriers to ectomycorrhizal colonizat ion are presented. In Chapter 4,1 explore the contribution to seedl ing growth of 7 variation in ectomycorrhizal colonizat ion levels, relative to genetic variation, in a host spec ies , lodgepole pine {Pinus contorta var. latifolia). Finally in Chapter 5,1 experimental ly examine the response of Douglas-f ir (Pseudotsuga menziesii var: glauca) to variation in ectomycorrhizal community composi t ion and in soil fertility and moisture character ist ics. The relative importance of variation in the abiotic versus symbiot ic environment for both host growth and ectomycorrhizal communi ty is separated statistically using a multivariate approach. Both spec ies used in exper iments are common , widely distributed trees in British Co lumb ia . I end the thesis with genera l conc lus ions and suggested future research in Chapter 6. References Agerer R. 1987 -98 . Co lour atlas of ectomycorrh izae. E inhorn-Ver lag Eduard Dietenberger, Munich Ge rmany Baxter J W , Dighton J . 2001 . Ectomycorrhizal diversity alters growth and nutrient acquisit ion of grey birch {Betula populifolia) seed l ings in host-symbiont culture condit ions. New Phytologist 152: 139-149 Bend ing G D , Poo le E J , Wh ipps J M , R e a d D J . 2002. Character isat ion of bacter ia from Pinus sylvestris-Suillus luteus mycorrhizas and their effects on root-fungus interactions and plant growth. F E M S Microbiology Eco logy 39: 219-227 Bidartondo M l , Ek H, Wal lander H, Soderst rom B. 2001 . Do nutrient addit ions alter carbon sink strength of ectomycorrhizal fungi? New Phytologist 151: 543-550 Bidartondo M l , Bruns T D . 2005. 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Myco log ia 95: 584-589 13 2 The mutualism-parasitism continuum in ectomycorrhizas: A quantitative assessment using meta-analysis1 Introduction Host plants do not a lways respond positively to mycorrh izas; thus, defining mycorrh izas as mutualists has been chal lenged (Francis and R e a d 1995, J o h n s o n et a l . 1997, Brundrett 2004, J o n e s and Smith 2004). Mycorrhizal fungi benefit from these assoc ia t ions because fungal reproduction is dependent on symbios is with a plant host (Jones and Smith 2004). Data from large, single studies of arbuscular mycorrhizal assoc ia t ions indicate that host plants have a continuum of posit ive to negative responses to mycorrh izas (e.g. K l i ronomos 2003), but data for ectomycorrhizal assoc ia t ions are scattered among many smal l studies. Hence we have much less understanding of the range of host responses to ectomycorrhizal assoc ia t ions. The validity of altering the definition of mycorrhizas to remove the requirement for mutualistic responses can be quantitatively evaluated by measur ing the mean and variation of host response over many pairwise combinat ions of host and fungus. The mean indicates whether hosts have a positive, neutral or negative response to mycorrh izas, and variation around the mean indicates whether there is a range of host responses and ou tcomes are dependent on the context of the associat ion. Var iat ion in host response from positive to negative outcomes would support the cont inuum concept . The absence of variation around the mean indicates that regardless of the biotic or abiotic environment of the associat ion, host responses are consistent. Ectomycorrhizal plants, which include many tree spec ies in the northern hemisphere, exper ience two main kinds of variation in ectomycorrhizal assoc ia t ions : the identity and abundance of fungal spec ies . The manner by which a host responds to variation in mycobiont identity has important evolutionary consequences . If there has been select ion for special izat ion among mycobionts, we predict that growth responses of hosts will depend upon the taxonomic identity of the fungus. Funct ional variat ion among taxa of ectomycorrhizal fungi is well documented for character ist ics including nutrient uptake (e.g. Abuz inadah and R e a d 1989, Dighton et a l . 1990, Jongb loed et a l . 1991, Li l leskov et a l . 2002), and drought (Parke et a l . 1983, Boyle and Hel lenbrand 1 A version of this chapter has been submitted to Ecology as: Karst J , Marczak L, Jones MD, Turkington R. The mutualism-parasitism continuum in ectomycorrhizas: A quantitative assessment using meta-analysis. 14 1991, Dixon and Hiol-Hiol 1992) and pH tolerance (Wal lander 2002, Y a m a n a k a 2003 , Dunabei t ia et a l . 2004). E a c h ectomycorrhizal root tip represents a conduit for resource exchange. A s such , the extent to which a root sys tem is co lonized could a lso influence response of the phytobiont to mycorrhizat ion. If ectomycorrhizs are mutual isms, we would predict that higher levels of colonizat ion are positively correlated to growth of the host; however, this relationship is not consistent (e.g. J o n e s et a l . 1990, Thompson et a l . 1994). A s well , T reseder and Al len (2002) predict a unimodal relationship between increasing nutrients in the soil and mycorrhizal b iomass, although how this relat ionship affects host growth is uncertain. Nutrient status of the soil is hypothesized to be a key factor in determining host posit ion on the mutual ism-parasit ism cont inuum (Johnson et a l . 1997). The past few decades have generated sufficient individual studies on plant host responses to ectomycorrhizal assoc ia t ions that some general izat ions can now be made about the nature of the associat ion (mutualistic to parasitic) ac ross different host- fungus pair ings. But, to date there has been no quantitative synthesis that al lows us to determine the general direction or magnitude of this phenomenon, or the variation in these responses . Meta-analys is is an increasingly common analyt ical tool used by ecologists to quantitatively summar ize the results of multiple independent studies (e.g. Gurevi tch et a l . 2000, T reseder 2004, Card ina le et a l . 2006, Lortie and Ca l laway 2006) , and is particularly useful when publ ished studies have conflicting results. Me ta -ana lyses have a lso been used to highlight gaps in the data and to identify c o m m o n methodological problems or constraints. More importantly, by treating separate empir ical studies as independent data points weighted by their replication and precis ion, meta-analys is al lows us to d iscern general patterns already existing in the data that might not be otherwise evident. W e used meta-analysis to determine: 1) how hosts respond to different ectomycorrhizal fungi; 2) if the response is host or fungal speci f ic ; 3) if levels of colonizat ion modify the response; 4) if soil nutrient condit ions modify host growth responses , and 5) if the perception of mycorrhizas as mutual isms has b iased publication of results. W e posed two additional quest ions about the role of exper imental condit ions in modifying host response: 6) does contamination of controls modify detectable host response to ectomycorrh izas? and 7) does host response change with the length of associat ion between host and fungus (i.e. experiment length)? 15 Methods Data col lect ion W e searched ISI W e b of Sc ience (1965 - present) using the keyword 'ectomycorrhiza' . Of the 3591 hits, we selected papers written in Engl ish reporting either total b iomass (g), shoot height (cm) or shoot roo t ratio of tree seedl ings inoculated with ectomycorrhizal fungi paired with non-inoculated control seedl ings. W e a lso checked the "literature cited" sect ion of these papers for addit ional references. Total b i omass is a measure of productivity. Shoot height may be indicative of competit ive ability in the seedl ing establ ishment phase, where tree seedl ings have to compete with rapidly growing herbs. C h a n g e s in shoot:root ratio may identify factors that increase seedl ing survival in nutrient limited environments or that control the potential carbon supply to ectomycorrhizal fungi, the currency mediating the associat ion. For each study, we recorded the mean, standard deviat ion and sample s ize for both inoculated and control seedl ings. W h e n necessary , we digit ized graphs to obtain this information. W h e n experimental treatments involved severa l combinat ions of host spec ies with ectomycorrhizal fungal spec ies or fungal isolates, we treated e a c h combinat ion as a separate study, although not all studies were completely independent. Inclusion of severa l studies from one paper tends to reduce the overal l heterogeneity in effect s i zes , but excluding multiple results from a paper could underest imate effect s i zes (Gurevitch and Hedges 1999). W h e n results from papers involved inoculat ion trials in combinat ion with explicit manipulat ions of the environment, other than nutrient levels (e.g. p H , pathogen abundance, nematode density, salinity, soil moisture, C 0 2 ) , we used data from "ambient treatments". For example, we recorded data for inoculated and control seedl ings from ambient C 0 2 levels while excluding data from treatments featuring elevated C 0 2 levels. A m o n g those papers that manipulated fertilizer types and amounts , only the manipulation of inorganic phosphorus levels w a s reported in a sufficient number of studies to merit further analys is. W e converted phosphorus addit ions to a common unit, mg P kg" 1 substrate, and treated it as a cont inuous predictor with va lues ranging from 0 to 136 mg kg" 1 substrate. W e did not include studies where inoculation resulted in no colonizat ion, or where there were no control data (non-inoculated treatments). W h e n repeated measures were taken in a study, we used data from the last sampl ing period to capture the maximal length of assoc ia t ion between host and fungus. 16 Host and fungal identity were treated a s categorical explanatory var iables in the meta-analyt ic model . W e recorded the spec ies of host and, when given, ectomycorrhizal fungus (in some c a s e s the fungus was an unknown isolate, or the spec ies epithet w a s not provided). W e then grouped spec ies into genera for both host and fungus, and when testing for di f ferences among genera, we included only those that were represented by at least 10 studies. Duration of associat ion and colonizat ion level (percent of root t ips co lon ized or percent root length colonized) of inoculated seedl ings were investigated a s possib le cont inuous explanatory var iables in the model . W h e n colonizat ion level w a s given a s a range, we used the median value. Contaminat ion of non-inoculated seedl ings reduces di f ferences in colonizat ion levels between control seed l ings and inoculated seedl ings. Consequent ly , the perce ived response of hosts to ectomycorrhizal inoculation may be reduced as a result of contaminat ion. W e determined the magnitude of contaminat ion by calculat ing the level of colonizat ion on control seedl ings relative to that measured on inoculated seed l ings accord ing to the proportion: C C / ( C C + C T R ) where C T R is the percent colonizat ion of target fungi on inoculated seed l ings, and C c is the percent colonizat ion of contaminant fungi on control seedl ings. W e quantif ied the duration of the associat ion by recording the number of w e e k s e a c h experiment ran. Th is measure was the only consistent proxy to evaluate the influence of experimental duration on host outcome to ectomycorrhizal assoc ia t ions; however, we recognize that extreme dif ferences in growth rates among host spec ies would render absolute length of time irrelevant. W e a lso examined the relationship between time and the variation among effect s i zes . To do so , residuals were ca lcu lated using the absolute difference of effect s i zes from the cumulat ive mean and weighted by their sample s izes . Res idua ls were then regressed against duration of assoc ia t ion. W e performed identical calculat ions to examine residuals for effects s i zes ac ross phosphorus levels. Data analysis The effect s ize of ectomycorrhizal inoculation for total b iomass , shoot height and shoot:root ratio was calculated as the natural log of the response ratio of inoculated to control seedl ings. The response ratio (R) is the ratio of the mean outcome in the 17 experimental (inoculated) group to that of the control (non-inoculated) group (Rosenberg et a l . 2000). On ly 1 2 % of the studies in our analys is reported measu res of variation around means . Consequent ly we weighted va lues by their sample s ize instead (Shurin et a l . 2002, La jeunesse and Forbes 2003, Marczak et a l . 2006), and while this increases the probability of Type II errors, it avoids underest imating effect s i zes (Gurevitch and Hedges 1999). Effect s i zes were cons idered significantly different than zero when 9 5 % conf idence intervals did not over lap zero; explanatory var iab les were cons idered significant at a = 0.05. Tes ts for homogeneity of effect s i zes were based on the statistic Q T , with larger va lues indicating greater heterogeneity in effect s i zes among compar isons (Rosenberg et a l . 2000). W e a s s e s s e d the importance of publication b ias using a non-parametr ic rank correlation test (Spearman 's rho). A significant correlation between effect s ize and sample s ize ac ross studies would indicate bias in the publication of extreme effect s i zes . Effect s i zes for all ana lyses were not normally distributed, s o we relied on randomizat ion tests (4999 iterations) to a s s e s s signi f icance levels. W e first tested the null hypothesis that all effect s i zes were equal , and if rejected, we examined the categorical (fungal and host genus identity) and cont inuous (colonization levels, magnitude of contaminat ion, and duration of associat ion) explanatory var iables descr ibed above. W h e n categorical predictors were signif icant, we a s s e s s e d dif ferences among groups based on 9 5 % bootstrapped conf idence intervals. W e then regressed effect s ize against all cont inuous predictor var iables. For any significant explanatory variable, we only report those explaining > 5 % of the variation in effect s i zes as est imated by Q M / Q T , where Q M is the variation in effect s i zes that is expla ined by a particular model (Rosenberg et a l . 2000). Al l data ana lyses were performed in MetaWin software version 2.1.4 (Rosenberg et a l . 2000). Results Seedling response to ectomycorrhizal inoculation Overal l we extracted 459 studies of inoculation response of total b iomass from 36 papers , 329 studies of shoot height from 24 papers, and 235 studies of shoot:root ratio from 20 papers (Appendix A) . A c r o s s all growth traits, we a s s e s s e d the ou tcome of 21 host genera inoculated with 31 fungal genera ; however, these inoculat ions were not 18 represented in all possible combinat ions. The mean age of seed l ings at the end of exper iments was 23 weeks (range = 10 to 104 weeks) . O n average, seedl ings increased in total b iomass and shoot height, but did not change in shoot:root b iomass al location when inoculated with ectomycorrhizal fungi (mean cumulat ive effect s i zes = 0.208, 0.113, -0.0174, respectively; F ig . 2.1). However , there was significant heterogeneity in the data ( Q T = 10152, df = 458; Q T = 95389, df = 328; Q T = 705, df = 234, respectively; all p < 0.001) to indicate that further structure existed. The identity of the host genus was significant in explaining variation in effect s i zes for both total b iomass (p = 0.028, df = 4, 409, Q M / Q T = 0.18) and shoot:root ratio (p < 0.001, df = 3, 191, Q M / Q T = 0.22). In particular, inoculated seedl ings of the genera Quercus, Pseudotsuga and Eucalyptus increased in total b iomass more than those of Pinus and Picea (Fig. 2.2a), while Picea seedl ings al located more b iomass to shoots than seedl ings of Quercus, Pseudotsuga and Pinus when inoculated (Fig. 2.2b). Al though there was a positive relationship between total b iomass and shoot height (p < 0.001, df = 1, 567, r2 = 0.37), neither categorical nor cont inuous predictors exp la ined variation in effect s izes of shoot height. Fungal genus inf luenced al location of b iomass to shoots versus roots (p < 0.001, df = 5, 199, Q M / Q T = 0.26), but did not expla in variation in effect s i zes for total b iomass or shoot height. Seed l ings inoculated with fungi from the genus Scleroderma al located more b iomass to roots than that observed for other genera (Fig. 2.3). Level of colonizat ion of inoculated seedl ings, ranging from 0.5 to 9 8 % , w a s not important in explaining variation in effect s izes for total b iomass (p = 0.043 df = 1, 349, Q M / Q T = 0.03), shoot height (p = 0.30, df = 1, 220) or shoot:root ratio (p = 0.03, df = 1, 211 , Q M / Q T = 0.03) (note that although level of colonizat ion w a s significant, Q M / Q T < 0.05 for both total b iomass and shoot roo t ratio [see Methods])(Fig. 2.4). Heterogeneity in effect s i zes was unrelated to the magnitude of contamination for total b iomass (p = 0.20, df = 1, 324), shoot height (p = 0.48, df = 1, 211) and shoot:root ratio (p = 0.063, df = 1, 197)(Fig. 2.5). Contaminat ion levels were highest in those exper iments performed in nurser ies and in the field, and lowest in those in growth chambers (p < 0 .001, F3,416 = 76.9) (Table 2.1). The average length of exper iments was 21 weeks (range = 8 to 104 weeks) , slightly less than the average age of seedl ings used in exper iments. Duration of 19 associat ion between host plant and fungus did not explain variation in effect s i zes for total b iomass (p = 0.86, df = 1, 457) or shoot height (p = 0.97, df = 1, 327) (F ig.2.6a, b). O n average, seedl ings al located more b iomass to roots than shoots, with increasing duration of associat ion (p < 0.001, df = 1, 233, Q M / Q T = 0.06) (Fig.2.6c). The magni tude of contaminat ion was positively related to duration of experiment (p < 0 .001, df = 1, 418 , r2 = 0.14) (Fig. 2.7). Variability among effect s i zes dec reased with duration of associat ion for both total b iomass (p < 0.001, df = 1, 457, r2 = 0.12) and shoot height (p < 0.001, df = 1, 327, r2 = 0.25), but was unrelated to duration for shoot:root ratio (p = 0.033, df = 1, 233 (Fig. 2.8). That is, longer running exper iments had effect s i zes more similar to the cumulat ive mean. In particular, for measures of total b iomass and shoot height, variation among effect s i zes decl ined to nearly zero (effect s i zes converged on the cumulat ive mean) at approximately 30 weeks (Fig. 2.8). The level of contaminat ion for control seedl ings was predicted to increase by 8 4 % for this time period (Fig. 2.7). There was ev idence for significant publication b ias in data for total b iomass ; Spea rman ' s rho for the correlation between effect s ize and sample s ize was -0.28 (p < 0.001), indicating that there was an over-representat ion of studies with posit ive effect s i zes at low replication. There was no ev idence of publication b ias in data for shoot height (Rs = 0.054, p = 0.33) or shoot roo t ratio (Rs = -0.105, p = 0.109). Seedling response to ectomycorrhizal inoculation and phosphorus addition W e analyzed 234 studies (6 host and 15 fungal genera) from 10 papers for changes in total b iomass of seed l ings inoculated with ectomycorrhizal fungi under phosphorus (P) addit ions ranging from 0 to 136 mg P kg" 1 (Appendix B). T h e cumulat ive effect s ize was positive (0.0769), but the 9 5 % conf idence intervals over lapped zero , indicating there w a s no average change in total b iomass of seedl ings inoculated with ectomycorrhizal fungi subjected to manipulated phosphorus levels when all levels of substrate P, including no addit ions, were included. There was underlying structure in the data (p < 0.001, df = 232, Q T = 1236); however, of the explanatory var iables, only host genus expla ined a significant amount of variation in effect s ize (p < 0.001, df = 3, 24 , Q M / Q T = 0.31). Specif ical ly, seedl ings of the genera Eucalyptus, Pinus and Larix had relatively less b iomass than those of Picea when inoculated, regardless of phosphorus level. There was a negative relationship between the residuals of effect s ize and amount of phosphorus added , indicating that variation among effect s i zes d e c r e a s e d 20 with levels of phosphorous (Fig. 2.9). Publ icat ion bias was a lso evident in these data (Rs = 0.31, p < 0.001), i.e. there were a lack of studies with posit ive effect s i zes at low sample s izes . Discussion Seedling response to ectomycorrhizal inoculation A c r o s s the studies included in our analys is , it appears that on average, hosts respond positively to ectomycorrhizal inoculation; both total b iomass and shoot height are greater in inoculated seedl ings. However, when all avai lable studies are cons idered and weighted by their sample s izes , the ev idence in support of positive growth outcomes through ectomycorrhizal inoculation is considerably weaker than many single studies suggest. Addit ionally, factors unrelated to inoculation perse have inf luenced interpretation of host responses to ectomycorrhizal inoculat ion, namely publication b ias towards large positive effects, the duration of exper iments and artificial pairing of host and fungal symbionts. The presence of these factors effectively reduces and distorts the spectrum on which host responses to ectomycorrhizal inoculation are evaluated. The spectrum is reduced: Publication bias inflates measures of effect sizes Under a model of no publication bias, est imated effects should be distributed around the unknown true effect, with the spread of the effects representing their var iances. A s sample s i zes increase, the spread of the distribution should dec rease resulting in a funnel shaped distribution of effect s i zes . Publ icat ion b ias against s tudies with negative results will produce a negative correlation between sample s ize and the magnitude of effect (Begg and Mazumdar 1994) and this inflates the magnitude of overal l effect s i zes calculated in a meta-analysis. W e detected publication b ias for measures of total b iomass but not for shoot:root ratio or shoot height responses to inoculation. Shoot height increases with ectomycorrhizal inoculat ion, but it is independent of the identity of host and fungal genus, colonizat ion levels and duration of assoc ia t ion. B e c a u s e the lower limit of the cumulat ive effect on total b iomass is well above zero, there may indeed be a change in seedl ing b iomass upon inoculat ion. A m o n g the papers used in this meta-analys is, Dixon et a l . (1984) and Hung and Mol ina (1986) explicitly reported that data had been omitted due to non-signif icant di f ferences between control and inoculated seedl ings. It is unlikely that these particular 21 omiss ions alone caused publication bias in our dataset, but they may be symptomat ic of b ias in the select ion of data reported in publ ished papers. At the other extreme, although they did not affect the results of the meta-analysis, host-fungus pair ings extracted from Burgess et a l . ([1994]; identified as outliers in F ig . 2 .4a and b) were irregularities in our dataset, reporting highly positive responses to ectomycorrhizal inoculation by var ious strains of Pisolithus. Due to the tradition of categoriz ing mycorrhizal fungi as mutualists, such extreme positive results are unlikely to go unpubl ished. Negat ive results in mycorrhizal research may be more likely to go unpubl ished compared to other f ields in which no a priori expectat ion exists of the magnitude or direction of the outcome of spec ies interactions. From a silvicultural perspect ive, interest primarily in positive growth responses to ectomycorrhizal inoculation may be warranted, but it has hindered our ability to evaluate the full spectrum of responses. Moreover, negative responses are not aberrant ou tcomes when we cons ider that hosts are evolutionarily compat ib le with both mutualistic and parasit ic modes of symb ioses . For example, the pathways and physiological machinery involved in arbuscular mycorrhizal development are conserved among symbios is types, including those that are parasit ic (Mathesius 2003 , Paszkowsk i 2006). A s arbuscular mycorrh izas are cons idered to be ancestra l to all other mycorrhizal types (Wang and Qiu 2006), there is no biological bas is to presume that responses to ectomycorrhizal inoculation should be solely positive. Chang ing our definition of ectomycorrh izas (Johnson et a l . 1997, Brundrett 2004, J o n e s and Smith 2004) will become necessary as ev idence accumulates on their evolutionary origins (Hibbett et a l . 2000) and on variation in the outcomes of ectomycorrhizal assoc ia t ions ( S a c h s and S i m m s 2006). Th is will a lso broaden the view of their ecological role. The spectrum is distorted: Factors that covary with time may cause spurious effects Not surprisingly, levels of contamination were highest on seedl ings grown in either the field or in nurser ies, although most of the exper iments from which the da ta were extracted were done in g reenhouses. The magnitude of contaminat ion w a s positively correlated to the duration of the experiment (Fig. 2.7). The problem of increased contamination could be al leviated if measurements were made earlier. Th is approach , however, is not recommended. Variat ion among effect s i zes for both total 22 b iomass and shoot height significantly decl ined with the duration of the experiment. Factors such as maternal effects (Weiner et a l . 1997), substrate di f ferences, temperature and light condit ions may all obfuscate the role of ectomycorrh izas in influencing seedl ing growth in shorter exper iments. Seed l ings a lso vary in the time it takes to develop ectomycorrhizal assoc ia t ions. O n roots of Eucalyptus globulus, ectomycorrh izas formed by Pisolithus tinctorius and Paxillus involutus deve loped in 4 days when in direct contact (Horan et al . 1988). Converse ly , colonizat ion w a s not observed until 4 weeks on roots of Eucalyptus coccifera inoculated with Thelephora terrestris or Laccaria bicolor (Jones et a l . 1990). Early measurements (prior to 30 weeks) may preclude detection of a mycorrhizal "s ignal" as the strength of this s ignal is likely to be weak compared to other factors influencing seedl ing growth. The spectrum is distorted: Effects of crossing hosts and ectomycorrhizal fungi not known to co-occur remain poorly understood Often inoculation trials are performed using artificial pair ings of host and fungus (e.g. C h e n et a l . 2006) and rely on ectomycorrhizal fungi that are amenab le to experimentat ion. Choos ing fungi based upon character ist ics that render them e a s y to work with in laboratory condit ions may also have se lected for uniformity in other traits. Until techniques become avai lable to represent the diversity of ectomycorrhizal fungi observed in natural sys tems, interpretations of host response to ectomycorrhizal inoculation will be limited. Moreover, the geographic origin of fungi and hosts used in trials may affect inoculation responses in unpredictable ways . Simi lar to plants, some but not all spec ies of ectomycorrhizal fungi are cosmopol i tan in their distribution. O n e corollary to this pattern is that not all host and ectomycorrhizal fungal spec ies will interact and that at any given location a host spec ies will encounter a subset of the global pool of ectomycorrhizal fungi. Th is geographic variation in plant-mycorrhizal community structure has likely resulted in a mosa ic of coevolut ion between plants and mycorrhizal fungi (Thompson 2005), but we still have very few data on the consequences of this mosa ic on mycorrhizal inoculation responses (but s e e H o e k s e m a and Thompson 2007, Kl i ronomos 2003, Monzon and A z c o n 1996, Sy lv ia et a l . 2003). Th is lack of knowledge of the range of host responses to exotic symbionts a lso carr ies over to conservat ion research; the ecological consequences of mycorrhizal fungal spec ies ' introductions are unpredictable (Schwartz et a l . 2006). 23 In our meta-analys is , we could not categor ize each host/fungus pairing a s " local" or "foreign", as such information was either unavai lable, or it was not c lear at what sca le we should cons ider a host and fungal spec ies to co-occur (e.g. within a forest s tand, region or country). Stud ies on arbuscular mycorrhizas have shown that c ross ing local plants and fungi produces a greater range in responses measured by plant b iomass than for c rosses involving foreign symbionts (Kl i ronomos 2003). Converse ly , variation in plant growth was independent of fungal isolates when different geographic populat ions of 3 host plant spec ies were c rossed with 4 populat ions of the ectomycorrhizal fungus Rhizopogon occidentalis (Hoeksema and Thompson 2007). Origin of fungal isolate w a s a lso not found to be important in modifying growth of Eucalyptus globulus (Thompson et a l . 1994). T h e s e findings are consistent with our results that variation in fungal identity bears little consequence to variation in shoot height or seedl ing b iomass . The role of variation in fungal properties in host response to ectomycorrhizal inoculation The magnitude of effect s ize for seedl ing b iomass and shoot height for the most part did not covary with var iables related to ectomycorrhizal fungi, namely colonizat ion level and genus identity. Our results suggest that colonizat ion levels are not an ecological ly useful measure of host response to ectomycorrhizal inoculat ion (Fig. 2.4). Moreover , we suggest that focus on colonizat ion levels has distracted investigation from other possib le mechan isms that may be more critical determinants of host response to ectomycorrh izas. Character is t ics of fungi s u c h a s those assoc ia ted with the deve lopment and differentiation of extramatrical mycel ium may correlate better to the magnitude of host response as they represent a potential increase to the absorb ing sur face a rea of roots (Jones et a l . 1990, Agerer 2001). Th is type of measurement relies on physical mechan isms underlying host benefits of being mycorrhizal . Our results suggest that these benefits may be equal ly expressed through colonizat ion levels ranging from 0 .5% to 9 8 % . It is unlikely that similar resource transfers could occur at low (0.5%) and high (98%) levels of colonizat ion that result in a comparable cumulat ive posit ive effect to inoculation among seedl ings. Never the less, there are many examples of growth response to very low levels of colonizat ion. It is possib le that the presence of growth promoting hormones may be responsible for increases in seedl ing b iomass and height 24 with inoculat ion. It is well establ ished that p lant-associated microorganisms are capab le of synthesiz ing phytohormones that are used for communicat ion between a host and its microflora (Tsavkelova et a l . 2006). For example , smal l amounts of aux ins increase shoot elongation and dry weight of wheat inoculated with rhizobacter ia (Khal id et a l . 2004). Aux ins , which are involved in a wide variety of physiological responses that influence growth of woody plants (Kozlowski and Pal lardy 1997), are a lso produced by ectomycorrhizal fungi (Barker and Tagu 2000). Though s o m e research has been conducted on the effects of auxins on ectomycorrhizal development (e.g. N iemi et a l . 2002, R incon et a l . 2003), its role at the level of the host has been neglected. G iven that posit ive effects of fungal inoculation are often observed at low levels of colonizat ion for both seedl ing b iomass and shoot height, we suggest that chemica l mechan i sms may often underlie host responses to ectomycorrhizal inoculat ion. W e determined that on average, seedl ings across multiple host spec ies had more b iomass when inoculated with any ectomycorrhizal fungus, regardless of the identity of the fungal assoc ia te . Th is supports f indings from research on non-symbiot ic interactions; for example, host plants are often general ists with response to different poll inators (Zamora 2000). Th is result conforms to theory predicting the outcome of multi-specif ic plant-pollinator sys tems, i.e. interactions involving many spec ies tend to result in the evolution of general ists because reciprocal specia l izat ion is unlikely (Howe 1984). In forest s tands, the number of spec ies of ectomycorrhizal hosts is typically an order of magnitude less than that of its fungal symbionts (Bruns 1995). Rec iproca l special izat ion is unlikely in this sys tem due to the changing composi t ion of ectomycorrhizal fungi both spatially (Izzo et al . 2005, G e n n e y et a l . 2006, Tol jander et a l . 2006) and temporal ly (Izzo et a l . 2005, Koide et a l . 2007). Thus , hosts may adapt to " landscapes" (sensu Howe 1984) of ectomycorrhizal fungi where fungal spec ies diversity dif fuses select ion from one source. Nonethe less , we cannot definitively conc lude that the identity of the fungus has no role in modifying host response for two reasons. First, al though it is evident that inoculation with most fungal genera results in increased b iomass al location to shoots, those fungi from the genus Scleroderma are an except ion. Seed l ing al locat ion to roots increased by almost three t imes when inoculated by fungi from this particular genus . Diedhiou e t a l . (2004) conc luded that Scleroderma dictyosporum has a higher requirement for g lucose relative to thelephoroid spec ies , perhaps related to construct ion 25 costs of its network-like mycel ium (Newton 1991). Plants growing in nutrient-depleted soi ls al locate more b iomass to roots than shoots (Gedroc et a l . 1996). If assoc ia t ion with fungi from this taxon is perceived by the host as equivalent to growing in nutrient-depleted soi ls this would explain al location patterns. S e c o n d , there appears to be a difference between those fungi that contaminate seedl ings and those used to inoculate seedl ings. B e c a u s e there was no effect of magnitude of contaminat ion on all three growth measures despite a cumulat ive positive effect, contaminant fungi are likely neutral in their effects. S p e c i e s of contaminant fungi were for the most part unidentif ied but included those from the genera Thelephora and Cenococcum. T h e s e fungi are common , w idespread, and widely d ispersed via airborne spores ; whether such character ist ics of fungi and magnitude of host response covary should be further studied. Al though a positive growth response was expressed by the most c o m m o n host genera in our analys is , hosts differed in the magnitude of response. In particular, Quercus seedl ing b iomass and b iomass al location to roots ranked highest, and Picea lowest, with ectomycorrhizal inoculation. W h e n phosphorus condit ions were manipulated (i.e. the subset of studies that explicitly altered phosphorus levels), Picea ranked highest in increased seedl ing b iomass with inoculation. W e cannot say whether these ou tcomes are taxon or trait-specific, due to the relatively few genera included in the analys is . For example, mycorrhizal dependency has been hypothesized to relate to var ious root morphological traits such as root th ickness, surface a rea and inc idence of root hairs (Brundrett 2002). In addit ion, dependency on arbuscular mycorrh izas s e e m s to be higher for hosts that have smal l s e e d s or have had seed reserves experimental ly reduced (Janos 1980, A l lsop and Stock 1995, S iquei ra et a l . 1998, Zangaro et a l . 2003) . Our results contrast with those observed for arbuscular hosts; s e e d s of Quercus are general ly larger than those of Picea, yet are more responsive to ectomycorrhizal inoculation. Root morphology is sensit ive to abiotic condit ions of the soi l , thus its role in determining mycorrhizal dependency is unclear. Go ing beyond taxonomic correlat ions with inoculation responses, and identifying those specif ic host traits that correlate to speci f ic ou tcomes will enrich our understanding of ectomycorrhizal interactions. In particular, further research within a broad framework, such as that which has deve loped for leaf traits (Wright et a l . 2004), would be especial ly fruitful to understand trade-offs among plant traits and mycorrhizal respons iveness. 26 Mycorrhizal assoc ia t ions are predicted to confer most benefit to the host plant in condit ions of low nutrients. A s such , we would expect a negative relationship between the magnitude of effect s ize and increasing phosphorus addition but our results do not support this prediction. The range of host responses appears to be environment speci f ic; variation among effect s i zes was high for studies with low phosphorus addit ions. Bougher et a l . (1990) have indicated there is an interaction between the effects of fungal taxa and P addit ions. Specif ical ly, at low P addit ions (2-12 mg P kg" 1 soil), di f ferences among Desoclea maculate, Laccaria laccata and Pisolithus tinctorius in host dry mass production are apparent, but these dif ferences are not apparent at greater than 16 mg P kg" 1 soi l . A similar interaction was reported for seed l ings co lon ized by Laccaria bicolor or Thelephora terrestris a long a P gradient (Jones et a l . 1990). Our meta-analysis could not detect such an interaction because not all host/fungi combinat ions were present ac ross the range of P addit ions. Whether the response to ectomycorrhizal fungi is taxon- or environment-specif ic (or both) warrants further study as it has implications for the strategies plants may use to maintain ectomycorrhizal assoc ia t ions that confer benefits to the host (Hoeksema and Kummel 2003). Conclusions and future directions Publ icat ion bias c louds our ability to conclusively determine general principles of host response to ectomycorrhizal inoculat ion. With recognition that mycorrhizal assoc ia t ions could fall on a cont inuum of possib le outcomes and that this range of responses is ecological ly significant, the tendency not to report negative results must be reduced. Our crit icism of methods employed to test host response to ectomycorrhizal inoculation is not one of mycorrhizal research in general , but instead reveals the limits of some of the methods used. In particular, there is tension between assess i ng the response too early when the mycorrhizal s ignal can be masked , and running the experiment too long and increasing the l ikelihood of contaminat ion. W e see no e a s y remedy to this problem. The use of mycorrhizal defective mutants, such a s those used by Gavagnora et a l . (2004) may offer a way to circumvent the issues highlighted with current methods. Even so , the rel iance on compar isons between mycorrhizal and non-mycorrhizal individuals, beyond its heuristic purpose, is somewhat artifactual because in nature, non-mycorrhizal phenotypes do not occur, except in very young seedl ings. Finally, a s recommended in non-symbiot ic sys tems (e.g. Stanton 2003, S t rauss and 27 Irwin 2004), a departure from focusing on pairwise spec ies interactions and moving to considerat ion of host responses to variation in the composi t ion of ectomycorrhizal fungal communi t ies may encourage a broader perspect ive on the ecological and evolutionary consequences of ectomycorrhizal assoc iat ions. 28 Tab le 2 .1 :Means and standard errors (SE) for the influence of location of exper iment on the magnitude of contaminat ion. Sou rce n M e a n * S E Field 43 0 .33 a 0.0251 Nursery 28 0 .43 a 0,0311 Greenhouse 243 0 .10 b 0.0105 Growth chamber 106 0.0026° 0.0160 * M e a n s fol lowed by the s a m e letter are not statistically different (Tukey-Kramer H S D , a = 0.05). 29 Figure 2 .1 : Cumulat ive mean effect s i zes for total b iomass, shoot height and shoot:root ratio. Error bars are 9 5 % bootstrapped conf idence intervals. 0.3 -j 0.25 -0.2 -0.15 0.1 -0.05 -0 -0.05 -0.1 -0.15 -I CO — <f> eg cc o E I— o JQ O O sz CO sz O) 'CD SZ o e o o sz CO 30 Figure 2.2: M e a n effect s ize for a) total b iomass and b) shoot:root ratio by host genus . M e a n s with 9 5 % bootstrapped conf idence intervals are shown. M e a n s fol lowed by the s a m e letter are not statisticaly different (95% bootstrapped conf idence intervals overlap). For b), posit ive va lues indicate al location of b iomass to shoots w a s higher than al location to roots. a) 0.6 0.5 0.4 0.3 0.2 0.1 0 f CD o co cz CO D ) o •o CD CO CL I CO 3 -*—< Q. >> CO O LU I CO CD o b) 0.4 0.3 0.2 0.1 0 -0.1 -0.2 -0.3 CO CD o CO c CO D ) 13 CO •I—' o T J Z5 CD CO Q_ CO 2 CD o 31 Figure 2.3: M e a n effect s ize for shoot:root ratio by fungal genus. M e a n s with 9 5 % bootstrapped conf idence intervals are shown. M e a n s fol lowed by the s a m e letter are not statistically different (95% bootstrapped conf idence intervals overlap). Posi t ive va lues indicate al location of b iomass to shoots was higher than that al located to roots. 32 Figure 2.4: Relat ionship between mean effect s i zes and level of ectomycorrhizal fungal colonizat ion of inoculated seedl ings for a) total b iomass, b) shoot height and c) shoo t roo t ratio. Outl iers (those data points falling above the 9 7 t h percenti le of the distribution) are indicated as tr iangles; these were retained in the analys is . a) 3 CC c ' 2 CD N CO 1 •ft?* 1 100 Ectomycorrhizal fungal colonization level of inoculated seedling (%) 33 b) _ 3 rr £ 2 .— 1 (/> -t—' o n a) 0 LU -1 0 20 60 80 100 Ectomycorrhizal fungal colonizat ion level of inoculated seedl ing (%) c) 3 rr c ' 2 •— 1 CO O O J 0 L U • • • • 2 0 * * 0 *# / ^ . « 0 * * 80 . • 100 Ectomycorrhizal fungal colonization level of inoculated seedl ings (%) 34 Figure 2.5: Relat ionship between mean effect s i zes and magnitude of contaminat ion for a) total b iomass, b) shoot height and c) shoo t roo t ratio. a) 4 n -2 J Magn i tude of contaminat ion 4 i 3 -0 . £ * 0.4 0.6 0.8 1 -2 Magn i tude of contaminat ion 35 c) 4 -1 3 -ln(R) 2 -0 N 1 -it CO Effecl • t 0.6 0.8 -2 Magnitude of contamination 36 Figure 2.6: Relat ionship between effect s i zes and duration of associat ion of ectomycorrhizal fungus and host for a) total b iomass, b) shoot height and c) shoot:root ratio. QM/QT is the amount of total heterogeneity in the data due to variation in effect s i zes expla ined by the model . Statist ics are reported for significant mode ls only. a) 4 -, i 60 80 100 Durat ion of assoc ia t i on (weeks) b) 40 60 80 100 Durat ion of assoc ia t i on (weeks) Durat ion of assoc ia t i on (weeks) Figure 2.7: Relat ionship between magnitude of contaminat ion and duration of associat ion of ectomycorrhizal fungus and host. 39 Figure 2.8: Relat ionship between effect s ize residuals and duration of assoc iat ion of ectomycorrhizal fungus and host for a) total b iomass, b) shoot height and c) shoot:root ratio. Statist ics are reported for significant models only. b) w 3.5 CO • g w 2 5 N o CD i t LU 3 -2 w 1.5 1 0.5 0 0 'bun. y=1.751-0.0687x p<0.0001 i-*=0.25 2 0 40 60 80 Duration of assoc ia t ion (weeks) 100 w 3.5 | 3 1 2.5 CD N 2 r? 1-5 1 o CD LU 0.5 0 0 20 40 60 80 Duration of associat ion (weeks) 100 41 Figure 2.9: Relat ionship between effect s ize residuals for total b iomass and amount of phosphorus added . y=1.25-0.0232x p<0.0001 1^=0.18 50 100 Amount of P a d d e d (mg/kg) References Abuz inadah R A , R e a d D J . 1989. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. IV. The utilization of peptides by birch (Betula pendula L.) infected with different mycorrhizal fungi. New Phytologist 112: 55-60 A l lsop N, Stock D. 1995. Relat ionship between s e e d reserves, seedl ing growth and mycorrhizal responses in 14 related shrubs (Rosidae) from a low nutrient environment. Functional Eco logy 9: 248-254 Agerer R, 2001 . 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Journa l of Tropical Eco logy 19: 315-324 48 3 Methods to control ectomycorrhizal colonization: Effectiveness of chemical and physical barriers1 Introduction In mycorrhizal research, evaluation of mycorrhizal effects on plant per formance often requires compar isons between mycorrhizal and non-mycorrhizal plants. Creat ing effective, yet feasible methods to control mycorrhizal colonizat ion in the field has become of utmost importance as there has been a recent demand to increase the ecological re levance of mycorrhizal research (Read 2002). Th is requires moving away from laboratory based work to 'exper iments conducted in natural envi ronments. Currently, most studies have obtained non-mycorrhizal plants by employ ing one of three methods: substrate steril ization (via autoclaving, s team steril ization or g a m m a irradiation), the creation of mutant plants unable to form mycorrh izas, or the use of fungicides appl ied to soil around plant roots. Steri l izing soi l can result in substant ial changes in its chemica l and physical properties (Lenis et a l .1991, C h a m b e r s and Attiwill 1994, Sheremata et a l .1997, S h a w et al.1999); moreover, its appl icat ion in the field is futile because contamination is certain. The development of plants that lack the ability to form mycorrh izas has been limited to a few plant spec ies associat ing with arbuscu lar mycorrhizal fungi (AMF) (Marsh and Schul tze 2001). More research is a lso required to determine whether the functioning of mutants is otherwise identical to non-mutant plants (Kahiluoto et a l . 2000). Of the fungicides, benomyl has been used effectively to reduce arbuscular mycorrhizal colonizat ion of plants in the field by as much as 8 0 % (Hartnett and Wi lson 1999, Wi lson et a l . 2001 , Ca l laway et a l . 2004, Dhill ion and Gards jord 2004). Benomyl , no longer l icensed for use in some countr ies and relatively ineffective against bas id iomycetes, is however, not an option to control most ectomycorrhizal fungi. Fungic ides have general ly not been employed in ectomycorrhizal sys tems (but s e e Page -Dumroese et a l . 1996, Manninen et a l . 1998). Ectomycorrhizal fungal communi t ies are more taxonomical ly diverse than arbuscular mycorrhizal fungal communi t ies, thus requiring a broad spectrum fungicide to adequately dec rease ectomycorrhizal colonizat ion. Of the three methods currently employed to control mycorrhizat ion, the use of fungicides appears the most feasible for field research in 1 A version of this chapter has been published as: Teste F, Karst J , Jones MD, Simard S W , Durall DM. 2006. Methods to control ectomycorrhizal colonization: effectiveness of chemical and physical barriers. Mycorrhiza 17: 51-65 49 Two fungicides, Topas® and Senator®, have been suggested by g reenhouse managers for control of ectomycorrhizal hyphal growth. Prop iconazo le , the act ive ingredient in Topas® (25% a.i.), interferes with ergosterol b iosynthesis, which is critical to the formation of fungal cel l membranes (Kendrick 2000). The lack of normal sterol production s lows or stops the growth of the fungus, effectively preventing further infection and/or invasion of host t issues (Kendrick 2000). Prop iconazo le incorporated into agar med ia at 1 ppm or higher inhibited growth of many ectomycorrhizal fungal strains (Zambonel l i and lotti 2001 , Laat ikainen and Heinonen-Tansk i 2002). Colonizat ion of Pinus sylvestris roots by ectomycorrhizal fungi dec reased by approximately 2 0 % , with some morphotypes affected more than others, when propiconazole was appl ied for two consecut ive years in the field at a rate of 250g I"1 every two weeks (Manninen et a l . 1998). Thiophanate-methyl , the active ingredient in Senator® (70% a.i.), interferes with the functioning of microtubules, so that treated cel ls cannot divide. Thiophanate-methyl targets the cel ls of ascomyce tes (Kendrick 2000) , but to our knowledge has not been used to control ectomycorrhizal fungi. Stud ies of common mycorrhizal networks ( C M N s ) in plant communi t ies form a unique subset of studies on mycorrhizal ef fect iveness (Simard and Durall 2004) . They require compar isons between plants that are l inked with those that are not l inked by a C M N (Simard et a l . 1997, Booth 2004). In these studies, control plants may be mycorrhizal , but hyphal l inkages between plants must be absent . Whi le non-mycorrh izal or non-l inked controls are easi ly establ ished in the laboratory using substrate steril ization techniques, this is more problematic in the field where seed l ings are grown in native soi ls. M e s h barriers constructed of either steel or nylon have been used to prevent formation of ectomycorrhizal connect ions between plants (e.g. Franc is and R e a d 1984, Schuepp et a l . 1992, Booth 2004, Kranabetter 2005), or provide root-free compartments where mycorrhizal hyphae can explore and grow. To restrict penetrat ion of roots and hyphae, mesh with pores 1 um or smal ler has been used (Rob inson and Fitter 1999, Johnson et a l . 2001, Zabinsk i et a l . 2002, Ca rdoso et a l . 2004) , however , given that hyphal width var ies (from 1.5 to 9 um), a mesh with pore s i zes larger than 1 um may restrict penetration of some mycorrhizal fungal spec ies but not others. Consequent ly , the mesh pore s ize could alter the ectomycorrhizal fungal communi ty composi t ion. Ectomycorrhizal fungi vary in their ability to absorb and transport nutrients 50 and water (Simard and Durall 2004); therefore, any alteration of the communi ty may affect transport within the C M N . The objective of this study was to examine the ef fect iveness of chemica l and physical methods at controll ing formation of ectomycorrh izas on Douglas-f i r seed l ings . W e tested the ef fect iveness of the fungic ides, Topas® and Senator®, at var ious concentrat ions and application f requencies. W e predicted that both fungic ides would reduce ectomycorrhizal colonizat ion, however, we expected that colonizat ion of ascomycete fungi would be particularly reduced with the application of Senator®. Thus , the composi t ion of the ectomycorrhizal fungal community would be altered compared to untreated controls, In addit ion, we tested the ef fect iveness of nylon mesh with var ious pore s i zes at preventing hyphal penetration, and its effects on ectomycorrhizal community composi t ion of neighboring seedl ings. W e predicted that percent colonizat ion and similarity of ectomycorrhizal communi t ies between seed l ings on opposite s ides of the mesh barrier would dec rease with decreas ing mesh pore s ize . Materials and methods Field soil collection O n August 27-28 of 2003, we col lected 600 L of soil from the B lack P ines variable retention cut (also known a s a green-tree retention cut where s o m e trees are not harvested) and adjacent forest approximately 50 km northwest of Kamloops , British Co lumb ia (120°26'W, 50°42'N). The Black P ines variable retention cut occurs in the dry cool subzone of the Interior Douglas-f ir (IDFdk) biogeocl imatic zone (Meidinger and Pojar 1991). It has an elevation of 1180 meters above s e a level (masl) and loamy Gray Luvisol ic soil (Krzic et a l . 2004). The plant community is dominated by residual Doug las-fir Pseudotsuga menziesii war. glauca (Beissn.) Franco) and subalp ine fir {Abies lasiocarpa (Hook.) Nutt.) trees and advanced regeneration (sapl ings), with shrub and herbaceous layers dominated by soopolal l ie (Sherpherdia canadensis (L.) Nutt.) and p inegrass (Calamagrostis rubescens Buckley), respectively. W e col lected forest floor (30 cm x 30 cm) together with mineral soi l (to 40 cm depth) from 15 random locat ions in 1 h a of the B lack P i n e s forest. Th i s soi l w a s u s e d for both exper iments. The fifteen samp les were combined and thoroughly mixed, then stored at room temperature until needed (see below). 51 Plant material Interior Douglas-f ir seedl ings (seedlot #48520, British Co lumb ia Ministry of Forest Tree S e e d Center , Surrey, British Co lumb ia , Canada ) were grown at the University of British Co lumb ia (Vancouver, Canada ) greenhouse (temperature minimum 20°C, temperature maximum 24°C, average humidity 60%). S e e d s were moist-stratif ied at 4°C for 21 days. S e e d s were then steri l ized in constantly mixed 3 % H2O2 for two hours. Styroblock™ 512B trays (Beaver Plast ics Ltd., Edmonton, Alberta, Canada ) were cut in half horizontally and filled with autoclaved peat and sawdust (3:1, v:v). Three s e e d s were sown in each cavity and 4 weeks later were thinned to one seedl ing per cavity. The trays were p laced under a mist tent for 12 days and then moved to a g reenhouse bench for the remaining time. To improve seedl ing vigor and d iscourage mycorrhizal colonizat ion, we appl ied 1.9 g L"1 water soluble R o s e Plant Food (Mirac le-Gro, Scot ts C a n a d a Ltd., M iss i ssauga , Ontario, Canada ) (18:24:16 N:P:K) once per week for 4 weeks following germination. Afterwards, we fertilized with 4 ml L"1 Peter 's solut ion (Plant-Prod ®, Plant Products C o . Ltd., Brampton, Ontario, Canada ) (20:20:20 N:P :K) once per week until the seedl ings were transplanted into the treatment pots. For the duration of the two concurrent exper iments (five months), natural daylight in the greenhouse was supplemented by 400 W high pressure sodium lamps to maintain an 18 hour photoperiod. Fungicide experiment Experimental design and treatments On September 16, 2003, 14-week-old seedl ings were transplanted into 3.2 L pots (175 mm x.180 mm) (Listo Products Ltd., Surrey, British Co lumb ia , Canada ) with drainage holes. The pots contained field soil mixed with perlite (3:1, v:v). A 3 x 3 x 3 factorial set of treatments with a separate control group was replicated 10 t imes in a completely randomized des ign, where the factors were fungicide type, rate of appl icat ion, and frequency of application (270 seedl ings + 10 controls = 280 total). The three fungicide types were Senator®, Topas®, and a combinat ion of the two fungic ides (both from Engage Agro Corporat ion, Gue lph , Ontario, Canada ) . The three rates of application were: 0.5,1 or 1.5 ml L"1 of Senator®; and 0.5,1 or 1.5 g L"1 of Topas®. R e c o m m e n d e d concentrat ions of Senator® and Topas® are 0.5 ml L"1 and 0.5 g L" \ respectively. To our knowledge this is the only study assess ing the effect of these 52 fungicides on ectomycorrhizal fungi thus, we dec ided as a starting point to use the above rates. The fungicide was mixed with water and added at a constant vo lume of 600 mL pot"1; therefore, seedl ings that were treated with Senator® and Topas® in combinat ion received 300 mL of each fungicide-water mixture. The three f requencies of application were: once at the beginning of the experiment, every two months (three appl icat ions total), or every month (five appl icat ions in total). For each fungicide appl icat ion, we drenched the soil around the seedl ings, avoiding contact with fol iage. Addit ionally, ten control seedl ings were grown in pots to which only water w a s appl ied. O n September 30, 2003, initial height was recorded for all seedl ings. The seed l ings were watered as necessary and their locations re-randomized monthly. Seedling measurements O n February 10, 2004, the height of all surviving seedl ings was measu red . Shoots were removed, dried at 65° C for 48 hours and weighed. The roots and intact soil of up to seven replicates were stored at 4°C for 45 days before process ing. E a c h root system was soaked in tap water, r insed c lean of soi l , and cut into 1 cm fragments. The sample was then divided approximately in half, and one half was dried and weighed. W e used this measurement to estimate dry weight of the remaining roots, which were weighed wet, and then c leared and stained following the methodology of Phi l l ips and Hayman (1970) to a s s e s s percent ectomycorrhizal colonizat ion. For a given seedl ing, percent ectomycorrhizal colonizat ion was calculated as : Percent ectomycorrhizal colonizat ion = (Active ectomycorrhizal root tips / Act ive ectomycorrhizal root tips + Act ive non-ectomycorrhizal root tips) x 100 A root tip surrounded by a mantle was c lassi f ied a s mycorrhizal . In addition to assess ing percent colonizat ion, we recorded the abundance and r ichness of ectomycorrhizal morphotypes in each of the treatments. Root sys tems of the remaining three replicates from each of the ten treatments were carefully w a s h e d under running tap water and then cut into approximately 1 cm p ieces. Al l root f ragments were p laced in a baking dish containing water and thoroughly mixed. W e randomly subsampled and counted up to 100 ectomycorrh izas, or 100 non-ectomycorrhizal root 53 tips, whichever c a m e first. General ly , ectomycorrhizal tips were turgid and smooth, had emanat ing hyphae or rh izomorphs (Harvey et a l . 1976), and had a Hartig net. A root tip that was dark and wrinkled, or was somewhat hollow and fragmented under minimal pressure was c lassi f ied a s 'dead ' . G r o s s morphology of ectomycorrhizal roots and rhizomorphs were descr ibed using a s tereomicroscope, while the mantle, cyst idia, and emanat ing hyphae were descr ibed using a compound microscope under 400x or 1000x magnif ication. W h e n possib le, mant les were peeled by separat ing the fungal t issue from the root with forceps and micro-scalpels , and then descr ibed. Morphological descr ipt ions were made with reference to Agerer (1985-1998) , Ingleby et a l . (1990), G o o d m a n et a l . (1996), and Hagerman et a l . (2001). Morphotyped roots were then dried and weighed. Mesh barrier experiment Experimental design and treatments To test the effect of pore s ize on penetration by ectomycorrhizal fungi, we grew seedl ings in 3.2 L pots divided vertically by nylon mesh barriers with different pore s izes . The pore s i zes of the four m e s h e s were: 0.2 um (catalogue number 25007 , polyamide type 250 membrane, Sartor ius A G , Goet t ingen, Germany) , 1 um (catalogue number 03-1/1 Nitex, Sefar Amer i ca Inc., Depew, N Y , U S A ) , 20 pm (catalogue number 03-20/14 N i tex ) , and 500 um (catalogue number 06-500/47 Nitex). Control pots were divided by an impermeable acetate sheet to test for ectomycorrhizal contaminat ion through insufficient steri l ization, or water and airborne ectomycorrhizal propagules. E a c h of the five barrier treatments was replicated 12 t imes in a completely randomized des ign. The pots were first steri l ized in a 2 0 % bleach solution for at least one hour, cut in half vertically, and then reassembled using non-toxic adhes ive si l icone sealant (catalogue number 3145 -Grey -RTV ; mi l -A-46146, Dow Corn ing Mid land, M l , U S A ) to attach the mesh and hold the two halves of the pot together. E a c h pot had two compartments. O n August 30, 2003 one compartment was filled with field soi l mixed with perlite (3:1, v:v), watered, and planted with14-week-old seedl ings (see Plant Material for growth condit ions). Three weeks after the seedl ings were transplanted into the unsteri l ized soi l , the second compartments were filled with steri l ized field soi l . Soi l was steri l ized by autoclaving at 15 p.s.i for 90 minutes, repeated 24 hours later. Unco lon ized 17-week-old seedl ings were then transplanted into the steri l ized soil and 54 watered. The purpose of transplanting seedl ings into the unsteri l ized field soi l 3 weeks prior to the introduction of seedl ings into the other half of the pot w a s to insure that the seedl ings were already co lon ized by ectomycorrhizal fungi when the exper iment w a s started. W e refer to the initially transplanted seedl ings a s "source seedl ings" . If hyphae from the source seedl ings were able to penetrate a mesh of a given pore s ize , we expected to see mycorrhizal root tips on "recipient" seedl ings grown in steri l ized field soi l . O n c e all source and recipient seedl ings had been transplanted into the pots, the seedl ings were watered as necessary . Jus t prior to transplanting, we destructively subsampled fifteen source seedl ings to quantify ectomycorrhizal colonizat ion fol lowing the methodology of Phi l l ips and Hayman (1970). Afterwards, pot location on the greenhouse bench was re-randomized monthly. Initial shoot height w a s measured shortly after transplanting, on September 30, 2003. Seedling measurements At harvest, January 11, 2004, shoot height and b iomass (dried at 65°C for 48 hours) were measured . During the harvest, we also inspected mesh barriers for s igns of hyphal penetration using a s tereomicroscope. W e chose to randomly select ten repl icates per mesh barrier treatment for morphotyping using similar methods outl ined above (5 treatments x 2 seedl ings per pot x 10 replicates = 100 seedl ings) . Three replicate sets of one root tip per morphotype from different seedl ings were lyophi l ized prior to storage for subsequent molecular analys is. On average, 3 % of the total roots tips per morphotype examined were sent for molecular analys is. The remainder of the morphotyped roots were dried and weighed with the remainder of the root samp le . Molecular confirmation of ectomycorrhizal fungal species identification Total genomic D N A was extracted from single ectomycorrhizal tips by pulver iz ing them for 45 seconds at a speed of 5.0 units using a B i o l 01 Sys tems Fast Prep F P 1 2 0 high f requency shaker (Q-biogene, Ca r l sbad , C A , U S A ) . D N A was isolated us ing the procedure of Baldwin and Egger (1996). The final D N A pellet was dried using a s p e e d vacuum concentrator and then re-suspended in 50 u.L E D T A - T E buffer. Fol lowing D N A extraction and isolation, the internal t ranscribed space r (ITS) region of the fungal nuclear rDNA was specif ical ly amplif ied by the primers NSI1 and 55 N L C 2 (Martin and Ryg iewicz 2005). P C R reactions typically included 1 uL template D N A , 18.6 uL sterile purified water (Barnested Nanopure D iamond water purifier), 0.2 m M deoxyr ibonucleot ies (dNTPs) , 2.5 ul 10x P C R buffer, 1.5 m M M g C I 2 , 0.48 m M each primer, 1.6 mg mL" 1 bovine serum albumin (BSA) , and 0.25 U uL" 1 Amp l iTaq Go ld™ (Appl ied B iosys tems, Foster City, C A , U S A ) . S a m p l e s were amplif ied using a P T C - 2 0 0 thermal cycler (MJ Resea rch Inc., Wa l tham, M A , U S A ) . A 10 min hot start w a s fol lowed by P C R cycl ing a s fol lows: 45 seconds at 94°C fol lowed by 34 cyc les of denaturat ion at 94°C for 45 seconds , anneal ing at 54°C for 45 seconds , ramping 72°C for 1 minute with a 1 second extension after each cyc le , and extension at 72°C for 10 minutes, and then the temperature was held at 4°C. The P C R products were v isual ized on 1.5% agarose gels using a G e l Log ics 440 (Kodak Instruments, Rochester , N Y , U S A ) . T h e P C R product was c leaned using the QIAquick P C R Purification kit (Qiagen Inc., Va lenc ia , C A , U S A ) . Prior to sequenc ing , the large ITS fragment produced above, w a s re-amplif ied in a nested P C R reaction using the primers ITS 1 and ITS 4 (White et a l . 1990). P C R products were quantif ied and then sequenced using a 3730 D N A Capi l lary Sequence r (Appl ied Biosystems) at the University of British Co lumb ia Nuc le ic Ac id and Protein Serv ices Unit. Al l unique morphotypes were sequenced and then al igned using Sequenche r software (Gene C o d e s Corporat ion, Ann Arbor, M l , U S A ) . Taxonomic matches were based on B L A S T results with >98% sequence similarity. Statistical analysis The fungicide experiment examined a 3 x 3 x 3 factorial set with a separate control group of treatments (i.e., separate from the factorial but combined in the layout) in a completely randomized design (Bergerud 1989). W e used percent colonizat ion data obtained from the c leared and stained roots and normal ized the data with a square root transformation for analys is of var iance ( A N O V A ) . W e ana lyzed ectomycorrhizal fungal community data (r ichness and diversity, relative abundance of morphotypes with >5% of ectomycorrhizal root tips), seedl ing growth, and square root of percent colonizat ion, first by using the G L M procedure in S A S ( S A S Institute Inc. 1999). W e then ran a second G L M procedure with a contrast statement to compare the control treatment against all other treatment combinat ions. Ana l yses on data col lected from c leared and sta ined roots and morphotyped root tips were done separately, and consequent ly graphed separately. A N O V A tables were constructed manual ly to obtain the proper exper imental 56 error terms and degrees of f reedom. W h e n significant main treatment effects occurred, we separated m e a n s us ing the Bonferroni multiple compar ison test. For the mesh barrier experiment, the percent colonizat ion and ectomycorrhizal r ichness for both seedl ings per pot were used to calculate the Ste inhaus index of ectomycorrhizal community similarity (Legendre and Legendre 1998) and to calculate the difference in morphotype r ichness (the number of morphotypes on the donor root sys tem minus the number on the receiver root system). The effects of mesh pore s ize on ectomycorrhizal fungal community data (r ichness difference and Ste inhaus index of similarity), percent ectomycorrhizal colonizat ion and seedl ing growth (shoot height, b iomass and root b iomass) were detected with a one-way A N O V A using the G L M procedure in S A S ( S A S Institute Inc. 1999). For both percent ectomycorrhizal colonizat ion and seedl ing growth, the difference in the response variable between source and recipient seedl ings within a pot was calculated and used in the analys is . Dif ferences were cons idered significant at a = 0.05. Where significant mesh barrier treatment effects occurred, we separated means using the Bonferroni multiple compar ison test. Effects of steril ization on seedl ing growth and total percent ectomycorrhizal colonizat ion were ana lyzed using the T T E S T procedure for each mesh s ize ( S A S Institute Inc. 1999). Results Fungicide treatments Approximately 3 0 % of the roots of control seedl ings (i.e., seed l ings receiving only water) were co lon ized after 21 weeks in the treatment pots. Appl icat ion of fungicide reduced ectomycorrhizal colonizat ion by up to 5 0 % , depending on fungicide type (p < 0.0001) but not appl icat ion concentrat ion (p = 0.9) (Table 3.1). The most effective treatment regime w a s Topas® appl ied alone or in combinat ion with Senator® (Fig. 3.1a). Senator® alone w a s less effective at decreas ing ectomycorrhizal colonizat ion, with only a 3 6 % reduction compared with 5 6 % reduction using Topas®. There were no di f ferences assoc ia ted with different application f requencies (Fig. 3.1b) and there were no significant interactions among any combinat ion of the three treatment factors (p > 0.05, Tab le 3.1). None of the fungicides appl ied at any concentrat ion or appl icat ion frequency, affected seedl ing height or shoot or root b iomass (Table 3.1). 57 A total of eight morphotypes were identified and descr ibed (Table 3.2). Two had > 9 8 % sequence matches of their ITS sequences to Wilcoxina rehmi and Thelephora terrestris access ions in Genbank . D N A from the other six morphotypes either did not amplify or had less than 9 8 % sequence homology with genotypes in Genbank . O n e morphotype was not identifiable and was classi f ied as undifferentiated. Only the Rhizopogon/Suillus-\ype formed rhizomorphs; the remainder had relatively smooth mant les (Table 3.2). O n average we observed more morphotypes on seedl ings that were subject to fungicides than those that were not (Fig. 3.2). However, neither ectomycorrh izal community r ichness (p = 0.2) nor diversity (p = 0.3) was significantly affected by the fungicide types. The abundance of Wilcoxina rehmii mycorrh izas (the most common ectomycorrhiza) as a percentage of all root tips examined was reduced by Topas® appl ied alone or in combinat ion with Senator®, when compared to Senator® alone or the control (Fig. 3.3). The abundance of Cenococcum geophilum, the other dominant ascomyce tous mycorrh iza, was not affected by application of fungic ides (p = 0.6, data not shown). Similarly, the abundances of Rhizopogon/Suillus- and Tomentella-type mycorrhizas, the most abundant basid iomycetes, were a lso not affected by fungicide treatment (p = 0.7, p = 0.8, respectively, data not shown). Mesh barrier treatments Source seedl ings had greater shoot height, shoot b iomass , root b iomass , and ectomycorrhizal colonizat ion than recipient seedl ings ac ross all mesh treatments except the 20 um pore s ize (Table 3.3), and mesh s ize did not affect the magni tude of these di f ferences (Table 3.4). A c r o s s all mesh s izes , on average, 50 and 2 1 % of roots of source and recipient seedl ings were co lonized by ectomycorrhizal fungi, respect ively. T h e s e colonizat ion levels contrast measurements at planting where colonizat ion of source seedl ings was less than 1%. W e found six distinct morphotypes on source seedl ings (Table 3.2). Mos t of the six morphotypes were represented in all mesh treatments (Fig. 3.4). Wilcoxina rehmii ectomycorrh izas compr ised >85% of the community on source and recipient seed l ings separated with mesh barriers of 1 um or larger (> 80%). By contrast, both the 0.2 um and 1 um pore-s ized m e s h e s blocked the formation of Rhizopogon/Suillus-Xype mycorrh izas on recipient seedl ings (Fig. 3.4). Th is type formed approximately 5 % of the 58 mycorrh izas on source seedl ings. MRA- type morphotypes were found on all source seedl ings, but were absent from recipient seedl ings of all mesh treatments. Thelephora terrestris ectomycorrh izas formed an increasingly high proportion of the communi ty on recipient seedl ings as mesh s ize dec reased , whereas they were not found on source seedl ings. The abundance of Cenococcum geophilum mycorrh izas w a s too low to be useful in detecting mesh effects. Ectomycorrhizal community similarity, which takes into account r ichness and relative abundance , between recipient seedl ings versus source seedl ings increased with mesh pore s i zes greater than 0.2 urn (p < 0.0001) (Fig. 3.5a). The ectomycorrhizal communi t ies separated by the full barrier (control) or by mesh of pore s ize 0.2 urn were significantly dissimi lar from those separated by mesh with pore s i zes 1 urn and larger (Fig. 3.5a). The difference in morphotype r ichness between source and recipient seedl ings was large in the full barrier treatment and general ly dec reased as mesh s ize increased (p = 0.09) (Fig. 3.5b). W h e n examined under the microscope, we observed hyphae penetrating pore s i zes of 1 urn and larger, and roots penetrating only 500 urn pores. Three of the mesh barriers were torn in pots of the 0.2 urn mesh treatment; these repl icates were omitted from the ana lyses . Discussion Fungicide effects on ectomycorrhizal colonization This study shows that fungicides can be used to significantly reduce ectomycorrhizal colonizat ion in controlled exper iments. Topas® was more effective than Senator® at reducing ectomycorrhizal colonizat ion levels. The manufacturer 's recommended concentrat ion was effective in reducing colonizat ion, and there was no advantage to applying Topas® repeatedly during the course of the experiment. In our study, ectomycorrhizal colonizat ion dec reased by as much as 5 6 % compared with the control. Douglas-f ir control seedl ings in this experiment had relatively low levels of colonizat ion (approximately 30%) but these levels are typical for greenhouse-grown interior Douglas-f ir (5-42%) (Hagerman and Durall 2004, Teste et a l . 2004). Our results are consistent with another study using propiconazole. Mann inen et a l . , (1998) found that 0.15 g of propiconazole appl ied to seedl ings in the field (versus 9.6 g at the highest application f requency in our study) caused a dec rease in ectomycorrhizal colonizat ion of 59 almost 3 3 % (from 67 to 4 5 % colonization) two years after 2 year-old nursery grown Pinus sylvestris seedl ings were outplanted. Al though the fungicides did not el iminate ectomycorrhizal colonizat ion altogether, we propose that Topas® reduces colonizat ion to an extent to be useful for field studies. Simi lar dec reases in arbuscular mycorrhizal colonizat ion following benomyl appl icat ion have resulted in substantial changes in structure of the plant community. For example , reductions in arbuscular mycorrhizal colonizat ion of 6 0 % have changed plant nitrogen and phosphorus concentrat ions and aboveground community productivity in Borea l grass land communi t ies (Dhillion and Gardsjord 2004). Hartnett and Wi lson (1999) found that a 7 5 % decrease in arbuscular mycorrhizal colonizat ion coinc ided with b iomass dec reases of dominant C 4 g rasses . Ca l laway et a l . (2004) reported that interactions between native grass land spec ies and the invasive Centaurea maculosa were substantial ly altered when experimental plots were treated with benomyl ; the fungicide dec reased arbuscular mycorrhizal colonizat ion by >80%, resulting in a C. maculosa b iomass dec rease when mixed with Koeleria cristata or Festuca idahoensia. A s s u m i n g reductions in arbuscular and ectomycorrhizal colonizat ion result in similar functional responses in plant communit ies, we expect that Topas® appl ied at the recommended rate once every five months will reduce ectomycorrhizal colonizat ion sufficiently to affect seedl ing performance in the field. The specificity of the fungicides for ascomyce tes and bas idomycetes differed from that expected. Senator® is reported to be more effective against ascomyce tes than bas id iomycetes, and yet it appeared to have no effect on Wilcoxina rehmii, a dominant ascomycete in this study. Mann inen et a l . (1998) reported that propiconzaole w a s a lso more effective at inhibiting ascomycete than basid iomycete symbionts and this is conf irmed by Laat ikainen and Heinonen-Tansk i (2002). The latter found that low concentrat ions of propiconazole (0.1 ppm) increased growth of Suillus bovinus and S . variegatus strains grown in vitro, and that these fungi were tolerant of concentrat ions up to 1 ppm. In our study, the effect iveness of propiconazole (Topas®) could not be predicted strictly by taxonomic status. For example , it caused a substantial reduction in colonizat ion by Wilcoxina rehmii, but not by Cenococcum geophilum, another important ascomycete . Colonizat ion by the bas id iomycetes forming Thelephora terrestris, Tomentella-type, and Rhizopogon/Suillus-type mycorrhizas either increased or w a s not affected by either fungicide however. In our study, Topas® targeted the most abundant 60 ectomycorrhizal fungi, Wilcoxina rehmii, so that the addit ional application of Senator® provided no further advantage. Other fungicides have had variable effects on ectomycorrhizal colonizat ion. O'Nei l l and Mitchell (2000) appl ied benomyl to Picea sitchensis seedl ings and found that colonizat ion was reduced from 6 0 % to 20%; however, only one morphotype, Wilcoxina mikolae, was observed on the nursery grown seedl ings. In another study, the percent of roots co lonized by Thelephora terrestris or Laccaria laccata dec reased when 0 .3% Dithane M-45 was appl ied to Pinus patula seedl ings grown in pouches , and similar reductions in hyphal dry weight occurred when the fungicide was appl ied to in vitro cultures (Reddy and Natarajan 1995). A wide range of responses were exhibited by 64 strains of ectomycorrhizal fungi grown in vitro and exposed to relatively low concentrat ions (<10 ppm) of five fungicides (benomyl, chorothaloni l , copper oxychlor ide, maneb and propiconazole) (Laatikainen and Heinonen-Tansk i 2002). Converse ly , in some other laboratory studies fungicides have increased ectomycorrhizal colonizat ion (Pawuk et a l .1980, Marx and Rowan 1981, de la Bast ide and Kendr ick 1990). Th is effect is likely due to the select ive inhibition of fungi that are competit ive towards ectomycorrhizal fungi (Summerbel l 1988). In our study, interactions among ectomycorrhizal fungi could have resulted in the increase of basidorhyctes observed . Wilcoxina rehmii, a rapid colonizer of nursery seedl ings (Mikola 1988) w a s supp ressed by the application of Topas®. Remova l of this rapid colonizer could have al lowed other ectomycorrhizal fungi to colonize seedl ing root tips. Surveys of the entire fungal community on a large number of replicate seedl ings is required to investigate this possibil ity. Our results suggest that Topas® should be effective at reducing morphotypes commonly found in greenhouse b ioassays of field soi ls, but there are two caveats . First, we could not a s s e s s the effects of fungicides on rare ectomycorrhizal fungal spec ies or those that do not colonize seedl ings in greenhouses. S e c o n d , Topas® may affect seedl ing physiology and/or other soil biota. These impacts are more difficult to identify and quantify by short term exper iments in a greenhouse setting. Prop iconazo le has been shown to have growth-regulator effects on plants in the S o l a n a c a e a e family (Kendrick 2000), and it has a lso been shown to affect soil fauna, such a s f lagel lates (Ekelund et a l . 2000), as well as soil respiration (Elmholt 1992). Topas® is recommended for prevention of a variety of foliar fungal d i seases , and its mode of 61 action by preventing ergosterol synthesis makes it likely to a lso affect non-target saprotrophic and parasit ic soil fungi. A change in this community would alter potential food substrates of soil fauna. In exper iments where reduction of ectomycorrhizal fungi is of primary concern , and side-effects on the soil biota is unimportant, then appl icat ions of Topas® can be an effective treatment regime. G iven that the active ingredient in Topas® is fungistatic, repeated appl icat ions may be required where there is high hyphal turnover, as would happen over a temperate growing s e a s o n , or where there is high fungal propagule pressure; both of these condit ions occur in field situations. Mesh barrier effects on hyphal penetration Our study indicates that mesh with pore s ize 0.2 urn is effective at reducing hyphal penetration and mycorrhizal colonizat ion of neighboring seedl ings. However we conc lude that the threshold for restricting ectomycorrhizal hyphal penetration l ies between 0.2 and 1 pm. Ectomycorrhizal r ichness tended to increase in steri l ized compartments where mesh s ize equaled or exceeded 1 urn, suggest ing hyphae from the source seedl ings compartment penetrated the mesh and co lon ized the recipient seedl ings growing in the steri l ized compartment. Of even greater s igni f icance, ectomycorrhizal community similarity between source and recipient seed l ings greatly increased in m e s h e s > 1 pm. If the recipient seedl ings were mycorrhiza-free, di f ferences in r ichness alone should have indicated mesh ef fect iveness at restricting hyphal penetration, regardless of abundance, but the smal l number of morphotypes may have rendered r ichness as a measure with little resolving power. The ectomycorrhizal community observed in our study w a s typical for interior Douglas-f ir seedl ings inoculated with field soil and grown in the g reenhouse (Jones et a l . 1997, S imard et a l . 1997, Hagerman and Durall 2004, Teste et a l . 2004) . The six morphotypes formed on the source seedl ings also represented a broad range of mantle types (texture and thickness), width of emanat ing hyphal forms (width and extension 3 to 7 pm), as well as the presence or absence of rhizomorphs. Their p resence al lowed us to test the effect iveness of the pore s i zes at preventing hyphal penetration by ectomycorrhizal fungi with different character ist ics. W e might predict, for example , that a mesh with a smal ler pore s ize would be required to prevent penetration of s ingle hyphae, compared to the s ize required to stop penetration of rh izomorphs. Our f indings support this prediction s ince we found that the rhizomorph-forming Rhizopogon/Suillus-62 type morphotype was restricted by a mesh s ize between 1 to 20 um. W e propose that m e s h e s with pore s i zes smal ler than 1 um would be adequate in field situations. Al though mesh with 0.2 um pores was the most effective at reducing hyphal penetration, it w a s very fragile. Th is characterist ic of nylon mesh with pore s i zes smal ler than 1 um has been noted previously (Tarafdar and Marschner 1994). Our results suggest that field exper iments requiring fine mesh (0.2 um) should use more durable nylon (i.e. mesh th ickness > 115 um) or metal based mesh . Our finding that mesh with pore s i zes between 0.2 um and 1 um are most effective at inhibiting ectomycorrhizal colonizat ion must be interpreted caut iously because some ectomycorrh izas were found in steri l ized soi ls with a 0.2 um mesh barrier. Within the steri l ized compartment of these pots, the ectomycorrhizal communi ty was reduced but not el iminated. For example , Wilcoxina rehmii was on the recipient seedl ings, regardless of the mesh barrier type, but was not observed in control pots, suggest ing that hyphal penetration or spore d ispersal may have occurred. W e are uncertain why Wilcoxina rehmii was not found in the steri l ized compartment of the control pots. Further research is warranted on Wilcoxina rehmii propagat ing strategies in nurser ies (e.g., hyphal and spore) and morphological plasticity. W e a lso found that Thelephora terrestris had co lon ized root tips in one seedl ing of the control treatment (i.e. steri l ized soil with a full barrier), confirming previous studies that it is a common greenhouse contaminant. Statistical ana lyses were run without Thelephora terrestris (data not shown); however, results were similar, and did not change our conc lus ions about the hyphal restriction properties of the mesh treatments. MRA- t ype mycorrh izas were a lso only observed on source seedl ings ac ross all mesh treatments, suggest ing that chemica l changes induced by autoclaving may have inhibited this particular ectomycorrhizal fungus. Rh izomorphs were completely exc luded from steri l ized compartments separated by 1 or 0.2 um mesh . Conclusions The use of mesh barriers versus fungicides for controll ing ectomycorrhizal colonizat ion depends on the ecological p rocesses that must be maintained and those that can be compromised in the experiment. Future C M N research can benefit from the use of mesh barriers. M e s h barriers with a gradient of pore s i zes have the potential to tease out carbon and nutrient pathways (soil-only, hyphal-only, rhizomorph-only, etc.) in 63 resource shar ing C M N studies. However, installing mesh barriers will disrupt soi l structure and potentially reduce water flow through smal l pore s i zes . If the purpose of mesh is to exc lude mycorrhizal hyphae, and maintain non-mycorrhizal status of the enc losed host, the soil contained in the mesh barrier compartment will require steri l ization. M e s h with pore s i zes < 1 pm appear to reduce hyphal penetrat ion, however care will be required to exc lude fungal propagules arriving via air or water pathways. W e suggest that mesh barriers, apart from their disruptive installment, are a more promising method than fungic ides to completely exclude fungi. T a b l e 3.1: A n a l y s i s of v a r i a n c e for e f fec t o f f u n g i c i d e t y p e (F ) , c o n c e n t r a t i o n (C) , a n d app l i ca t i on f r e q u e n c y (A) o n s q u a r e root p e r c e n t e c t o m y c o r r h i z a l c o l o n i z a t i o n (%) a n d s i z e of D o u g l a s - f i r (Pseudotsuga menziesii var . glauca) s e e d l i n g s af ter f ive m o n t h s . Source of variation df MS F P MS Control vs. all others 1 13.55 9.67 <0001 0.03 Fungicide type 2 9.70 6.92 <0001 69.40 Concentration 2 0.23 0.16 0.85- 16.10 Application frequency 2 22.81 16.28 <0001 46.00 FxC 4 1.56 1.11 0.35 22.90 FxA 4 1.99 1.42 0.23 47.90 CxA 4 0.25 0.18 0.95 8.32 FxCxA 8 1.44 1.03 0.42 17.90 Error 135 1.40 33.70 Height Shoot biomass Root biomass F P MS F P MS F P 0.00 0.98 0.01 0.01 0.90 0.03 0.32 0.57 2.06 0.13 0.29 0.60 0.55 0.08 1.01 0.37 0.48 0.63 0.62 1.29 0.28 0.04 0.48 0.62 1.37 0.26 0.74 1.55 0.22 0.11 1.47 0.23 0.68 0.61 0.36 0.75 0.56 0.06 0.77 0.55 1.42 0.23 0.15 0.32 0.87 0.05 0.68 0.61 0.25 0.91 0.73 1.52 0.20 0.16 2.11 0.08 0.53 0.83 0.43 0.90 0.52 0.02 0.29 0.97 0.48 0.48 0.08 0) Table 3.2: Descript ion of morphological characterist ics of ectomycorrhizas observed on Douglas-fir {Pseudotsuga menziesii glauca) seedl ings grown in the fungicide (F) and mesh (M) study. Morphotype and Blast match Macroscopic description Mante type(s) Emanating hyphae Rhizomorphs Cystidia Rhizopogon/Suillus-type (R/S); F and M Unbranched to subtuberculate silvery white mycorrhiza with rough texture Outer: felt prosenchyma, hyphae 3-4 pm smooth, and thick-walled; inner: net synenchyma, thin, hyphae 2 pm 3 pm wide; no clamps, crystalline ornamentation, and elbow-like bends Compact brown with crystalline ornamentation and elbow-like bends Absent Thelophora-lype (T) Blasted to Thelephora terrestris, Accession No. U83486, 619/627 base pairs = 99%; F and M Unbranched or irregular bright orange to brown (sometimes whitish) mycorrhiza with smooth reflective texture Outer: net synenchyma, hyphae 3 pm wide; inner: incomplete interlocking irregular synenchyma, hyphae 4-5 pm wide Rare, 3 pm wide; clamps, smooth with occasional enlarged hyphal junctions Absent Common, 40-50 pm long and 3 pm wide with basal clamp Morphotype and Blast match Macroscopic description Mante type(s) Emanating hyphae Cenococcum geophilum (Cg); F and M Unbranched, black mycorrhiza with rough hairy texture Outer: net synenchyma in a stellate pattern, hyphae 6 pm wide; inner: net synenchyma 5-6 pm wide black, straight Rhizomorphs Absent Cystidia Absent W/Vcox/na-type (W) Blasted-to Wilcoxina rehmii Accession No. DO069C01, 510/519 base pairs = 98% Irregular dark brown to orangish mycorrhiza, often wrinkled, also called E-strain Outer: not seen; inner: patchy and incomplete net prosenchyma, hyphae 2 pm wide Absent Absent Absent Mycelium radicis atrovirens-type (MRA); F and M Unbranched black to brown mycorrhiza with curled hairy or very rough texture Outer: felt prosenchyma, hyphae 3 pm wide; inner: net synenchyma, hyphae 2-3 pm wide Rare, 5-7 pm wide, no clamps, smooth but becoming progressively more verrucose away from the mantle Absent Absent Undifferentiated (Undif); F and M Young orange mycorrhiza with no distinct characters Barely visible net synenchyma readily turning into Hartig net Absent Absent Absent Morphotype and Blast match TomentellaAype (Tom); F Macroscopic description Mante type(s) Emanating hyphae Swollen dark-brown sandy textured mycorrhiza Outer: squarish incomplete interlocking irregular synenchyma with thick-walled hyphae; inner: net synenchyma Absent Rhizomorphs Absent Cystidia Absent Piloderma-type (P ) ;F Yellow coarsely felty mycorrhiza with abundant rhizomorphs Not determined Absent Finely verrucose, Absent septa common, not clamped, approximately 3 urn wide Table 3.3: Effect of steril ization on growth and ectomycorrhizal (EM) colonization of Douglas-fir (Pseudotsuga menziesii var. glauca) seedl ings. A ser ies of t-tests were used to determine differences among source (S) and recipient (R) seedl ings grown each mesh barrier treatment. Height Shoot Root M e s h increment gain gain (pm) So i l n (cm) S E M * P n (g) S E M P n (g) S E M P S 10 15 2.6 10 0.91 0.137 9 0.32 0.056 control 0.0294 0.0061 0.6633 R 12 10 2.6 .12 0.42 0.137 10 0.29 0.056 S 12 14 0.8 12 0.97 0.084 10 0.44 0.064 0.2 <0001 <0001 0.0055 R 12 9 0.8 12 0.34 0.084 12 0.19 0.064 S 11 20 2.4 11 1.55 0.196 8 0.61 0.123 1 0.0195 <0001 0.0360 R 11 14 2.4 11 0.70 0.196 9 0.31 0.123 S 12 18 3.6 12 1.23 0.379 10 0.48 0.204 20 0.3541 0.4363 0.4843 R 8 12 3.6 8 0.86 0.379 5 0.29 0.204 S 12 21 4.1 12 1.51 0.319 11 0.50 0.074 500 0.0588 0.0333 0.0400 R 9 11 4.1 9 0.61 0.319 9 0.30 0.074 * S E M : standard error of the mean. Seedling growth is expressed as height and biomass measured after 5 months. Table 3.4: (continued) Effect of sterilization on growth and ectomycorrhizal (EM) colonization of Douglas-fir {Pseudotsuga menziesii war. glauca) seedl ings. A ser ies of t-tests were used to determine differences among source (S) and recipient (R) seedl ings grown for each mesh barrier treatment. Root: Percent E M Mesh Shoot colonization (um) Soi l gain S E M P n (%) S E M P S 0.33 0.155 9 50 7 control 0.0075 O . 0 0 0 1 R 0.88 0.155 9 3 7 S 0.44 0.056 7 43 12 0.2 0.0675 0.0195 R 0.56 0.056 9 11 12 S 0.49 0.145 9 57 8 1 0.6007 0.0113 R 0.41 0.145 9 33 8 S 0.42 0.077 10 47 9 20 0.5329 0.1308 R 0.37 0.077 5 29 9 S 0.35 0.083 11 51 10 500 0.0427 0.0434 R 0.57 0.083 9 28 10 * S E M : standard error of the mean. Seedling growth is expressed as height and biomass measured after 5 months. Tab le 3.5: Effect of mesh treatment on growth and ectomycorrhizal (EM) colonization of Douglas-fir {Pseudotsuga menziesii glauca) seedl ings. R e s p o n s e dif ferences between source and recipient seedl ings were calculated for each pot. Th is single number was used in the A N O V A for each response variable. Statistically significant mesh treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05). M e s h (um) n Height increment (cm) S E M * n Shoot gain (g) S E M n Root gain (g) S E M control 10 6 a ± 2.6 10 0.49 a + 0.220 9 0.03 a + 0.088 0.2 12 5 a + 2.4 12 0.63 a + 0.201 10 0.23 a + 0.084 1 10 7 a + 2.6 10 0.90 a + 0.220 7 0.33 a + 0.100 20 8 4 a + 2.9 8 0.31 a + 0.247 5 0.16 a + 0.118 500 9 9 a + 2.7 9 0.82 a ± 0.232 9 0.18 a + 0.088 * S E M : standard error of the mean. Table 3.6: (continued).Effect of mesh treatment on growth and ectomycorrhizal (EM) colonization of Douglas-fir {Pseudotsuga menziesii var. glauca) seedl ings. R e s p o n s e dif ferences between source and recipient seedl ings were calculated for each pot. This single number w a s used in the A N O V A for each response variable. Statistically significant mesh treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05). M e s h (pm) n Root :Shoot (g) S E M n E M colonization (%) S E M control 9 -0.55 a + 0.105 9 47 a + 8 0.2 10 -0.12 ab + 0.100 7 30 a ± 9 1 7 0.08 b + 0.120 8 26 a + 9 20 5 0.05 b + 0.142 5 6 a + 11 500 8 -0.21 ab + 0.112 9 29 a + 8 * S E M : standard error of the mean. 72 Figure 3.1: Effect of a) fungicide type and b) application f requency on percent ectomycorrhizal colonizat ion (determined by clearing and staining root tips) of Doug las-fir {Pseudotsuga menziesii var. glauca) seedl ings. Fungic ide abbreviat ions: S = Senator® and T = Topas®. Frequency abbreviat ions: A = once upon commencemen t of the experiment, B = every two months, and C = once a month. Statistically signif icant fungicide treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05). Error bars are one standard error of the mean . a) c o ••§ N 'c _o o O N O o E Q o LU 50 45 40 35 30 25 20 15 10 5 0 b - E - c Control S T Fungicide type S T b) 50 2^ 45 .2 40 co N 35 c o o o 30 25 20 0 1 3 1 1 0 ° C LU O 0 b b Control A B Appl icat ion frequency 73 Figure 3.2: A b u n d a n c e of morphotypes (Tomentella-type (Tom) Thelephora terrestris (T); Mycelium radicis atrovirens-type (MRA) ; Wilcoxina rehmii (W); Cenococcum geophilum (Cg) ; Rhizopogon/Suillus-type (R/S) ; Piloderma-type (P) and Undifferentiated (Undif) found on morphotyped Douglas-f ir {Pseudotsuga menziesii va.x. glauca) root sys tems grown in soi l treated with different a) fungicide types and b) appl icat ion frequency. Fungic ide abbreviat ions: S = Senator® and T = Topas®. F requency abbreviat ions: A = once upon commencement of the experiment, B = every two months, and C = once a month. a) 18 h Fungicide type 74 Figure 3.2 (continued): Abundance of morphotypes (Tomentella-Xwpe (Tom) Thelephora terrestris (T); Mycelium radicis atrovirens-type ( M R A ) ; Wilcoxina rehmii (W); Cenococcum geophilum (Cg) ; Rhizopogon/Suillus-type (R /S) ; Piloderma-type (P) and Undifferentiated (Undif) found on morphotyped Douglas-f i r (Pseudotsuga menziesii war. glauca) root sys tems grown in soil treated with different a) fungicide types and b) application frequency. Fungic ide abbreviat ions: S = Senator® and T = Topas®. Frequency abbreviat ions: A = once upon commencement of the experiment, B = every two months, and C = once a month. 75 Figure 3.3: Abundance of Wilcoxina rehmii ectomycorrh izas, as a percentage of all root tips examined on Douglas-f i r (Pseudotsuga menziesii war. glauca) grown in soi l treated with fungicides. Fungic ide abbreviat ions: S = Senator® and T = Topas®. Statistically significant fungicide type treatment effects detected by a Bonferroni multiple compar ison test are designated by different letters (p < 0.05). Error bars are one standard error of the mean . 20 18 e 16 o co 14 N c o o o CD O c CO c < 12 10 8 2 0 Control S T Fungicide type ST Figure 3.4: Abundance of morphotypes {Thelephora terrestris (T); Mycelium radicis atrovirens-type ( M R A ) ; Wilcoxina rehmii (W); Cenococcum geophilum (Cg) ; Rhizopogon/Suillus-type (R/S); and Undifferentiated (Undif), a s a percentage of all root tips examined on recipient (R) and source (S) soil seed l ings separated by a mesh barrier. 60 n Mesh (um) 77 Figure 3.5: Ectomycorrhizal community di f ferences, a) Ste inhaus similarity index for ectomycorrhizal communi t ies observed on source and recipient seed l ings separated by a mesh barrier, b) R i chness difference = number of morphotypes observed on source Douglas-f ir (Pseudotsuga menziesii var. glauca) root sys tems minus morphotypes present on recipient Douglas-f ir separated by a mesh barrier. 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Phosphorus uptake, not carbon transfer, expla ins arbuscular mycorrhizal enhancement of Centaurea maculosa in the presence of native grass land spec ies . Funct ional Eco logy 16: 758-765 Zambonel l i A , lotti M. 2001 . Effects of fungicides on Tuber borchiiand Hebeloma sinapizans ectomycorrh izas. Mycolog ica l Resea rch 105: 611-614 83 4 Ectomycorrhizal colonization and intraspecific variation in growth responses of lodgepole pine1 Introduction Phenotypic variation of any organism is a product of its genotype, environment and the interaction between these components . Both abiotic and biotic factors will be ecological ly signif icant components of an organism's environment. B e c a u s e many tree spec ies rely on ectomycorrhizal fungi for establ ishment and survival , variation in the identity and abundance of ectomycorrhizal fungi can impact seedl ing growth (Dickie et a l . 2002). Thus , the presence of ectomycorrhizal fungi in soi ls is a critical d imens ion to the biotic environment with which trees will interact. Quantifying levels of colonizat ion on root tips of host trees is one method to measure the extent of interaction with their ectomycorrhizal fungi; however, the relationship between host growth and colonizat ion level is inconsistent (see Chapte r 2). B e c a u s e exper iments on ectomycorrh izas have used different spec ies (both phyto- and mycobiont) and substrates, it is difficult to untangle which factors contribute to the poor overal l relationship between colonizat ion level and plant response. Within a host spec ies , however, we might expect the relationship between colonizat ion level and host growth to be less variable, especial ly in homogenous environments. Furthermore, within more genetical ly similar groups nested within a spec ies , the relationship between colonizat ion level and host growth is expected to be even less var iable. Resea rch approaches to study mycorrhizal fungi do not al low direct manipulat ion of the level of colonizat ion of an individual plant. Here we present results from a greenhouse experiment with seedl ings from seed famil ies within the spec ies Pinus contorta Dougl . ex Loud. var. latifolia Enge lm. , which naturally var ied in ectomycorrh izal colonizat ion levels; that is, colonizat ion levels were not manipulated. W e have ana lyzed the results to test the direction and consis tency of the relationship between colonizat ion level and growth responses across seed famil ies. Earl ier studies have cons idered the role of host genotype in determining level of colonizat ion (Tagu et a l . 2001 , 2005 ; Gehr ing et a l . 2006) and the composit ion of ectomycorrhizal fungal communi t ies (Korkama et a l . 2006), but to our knowledge this is the first study to test the relationship 1 A version of this chapter has been accepted by Canadian Journal of Botany as: Karst J , Jones MD, and Turkington R. Intraspecific variation in height of lodgepole pine is minimized with increased ectomycorrhizal colonization. 84 Methods Greenhouse experimental set-up The greenhouse experiment was set up to test the effects of five ectomycorrhizal fungal spec ies , plus a non-mycorrhizal control treatment on the variation in growth responses of ten seed famil ies of lodgepole pine seedl ings. E a c h fungal spec ies x family treatment was replicated twenty t imes. S e e d was produced during control led pollination trials by the British Co lumb ia Ministry of Forests using trees from s e e d planning units in the central interior of British Co lumb ia . S e e d from within each family was full s ib, i.e. genetic similarity was higher within than among famil ies. Relat ive wood density was the primary trait for which s e e d s had been se lec ted. S e e d s were soaked 24 hours in distil led water, then steri l ized in 3 0 % H 2 0 2 for 15 minutes, and 3 % H 2 O 2 for a further 2 hours. Al l solut ions were mixed constantly. The s e e d s were dried and kept at 4°C for 28 days. For each fungal spec ies x family replicate, we sowed two s e e d s into a S C 1 0 R Super cell Ray Leach cone-ta iner (Stuewe and S o n s , Inc., Corval l is , Oregon , U S A ) measur ing 3.8 cm in diameter and 21 cm in length, filled with 3:1 (v:v) autoclaved peat and perlite. Al l cone-ta iners were steri l ized previously in a 3 0 % bleach solution for 30 minutes. The cone-tainers were held in R L 9 8 trays (Stuewe and S o n s , Inc., Corval l is , Oregon , U S A ) and randomized monthly. W e covered the s e e d s with 0.5 cm of autoclaved sand and all s e e d s were watered every 4 days. Two weeks after germination, we thinned seedl ings to one per cone-tainer. For the next eight months, we watered the seedl ings as required and fertilized once every 2 weeks with V* strength Ingestad's solution (Pelham and M a s o n 1978). Natural daylight in the greenhouse was supplemented by 400 W high pressure sodium lamps for 18 hours daily. T h e average temperature ranged from 20 to 25°C and the average relative humidity was 5 3 % . At four weeks and again at four months we inoculated the seedl ings with 5 m L of mycel ial slurry of one of the five ectomycorrhizal fungi. The spec ies of fungi used were: Cenococcum geophilum, Rhizopogon roseolus, Wilcoxina mikolae, Hebeloma crustuliniforme and Paxillus involutus. Cul tures of these fungi were obtained from the Mycorrh iza Research Group, University of British Co lumb ia O k a n a g a n and maintained on sol id modified Mel in-Norkrans (MMN) media . To obtain the mycel ium, we p laced approximately twenty 0.5 c m 3 cubes of actively growing mycel ium in each f lask of liquid 85 M M N media . Liquid cultures were grown under sterile condit ions and shaken daily. No contamination occurred in liquid cultures. The mycel ial slurry used to inoculate seedl ings was produced by blending 150 mL of mycel ium with 1850 m L of distil led water. W e also produced a non-mycorrhizal slurry for control plants from sol id M M N media that had not been inoculated. W h e n seedl ings were harvested after nine months, v isual observat ions of morphotypes under a d issect ing scope showed that none of the inoculated fungi were present on the roots; however, seed l ings were mycorrhizal with other fungi. Hence , we harvested 45 randomly se lected seedl ings per seed family in order to test the relationships among percent colonizat ion, s e e d family and growth responses (height and b iomass) of lodgepole pine. W e measured the height of each harvested seed l ing and subsequent ly dried the shoots at 65°C for 72 hours. Roots were refrigerated at 4°C until examined (see below) and then were dried at 65°C for 72 hours. Assessment of ectomycorrhizal fungal colonization S e e d famil ies having less than 1 0 % of seedl ings survive were omitted from the analys is ; thus, we examined eight of the initial ten famil ies. Us ing these 360 seed l ings (45 seedl ings x 8 famil ies), a power analys is performed in J M P IN 5.1 (Sai l et a l . 2005) determined that 64 seedl ings were required to detect observed di f ferences in height due to s e e d family, with a 9 7 % probability of achieving a signi f icance of 0.05. S i nce we could not est imate variation in mycorrhizal colonizat ion in advance , we used variation in height to determine how many seedl ings to examine for colonizat ion. Consequent ly , we sub-sampled eight seedl ings randomly from each family for which mycorrhizal colonizat ion was measured . Entire root sys tems were carefully w a s h e d under running water and cut into approximately 1 -cm p ieces. Al l root fragments were p laced in a baking dish containing water and a random sub-sample was then distributed into a Petri plate. W e examined 300 (± 57) root tips per seedl ing under a s tereomicroscope. T ips were classi f ied as mycorrhizal if root hairs were absent. Examinat ion of a sub-set of these roots under high magnif ication (400x) conf irmed that this approach accurately dist inguished mycorrhizal from non-mycorrhizal roots. Two morphotypes were dist inguished based on the presence or absence of cyst idia and on character ist ics of the mantle and mycel ial strands. 86 Molecular analyses Total genomic D N A from three replicate tips of each morphotype identified was extracted by pulverizing the tips for 45 seconds at a speed of 5.0 units using a B i o l 01 Sys tems Fast Prep F P 1 2 0 high f requency shaker (Q-biogene, Ca r l sbad , C A , U S A ) . D N A was isolated using the procedure of Baldwin and Egger (1996). The final D N A pellet was dried using a speed vacuum concentrator and then re-suspended in 50 uL E D T A - T E buffer. Fol lowing D N A extraction and isolation, the internal t ranscribed spacer (ITS) region of the fungal nuclear rDNA was specif ical ly amplif ied by the primers NSI1 and N L C 2 (Martin and Rygiewicz , 2005). P C R react ions typically included 1 uL template D N A , 18.6 uL sterile purified water (Barnested Nanopure Diamond water purifier), 0.2 m M deoxyr ibonucleot ies (dNTPs) , 2.5 uL 10x P C R buffer, 2.0 m M M g C I 2 , 0.48 m M each primer, 1.6 mg mL" 1 bovine serum albumin (BSA) , and 0.25 U uL" 1 Amp l iTaq Gold™ (Applied B iosys tems, Foster City, C A , U S A ) . S a m p l e s were amplif ied using a P T C - 2 0 0 thermal cycler (MJ Resea rch Inc., Wa l tham, M A , U S A ) . A 10 minute hot start was fol lowed by P C R cycl ing as fol lows: 45 seconds at 94°C fol lowed by 34 cyc les of denaturation at 94°C for 45 seconds , anneal ing at 54°C for 45 seconds , ramping 72°C for 1 minute with a 1 second extension after each cycle, and extension at 72°C for 10 minutes, and then the temperature was held at 4°C. The P C R products were v isual ized on 1.5% agarose gels using a G e l Log ics 440 (Kodak Instruments, Rochester , N Y , U S A ) . The P C R product was c leaned using the QIAquick P C R Purif ication kit (Qiagen Inc., Va lenc ia , C A , U S A ) . Prior to sequenc ing , the large ITS fragment produced above, w a s re-amplif ied in a nested P C R reaction using the primers ITS 1 and ITS 4 (White et a l . 1990). P C R products were quantif ied and then sequenced using a 3730 D N A Capi l lary S e q u e n c e r (Applied Biosystems) at the University of British Co lumb ia Nucle ic Ac id and Protein Serv ices Unit. Al l unique morphotypes were sequenced and then al igned using Sequencher software (Gene C o d e s Corporat ion, Ann Arbor, M l , U S A ) . Taxonomic matches were based on B L A S T results with >97% sequence similarity. Statistical analyses W e used an analys is of covar iance ( A N C O V A ) to test the effect of seed family on seedl ing growth responses using level of ectomycorrhizal fungal colonizat ion (% root 87 tips colonized) as a covariate regressor. W e included an interaction term (seed family x % colonization) to determine if colonizat ion interacted with s e e d family (i.e. whether the s lope of the relationship between colonizat ion and a given growth response differed by s e e d family). To meet A N C O V A assumpt ions, we ensured that colonizat ion levels did not differ by s e e d family using an analys is of var iance (see Resul ts) . W e used a reciprocal transformation on shoot height to meet the assumpt ion of homogenei ty of var iance. A s shoot height was the only growth response to show unequal var iance across seed famil ies and Burgess and Mala jczuk (1989) demonstrated d e c r e a s e s in variation of height among individuals of Eucalyptus globulus Labi l l . with inoculat ion by ectomycorrhizal fungi, we further explored the effects of colonizat ion level on variation in shoot height. To do so , we used colonizat ion level to predict the residuals in seedl ing height. Res idua ls were calculated by taking the absolute value of the deviat ion of each seedl ing from its mean family value, s tandardized by that particular family average: seedl ing residual = | y i F - X F | / X F where y i F is the value for the ith seedl ing from family F and X F is the mean for that family. W e used the family m e a n s in contrast to the overall mean because s e e d family had a significant effect in explaining variation in height among seedl ings (see Resul ts) . In other words, we removed s e e d family effects to look at the independent contribution of colonizat ion level on the height response of each individual seedl ing. Al l ana l yses were performed in J M P IN 5.1 (Sail et a l . 2005). The relative abundance of e a c h ectomycorrhizal fungal spec ies was calculated as the percentage of the total number of ectomycorrhizal tips that were co lon ized by a given fungal spec ies . Results All seed l ings were mycorrhizal . The mean level of colonizat ion w a s 8 5 % (SD 15%), ranging from 39 to 100% per seedl ing. M e a n colonizat ion levels did not differ by seed family (df = 7, 56; F = 1.08; p = 0.39). The effect of colonizat ion on root and shoot b iomass varied by seed family (Table 4 .1 , F ig . 4.1). In particular, both posit ive and negative relat ionships between colonizat ion level and shoot m a s s were observed , although seedl ings in most famil ies did not show any response to colonizat ion levels (Fig. 4.1). For the majority of seed famil ies no relationship was observed between 88 colonizat ion level and root m a s s , however, two seed famil ies showed negative relat ionships (Fig. 4.1). Shoot height differed only by s e e d family (Tables 4 .1 , 4.2). Res idua l height variation ac ross seedl ings w a s weakly expla ined by colonizat ion levels. In particular, there was a negative relationship between the magnitude of seedl ing deviation from its family mean and level of colonizat ion (Fig. 4.2). Thus , increased colonizat ion lessened height dif ferences among seedl ings within famil ies. The mean coefficient of variation in height for each seed family was not related to mean colonizat ion level (df = 1, 6; F = 0.0024; p = 0.96). Colonizat ion levels were a lso not related to the deviat ions of mean family heights from the overall mean (df = 1, 6; F = 0.03; p = 0.87) indicating that colonizat ion levels did not diminish di f ferences among s e e d famil ies. The two morphotypes identified on seedl ing root tips had >97% sequence matches of their ITS sequences to Thelephora terrestris and Rhizopogon vulgaris access ions in Genbank . Neither fungus had been inoculated onto seedl ings, but rather were greenhouse contaminants. Thelephora terrestris was the most common ectomycorrhizal fungus to co lonize seedl ings. It was present on root tips of all seed l ings and had a mean relative abundance of 98%. Rhizopogon vulgaris co lon ized roots of 5 % of the seedl ings, with a mean relative abundance of 6 0 % on those seedl ings. R. vulgaris was found on seedl ings from two s e e d famil ies (Table 4.2). Discussion Seed family effects on the relationship between colonization level and host growth In this study the role of genet ics was c lear in determining seedl ing growth character ist ics: s e e d family affected height and b iomass of individual seed l ings. Resu l ts from provenance trials of lodgepole pine in British Co lumb ia , C a n a d a indicate that dif ferences in height of 20-yr trees are a lso, to some degree, under genet ic control (Rehfeldt et a l . 1999); adaptive dif ferences among populat ions that were related to their cl imate of origin were demonstrated among populat ions that had been transplanted to var ious test si tes across British Co lumb ia . In our study, responses in seedl ing b iomass were modif ied by colonizat ion levels, representing a seed family x ectomycorrhizal colonizat ion interaction. B e c a u s e ectomycorrhizal fungi are part of the biotic environment, their p resence should be v iewed as a component within the more general 89 framework of assess i ng genotype x environment interactions influencing seedl ing growth. A c r o s s different host plant spec ies , the relationship between mycorrhizal colonizat ion and host growth parameters var ies (Jones et a l . 1990, Thompson et a l . 1994). Resul ts from our study indicate that this inconsistency can be observed even at an intraspecific level. The environment of the seedl ings in our experiment was homogeneous , indicating that the identity of s e e d family a lone can be an important factor determining the relationship of ectomycorrhizal colonizat ion to seedl ing b iomass . Stud ies from other sys tems a lso confirm that genotypic effects can be such that they are as strong as spec ies effects. For example , the effects of genotypic diversity of Solidago altissima on arthropod diversity and community structure living on their leaves are comparab le to those from studies testing the effects of spec ies diversity manipulat ions (Crutsinger et a l . 2006). Manipulat ions of the genetic diversity of seag rass (Zostera marina) showed that increasing genetic diversity results in increased invertebrate community resi l ience and dec reased recovery time to d is turbances c a u s e d by goose herbivory (Hughes and Stachowicz 2004); this finding mirrors those reported in exper iments manipulat ing functional (species) diversity (Diaz and C a b i d o 2001). Ectomycorrhizal colonization and host phenotypic variation Ecolog ica l p rocesses may be drivers of population differentiation and d ivergence (Schluter 2001). The role of ecological p rocesses in population convergence has ga ined more attention with the introduction of neutral theory (Hubbell 2001 , 2006); nonethe less, equal iz ing mechan isms are usually invoked in the context of explaining spec ies coex is tence (Chesson 2000). To our knowledge, ours is the first study to show that increases in colonizat ion by ectomycorrhizal fungi tends to reduce intraspecif ic variability or, in other words, di f ferences in height tend to be equal ized a m o n g seedl ings. Our results indicate that a mycorrhizal s ignal , albeit a weak one (9% of the var iance in seedl ing height residuals was expla ined by colonizat ion level), w a s observed at the seed family level. S u c h low r2 va lues are not unusual given that the mean amount of var iance expla ined in ecological exper iments is only 2 .5 -5 .4% (Mol ler and Jenn ions 2002). Our results suggest that those seedl ings able to escape ectomycorrh izal fungal colonizat ion could benefit in terms of height ga ined. Nonethe less, roots of seed l ings of 90 lodgepole pine occurr ing under natural condit ions are heavily co lon ized by ectomycorrhizal fungi (Bradbury 1998; Krahabetter et a l . 1999; Bothwell et a l . 2001). W e offer two reasons to explain high levels of colonizat ion on seedl ings in natural condit ions. First, poorly co lon ized seedl ings have an equal l ikelihood of growing shorter than the average seedl ings. Hosts may invest more in the maintenance of ectomycorrh izas a s a strategy ana logous to insurance that buffers against extreme variation in performance. However, despite some theoretical deve lopments (Kummel and Salant 2006), the amount of control a host has on the composi t ion and abundance of its mycorrhizal fungal partners is uncertain. If host select ion of a fungal partner is pass ive, our results would suggest that, in a reas devoid of ectomycorrhizal fungi, stochasticity will inf luence seedl ing growth more s o than in a reas replete with ectomycorrhizal fungal inoculum. S e c o n d , there may be no advantage perse to reduced intraspecific variation. It may be present only as a byproduct of select ion pressures on hosts for ectomycorrhizal colonizat ion, which in turn, is se lec ted for to increase plant survival in low nutrient condit ions. Host benefits received from increased ectomycorrhizal colonizat ion in terms of nutrient uptake may outweigh the costs (e.g. carbon) of support ing ectomycorrhizal fungi. W e observed a negative relationship between colonizat ion level and intraspecif ic variation within, but not among famil ies. Other studies have reported that phenotypic variation among famil ies is minimized in the presence of ectomycorrhizal fungi. For example , thirty open-pol l inated famil ies of Picea abies grown with or without Laccaria bicolor showed striking reductions in var iance of shoot and root dry weight among famil ies when ectomycorrh izas were present (Mari et a l . 2003). Variat ion in root architecture, an important trait for nutrient acquisi t ion, a lso decl ined when ectomycorrh izas with Paxillus involutus were present on Picea abies (Boukcim and P lassa rd 2003). In particular, the number of lateral roots per seedl ing differed when the two famil ies when non-mycorrhizal , but not when they were mycorrhizal . Ou r results are an advance over these earlier studies, which cons idered colonizat ion as a categor ical variable only (i.e., p resence or absence) . Colonizat ion levels of seed l ings in the field are more likely to be cont inuous rather than a discrete property as commonly employed in exper iments. A s such , results from our study may be more reflective of naturally occurr ing colonizat ion levels. 91 Our results differ from exper iments that treat ectomycorrhizal colonizat ion level as a response variable. W e found that colonizat ion levels did not differ by s e e d famil ies; a power analys is indicated that at least 220 seedl ings would be required to detect significant di f ferences (a=0.05) in colonizat ion levels among famil ies 9 7 % of the t ime. Progeny obtained from c rosses between two spec ies , Populus deltoides and P. trichocarpa differed in the extent to which they were co lonized by Laccaria bicolor (Tagu et a l . 2001). Hence , Tagu et a l . (2005) conc luded that the ability to form ectomycorrh izas (measured by colonizat ion levels) is a quantitative trait under polygenic control. The few measures of broad sense heritability calculated for levels of ectomycorrhizal colonizat ion range from 0.09 to 0.81 (Rosado et a l . 1994; T a g u et a l . 2001 , 2005) indicating possibly high involvement of environmental factors in determining the level of colonizat ion, depending on the host and fungal spec ies . The genetic bas is to the response by hosts to ectomycorrhizal colonizat ion levels dese rves further study. The prevalence of contamination on seedlings None of the target fungi were success fu l in coloniz ing seedl ings. Al though we inoculated seedl ings twice in the experiment, it is likely that the aggress ive colonizat ion abilities of those ectomycorrhizal fungi common to g reenhouses facilitated their early establ ishment on seedl ings. Contaminat ion of seedl ings used in ectomycorrh izal exper iments is not uncommon. Nearly half of all studies extracted from papers used in the meta-analys is (Chapter 2) reported contamination of seedl ings. Whi le levels of contaminat ion are lower in exper iments performed in growth chambers , the smal l s ize of growth chambers necessi tates the use of young seedl ings, or running exper iments for short durations. Clear ly, the presence of contamination is problematic in exper iments where maintaining non-ectomycorrhizal controls is required. Conclusions The role of the environment in determining plant phenotypes is undisputed in ecology. Our results suggest that mycorrhizal fungi should be cons idered a s a component of the environment that can influence the amount of phenotypic variation in a populat ion. Moreover, we highlight the importance of intraspecific d i f ferences in determining the sensitivity between symbionts involved in mycorrhizal assoc ia t ions . A s 92 such , models of intraspecific interactions should cons ider ectomycorrhizal assoc ia t ions when assess i ng phenotypic variability. S ince we are unable to manipulate colonizat ion levels directly, future research should examine the effects of the presence, absence and spec ies of ectomycorrhizal fungi on the var iance among s e e d famil ies sc reened for high differentiation in growth traits. Addit ionally, the ecological re levance of dec reased intraspecific variation through mycorrhizal colonizat ion deserves further study. 93 Table 4 .1 : Ana lys is of covar iance for effects of seed family, percent ectomycorrh izal fungal colonizat ion of root tips (% colonization) and their interaction on growth responses of Pinus contorta Dougl . ex Loud. var. latifolia Enge lm. seedl ings. Shoot height (cm) t Shoot m a s s (g) Root m a s s (g) Source df F P F P F P Family 7 2.20 0.051 1.31 0.26 3.44 0.0046 % colonizat ion 1 0.65 0.43 0.48 0.49 2.42 0.13 Family x 7 0.70 0.67 2.33 0.039 3.084 0.0091 % colonizat ion t A reciprocal transformation was used on shoot height to meet homogenei ty of var iance assumpt ion. 94 Table 4.2: M e a n shoot height of full s ib famil ies of Pinus contorta Dougl . ex Loud . var. latifolia Enge lm. Seed l ings grown for 36 weeks (n=8). British Co lumb ia Ministry of Forests seed family identification fol lows in brackets seed family designat ion. Shoot height (cm) S e e d family M e a n S D A (2094 x 2065 C P RD5) 5 . 3 b 0.54 B (354 x 468 B V RD2) f 5 .2 b 0.79 C ( 1 6 5 9 x 4 7 9 B V RD2) f 6 .2 a 1.01 D ( 2 6 8 x 1 6 3 1 B V RD1) 5 . 1 b 0.54 E ( 1 8 1 7 x 2 2 0 P G RD5) 6 .2 a 0.76 F (253 x 236 P G RD2) 4 .8 C 0.87 G (466 x 502 B V RD2) 5 . 1 b 1.24 H (2076 x 1620 C P RD2) 5 . 3 b 0.67 * Family effects sharing the same letter are not statistically different (P< 0.05 Tukey-Kramer multiple comparison test). tRoot tips of seedlings colonized by Thelephora terrestris and Rhizopogon vulgaris; all other seedlings colonized by Thelephora terrestris only. Figure 4 .1 : The effect of ectomycorrhizal fungal colonizat ion by s e e d family on shoot (top panel) and root m a s s (bottom panel) of Pinus contorts var. latifolia seedl ings. Regress ion l ines are shown for only those s e e d famil ies showing a significant relationship between shoot or root m a s s and level of colonizat ion. S e e Tab le 4.2 for British Co lumb ia Ministry of Forests s e e d family identification. Family A: y=0.3+0.005x p=0.021; r=0.55 Family H: y=0.8-0.003x 30 40 50 60 70 80 90 100 % ectomycorrhizal colonization Family D: y=1.9-0.1x p=0.036; r=0.55 30 40 50 60 70 80 90 100 % ectomycorrhizal colonization 96 Figure 4.2: The contribution of ectomycorrhizal fungal colonizat ion to height variation in seedl ings of Pinus contorta Dougl . ex Loud. var. latifolia Enge lm. , independent of s e e d family effects. y=0.26-0.0016x p=0.014; 1^=0.09 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 % ectomycorrh iza l co lon izat ion References Baldwin Q F , Egger K N . 1996. Protocols for analys is of D N A from mycorrhizal roots. In: G o o d m a n D M , Durall D M , Trofymow J A , Berch S M (eds). C onc i se descr ipt ions of North Amer ican ectomycorrh izae. Myco logue Publ icat ions, Victor ia B C , pp 3 C . 1 -3 C . 2 Bothwell K S , Prescott C E , J o n e s M D . 2001 . 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A c a d e m i c P ress , London U K , pp. 3 1 5 - 3 2 2 100 5 Interactions among soil characteristics, host intraspecific variation and ectomycorrhizal fungal communities Introduction Partner spec ies in coevo lved interactions are expected to be sensit ive to intraspecific variation of each partner due to the intimate and interdependent nature of their interactions (Thompson 1994). Ectomycorrhizal assoc ia t ions are of particular interest because individual trees host communi t ies of fungi, and whether the composi t ion of these communit ies is sensit ive to intraspecific variation in hosts is poorly understood. Prev ious research has shown that host individuals provide a range, or gradient, of biotic variation, and that this gradient produces changes in ectomycorrhizal fungal communit ies. For example , variation among host individuals induced by defoliation (Saikkonen et a l . 1999, Cul l ings et a l . 2005a) or varying levels of parasi t ism will change the composi t ion of ectomycorrhizal fungal communi t ies (Cul l ings et a l . 2005b, Muel ler and Gehr ing 2006). Communi t ies of ectomycorrhizal fungi have a lso been reported to segregate depending on the assoc ia ted growth rate of their host (Korkama et a l . 2006). In addit ion to the variation provided by properties of host spec ies , variation in soil character ist ics can structure ectomycorrhizal fungal communi t ies. For example , di f ferences in soil nitrogen (Lil leskov et a l . 2002) and nutrient and moisture status (Gehr ing et a l . 1998, Robertson et a l . 2006) have been shown to alter the composi t ion of ectomycorrhizal fungal communit ies. Similarly, the ecological ampli tude of host plants (measured by height and b iomass performance) is clearly dependent on soil propert ies such as nutrient and moisture availability (Burns and Honka la 1990). Thus , variation in soil character ist ics acts in parallel to influence both the composi t ion of ectomycorrhizal fungal communi t ies and intraspecific variation in hosts. Th is p rocess a lone should create a correlation between the composi t ion of ectomycorrhizal fungal communi t ies and intraspecific variation among hosts, independent of a direct interaction between the two components . Identification of a correlation between environmental ly- induced intraspecif ic variation in hosts and composit ion of their assoc ia ted ectomycorrhizal fungal community is key to understanding how environmental gradients structure ectomycorrhizal fungal 11 communit ies. A correlation not only indicates that variation in one symbiont is synchronous with variation in the other, but a lso that the environmental propert ies influencing host variation may act indirectly to affect the composi t ion of ectomycorrhizal fungal communit ies. The possib le sources of variation that inf luence ectomycorrhizal fungal communi t ies would have to be extended to include those that affect host intraspecific variation. B e c a u s e host and ectomycorrhizal fungal communi t ies are interdependent (Kernaghan 2005), covar iance between host intraspecific variation and the composi t ion of the ectomycorrhizal community cannot be used to infer causat ion . Regard less , the presence of a correlation between the two components offers a way of determining the ecological re levance to ectomycorrhizal fungal communi t ies of variation present within both a host spec ies and the abiotic environment. The objective of this study was to identify environmental factors that directly influence composi t ion of ectomycorrhizal fungal communit ies, a s well a s those that may act indirectly through host intraspecific variation. W e character ized "the environment" by measur ing variation in soil character ist ics related to fertility and moisture. Host intraspecific variation was measured by properties including shoot height, total b iomass and rootshoot ratio. The composi t ion of ectomycorrhizal communi t ies was quantif ied in two distinct ways : 1) categor ical ; the presence or absence of individual ectomycorrh izal fungal spec ies compris ing a community and 2) cont inuous; the relative abundance of each spec ies . Materials and methods Overview W e grew Douglas-f ir (Pseudotsuga menziesii war. glauca (Beissn.) Franco) seedl ings in pots containing soi ls that var ied naturally in fertility. W e a lso implemented an artificial gradient of soil moisture on replicate subsamp les of these soi ls. Seed l ings became co lon ized with the ectomycorrhizal fungi present in the soi ls and both the seedl ings and fungi were subject to the variation in soil fertility and moisture. W e then used multivariate ana lyses to correlate variation in soil fertility and moisture to variation in host growth and ectomycorrhizal fungal community composi t ion. 1l Origin of soils Soi l was col lected from the Thompson and Okanagan Va l leys of the southern interior plateau of British Co lumb ia , C a n a d a . Th is a rea has a continental cl imate, with warm, dry summers and cool winters. In val ley bottoms, the average daily minimum for the winter months is -5°C; for the summer months, the average daily max imum is 25°C (Environment C a n a d a 2004). There is a strong elevat ional gradient in annual precipitation ranging from 300 mm at lower elevat ions (300-800 masl) to greater than 1000 mm at montane (1200-1400 masl) elevat ions (Meidinger and Pojar 1991). O p e n forests of Douglas-f ir mixed with Ponde rosa pine {Pinus ponderosa Dougl . Ex P. & C . Lawa.) and several spec ies of g rasses (Koeleria macrantha [Ledeb.] J . A . Schu l tes f., Poa pratensis L. and Calamagrostis rubescens Buckl.) occur at lower elevat ions, whereas at higher elevat ions, Douglas-f ir grades into hybrid spruce (Picea engelmanni Parry ex Enge lm. x Picea glauca [Moench] Voss ) and lodgepole pine (Pinus contorta Dougl . Ex. Loud. var. latifolia Engelm.) (Meidinger and Pojar 1991). Soil collection Sampl ing locations were distributed over a distance of 140 km and ranged in elevation from 360 to 1390 mas l (Table 5.1). Th is elevational range co inc ides with that of Douglas-f ir in this region of British Co lumb ia . After removing loose litter or moss , we col lected 50 x 50 x 10 cm deep vo lumes of soil from six locat ions within the rooting zones of Douglas-f ir trees at each of six approximately 400 m 2 si tes. W e s ieved the soi l through a 2.5 c m 2 mesh in the field to remove woody debris and s tones and afterwards refrigerated the soil at 4°C in plastic tubs. By removing soil from the field, only those fungal spec ies able to survive through resistant propagules will be retained in soil samp les . The spec ies pool of ectomycorrhizal fungi evaluated in this a s s a y will be substantial ly less than what occurs in the field because those spec ies requiring mycel ia l connect ions will be absent. To determine the nutrient status of soi ls, one subsample of mixed soil from each of the six si tes was ana lyzed (Soi lcon Laborator ies Ltd., R ichmond , British Co lumb ia , Canada ) for p H , % organic matter measured by loss on ignition, total organic C , ammonium N, nitrate and nitrite N, total N, avai lable P, and est imated C : N (Table 5.2). Ana l yses were performed using procedures descr ibed in Carter (1993) and M c K e a g u e (1978). 1 Plant material In mid-November , 2003, non-mycorrhizal Douglas-f ir seed l ings were grown in a greenhouse at the University of British Co lumb ia , Vancouver , from s e e d s (seedlot #48520, col lected at 850-950 masl) obtained from the B C Ministry of Forest T ree S e e d Center (Surrey, British Co lumb ia , Canada ) . S e e d s were moist-stratified at 4°C for 21 days, then steri l ized in 3 % H 2 0 2 and mixed constantly for 2 hours. W e sowed the s e e d s into #1206 bedding inserts (Kora Products, B rama lea , Ontario, Canada ) filled with an autoclaved 3:1 (v:v) mixture of peat and perlite. Two s e e d s were p laced into each cavity and covered with 0.5 cm of steri l ized sand . The trays were misted each day for six weeks , after which seedl ings were transplanted into 1.5 L pots. Just prior to transplanting, a random subsample of twenty seedl ings was harvested to determine the initial m a s s of seedl ings. W e a lso c leared and stained roots from fifteen addit ional seedl ings to confirm their non-mycorrhizal status. Throughout the experiment, natural daylight in the greenhouse was supplemented by 400 W high pressure sod ium lamps for 18 hours daily. The temperature ranged from 20 to 24°C and the relative humidity was maintained at 6 0 % . Maintenance of soil moisture In December 2003, the field soil was removed from cold storage and mixed with perlite (3:1 v:v). So i ls from each sampl ing site were c rossed with three levels of watering (10, 20, and 3 0 % volumetric soil moisture). The range in watering levels w a s based on field measurements of soil at 450 and 1200 masl taken over one week in Ju ly 2003 using a C S 6 2 0 Hydrosense soil moisture probe (Campbel l Scienti f ic, Inc., Utah, U S A ) . In total, 200 pots were prepared into which the seedl ings were t ransplanted. O n e hundred and eighty pots were prepared for the treatments (6 si tes x 3 water ing levels x 10 repl icates = 180) and 20 were prepared to establ ish allometric relat ionships between seedl ing height and b iomass to adjust the total pot weight due to increased seedl ing b iomass (based on seedl ing height) over the course of the experiment. Dry soi l w a s determined to be equivalent to 3 % soil moisture using a C S 6 2 0 Hydrosense soi l moisture probe (Campbel l Scientif ic, Inc., Utah, U S A ) . The weight of a pot required to maintain the designated soil moisture levels was then calculated based on this initial measurement . 104 The seedl ings were transplanted on January 6, 2004 and during the 8 months of the experiment we regularly weighed pots and added enough water to bring the pot weight up to the appropriate weight for the watering treatment imposed. It was not feasible to maintain the pots at constant soil moisture; we let the pots dry to 1 0 % below their designated soil moisture level before adding water. Th is required that the seedl ings be watered every three days at the beginning of the experiment and each day by the end of the experiment. Final harvest A final harvest was done August 18, 2004. Shoots were dried at 65°C for 48 hours and weighed. Roots were bagged along with their surrounding soi l and refrigerated at 4°C. For process ing, entire root sys tems were carefully washed under running water and then cut into approximately 1-cm p ieces. Al l root f ragments were p laced in a baking dish containing water and a random subsample was then distributed into a Petri plate. W e a imed to count at least 100 root tips per individual seedl ing. In c a s e s where seedl ings had fewer than 100 root tips, all tips were counted. General ly , ectomycorrhizal tips were turgid and smooth , had emanat ing hyphae or rh izomorphs, and a Hartig net. A root tip that was dark and wrinkled, or was somewhat hollow and fragmented under minimal pressure was classi f ied as dead . G r o s s morphology of ectomycorrhizal roots and rhizomorphs was determined under a s tereomicroscope while Hartig net, mantle, emanat ing hyphae, and other such features were observed with a compound microscope under 400 or 1000x magnif ication. W h e n possib le, mantle pee ls were made by separat ing the fungal t issue from the root with fine forceps and micro-sca lpe ls . Morphological descr ipt ions were made with reference primarily to Ingleby et a l . (1990) and G o o d m a n et a l . (1996). O n c e p rocessed , roots were dried at 65°C for 48 hours and weighed. Two root tips representing each morphotype were lyophi l ized, and total genomic D N A was extracted from single ectomycorrhizal tips following the methods of Teste et a l . (2006) (Chapter 3). W e were success fu l in amplifying fungal D N A from only one morphotype out of seven (see Resul ts) , possibly due to lyophilization techniques. Thus , we relied on morphological character ist ics to differentiate among ectomycorrhizal types. 105 Statistical analyses Four response var iables were measured or calculated for each seedl ing at the end of the experiment: percentage of root tips that were mycorrhizal , total dry weight, shoot height, and root:shoot ratios. Data were aggregated to obtain a mean for each site x watering level treatment. W e used the actual soil moisture content measu red in each pot and treated it as a cont inuous variable because the soi l moisture va lues of the initially des ignated categor ies over lapped. W e used multivariate ana lyses to test for correlat ions among seedl ing growth, soil character ist ics and the ectomycorrhizal fungal community. In particular, canon ica l cor respondence analys is ( C A N O C O 4 - ter Braak and Smi laurer 1998) was used to correlate variation in (i) seedl ing growth traits, (ii) soil moisture, and (iii) soi l fertility with variation in fungal community composi t ion. Fungal community composi t ion w a s cons idered in two, multivariate forms. Co lumns in each matrix represented individual morphotypes and rows represented soi ls of the var ious treatment combinat ions. W e first a s s e s s e d individual morphotypes in a categorical nature which resulted in a matrix of 0s and 1s indicating presence (1) or absence (0) of each individual morphotype. Next, we a s s e s s e d the abundance of individual morphotypes; this resulted in a matrix of cel ls with va lues ranging from 0 to 100 (% relative abundance) . Th is type of ana lys is a s s u m e s that spec ies have unimodal distributions along environmental gradients. Va lues for soil moisture and soi l fertility were in two separate matr ices due to asymmetr ical units of replication (soil moisture: n = 16; soil fertility: n = 6). W e a lso used redundancy ana lyses , which a s s u m e linear relationships between response and explanatory var iables, to correlate (i) soil moisture and (ii) fertility with seedl ing growth traits (total b iomass , root:shoot ratio and seedl ing height). Va lues for seed l ing growth traits were centered and standard ized. The signif icance level for all ordinat ions w a s determined by Monte Car lo permutation tests (999 permutations). Results Soi l properties affected shoot growth (Table 5.3). Speci f ical ly, 3 2 % of the var iance in seedl ing traits was expla ined by soil moisture (p = 0.018); both height and b iomass increased with soil moisture (Fig. 5.1). A substantial amount of var iance in seedl ing traits was expla ined by the ratio of carbon to nitrogen in the soil (r 2 = 0.63, p = 0.0090). W h e n examined individually, only height was posit ively correlated to C : N (Fig. 106 5.2) . On average seedl ings were 11.5 (± 3.08 SD) cm tall, weighed 2.2 (± 1.03 S D ) g and had root:shoot of 1.2 (± 0.31 SD) . In total, seven morphotypes were identified on the roots of the seedl ings (Fig. 5.3) with two Wilcoxina morphotypes being the most frequent. Fungal D N A from Wilcoxina mycorrh izas with abundant, smooth emanat ing hyphae matched that of Wilcoxina mikolae in a B L A S T search of Genbank (99% match; expected = 0.0). A second type of Wilcoxina mycorrhiza, which we refer to as Wilcoxina II, w a s clearly dist inguishable from the first because it had few, roughly verrucose emanat ing hyphae. Those matching descr ipt ions of mycorrh izas formed by Rhizopogon, Amphinema, Piloderma spp . a s well as Mycelium radicis atrovirens (MRA) - t ype mycorrh izas, as descr ibed by J o n e s et a l . (1997) and Hagerman et a l . (1999), were less frequent. The p resence or absence of each of the seven morphotypes was independent of variation in shoot growth traits (p = 0.27) (Table 5.3). L ikewise, variation in soil moisture levels did not explain variation in the presence or absence of the seven ectomycorrhizal fungal morphotypes (p = 0.34) (Table 5.3). Of the soil fertility character ist ics measured , only total amount of nitrogen expla ined significant amounts (31%) of the var iance in the presence or absence of ectomycorrhizal fungal morphotypes (p = 0.012) (Table 5.3). Piloderma, Rhizopogon and Amphinema- type morphotypes were present in low nitrogen soi ls, and both Wilcoxina morphotypes and Cenococcum geophilum occurred in soi ls with mid-range va lues of nitrogen (Fig. 5.4). The MRA- t ype morphotype was present only in high nitrogen soi ls (Fig. 5.4). No other soil fertility var iables (i.e. p H , % organic matter, % organic C , mg kg" 1 of N H 4 , NO3/NO2 and P, or C:N) correlated with presence or absence of morphotypes (minimum p > 0.41). Total amount of nitrogen w a s not correlated to C : N ratio in these samp les (p = 0.084). Variat ion in total colonizat ion (i.e., abundance measured by percent colonizat ion of all morphotypes combined) for each site x watering level combinat ion was not correlated to seedl ing b iomass or height (minimum p = 0.95), but was positively correlated to root:shoot ratio (p = 0.048, r = 0.52; F ig . 5.4). Total colonizat ion was not related to the soil moisture level (p = 0.090). Whi le neither soil moisture levels nor variation in seedl ing growth covar ied with the presence or absence of the individual fungal morphotypes, variation in seedl ing traits w a s related to the relative abundance of each of the seven morphotypes (Table 5.3). Speci f ical ly, Wilcoxina mikolae and Rhizopogon-type morphotypes were more abundant on tall seedl ings compared to other morphotypes (1^  = 0.22, p = 0.024) (Fig. 107 5.5). The abundances of each morphotype, however, were not related to soi l moisture (p= 0.45) or any of the soil fertility var iables (minimum p >_0.074). Discussion In our study, the composi t ion of the ectomycorrhizal fungal community w a s inf luenced both directly and indirectly by variation in the soil environment. The p resence or absence of each of the seven morphotypes was correlated with total soi l ni trogen, but this community metric was not correlated to any host shoot growth responses . In other words, of the pool of morphotypes sampled in our assay , occur rence of each morphotype was inf luenced by the soil environment. W h e n morphotypes were measured by their relative abundance , we found that the abundance of speci f ic morphotypes did not respond directly to any of the soil var iables, but instead w a s mediated by growth character ist ics in the host. B e c a u s e host growth was affected by soil moisture and C : N ratios, we suggest that the abundance of morphotypes samp led were indirectly affected by soil condit ions. It is to be expected that the presence or absence of individual morphotypes correlated directly to soil nitrogen. B e c a u s e we did not measure nitrogen status of host individuals, we cannot dist inguish whether fungi responded directly to nitrogen levels in the soil or indirectly v ia nitrogen status of the host (e.g. Ni lsson and Wal lander 2003). However, there have been numerous studies showing that in culture, ectomycorrh iza l fungal spec ies show distinct preferences for different forms and levels of nitrogen (e.g. Li l leskov et a l . 2002 and references therein). Niche segregat ion along nitrogen gradients has a lso been demonstrated in the field (see reviews by W a l l e n d a and Kottke 1998, T reseder and Al len 2000). Interestingly, those factors that affected the p resence or absence of individual morphotypes did not affect seedl ing growth traits. Nantel and Neumann (1992) a lso reported that factors affecting fungal spec ies distribution were different from those affecting the distribution of their tree hosts, namely humus character ist ics. More recently, Tol jander et a l . (2006) demonstrated that despi te variation in host identity a long a nutrient gradient, most variation in the ectomycorrh izal fungal community was attributable to soil characterist ics such a s extractable ammonium and base saturation. Overal l , f indings from previous literature suggest that beta diversity in ectomycorrhizal fungal communi t ies is somewhat controlled by the p resence or 108 absence of particular host spec ies , but soil environmental heterogeneity is a more important factor maintaining ectomycorrhizal fungal diversity. Our results were based on the responses of seedl ings grown from open poll inated s e e d s in which the effect of genetic diversity is expected to be consistent ac ross treatments. Thus , the nearly three-fold difference in seedl ing heights we observed was probably mostly due to soil environmental variation. The range of soil variation co inc ides with the elevational range of Douglas-f ir in this study area . However, because we had few samp les (n = 6) of soi ls within the study a rea , the resolving power of seedl ing sensitivity to variation in soil character ist ics is low. Nonethe less , this difference in seedl ing heights is more than that reported by Ko rkama et a l . (2006) who observed dissimi lar ectomycorrhizal fungal communi t ies between fast and slow-growing c lones of Picea abies. In our study the phenotypic gradient, a s measured by variation in shoot b iomass , height and root:shoot ratios, was not sufficient to promote partitioning among fungal morphotypes. It is possible that phenotypic variation expressed belowground could be amplif ied to the extent that the composi t ion of the ectomycorrhizal community would be affected. In particular, variation in quantity and quality of exudates, should be tested as possib le determinants of membersh ip within ectomycorrhizal fungal communit ies. The environmental gradients did not influence host intraspecific variation sufficiently to determine membersh ip within the fungal community, however they were important in modifying the abundance of morphotypes present. B e c a u s e it is unlikely that colonizat ion levels of individual morphotypes influence host intraspecif ic variation to the same extent as variation in soil characterist ics (e.g. C : N ratios expla ined 6 3 % of the var iance in seedl ing growth traits whereas only 2 2 % was related to morphotype abundance) , we suggest that C : N ratios and soil moisture levels may act indirectly to modify the abundance of individual morphotypes. Hogberg et a l . (2007) a lso reported the importance of indirect effects of soil chemistry (C :N ratio) on abundance of ectomycorrhizal fungi as measured by P L F A biomarkers. Of the soil characterist ics measured , it was surprising how little impact variation in soi l moisture had on the ectomycorrhizal fungal community. Soi l moisture has been shown to induce changes in the spec ies composit ion of ectomycorrhizal fungal communi t ies surveyed in intact forests (Shi et a l . 2002; Swaty et a l . 2004) and in part this is thought to reflect di f ferences in the drought tolerance of ectomycorrhizal fungi 1 (Parke et a l . 1983; Boyle and Hel lenbrand 1991). t h e relationship between ectomycorrhizal colonizat ion and soil moisture has been previously reported but the direction of the response var ies (Lodge 1989; Gehr ing and Whi tham 1994; Runion et a l . 1997; Ni lsen et a l . 1998; Swaty et a l . 1998; Va ldes et a l . 2006). The portion of the soi l moisture gradient studied clearly inf luences the response of ectomycorrh izas to soi l moisture, but despite a four-fold difference in imposed soil moisture va lues, ectomycorrhizal fungi did not sort a long this particular gradient. Poss ib ly , plasticity of colonizat ion levels present at a fungal spec ies level accommoda tes variation in soi l moisture. B e c a u s e we removed soi ls from the field, the avai lable ectomycorrhizal fungal spec ies pool should have been similar ac ross soi ls as we samp led only those spec ies with resistant propagules. Resul ts from both the field (e.g. Bidartondo et a l . 2001) and greenhouse studies document that the resistant propagule community (sensu Taylor and Bruns [1999]) is often spatially homogeneous . For example , Wilcoxina spp . and Cenococcum geophilum were reported to be abundant and spatially homogeneous in soi ls col lected from mixed-conifer forest b ioassayed with two host spec ies (Izzo et a l . 2006). C l ine et a l . (2005) demonstrated that ectomycorrhizal fungal communi t ies on Douglas-f i r seedl ings planted at var ious d is tances outside mycel ium networks were similar to those on greenhouse seedl ings grown in field soi l . W e cannot rule out however, that those fungal spec ies widely distributed via resistant propagules may a lso be "general ists" when responding to intraspecific host variation and soil moisture. In conc lus ion, individuals of a host spec ies and spec ies within their assoc ia ted ectomycorrhizal fungal community respond to different environmental gradients. Whi le host traits were controlled mostly by variation in soil moisture and C : N ratio, the occurrence of particular ectomycorrhizal morphotypes was structured by levels of total nitrogen. Host variation did not directly affect the presence or absence of individual ectomycorrhizal fungal morphotypes. However, host variaiton was correlated to the relative abundance of each of the ectomycorrhizal fungal morphotypes, suggest ing that the abundance of morphotypes may be modif ied by those gradients affecting intraspecif ic host variation. At the seedl ing stage, soil nitrogen and host growth character ist ics influence composit ion of ectomycorrhizal fungal communi t ies. Table 5.1: Site coordinates and elevation of soil sampl ing locations. Site Label Latitude Longitude Elevat ion (masl) B B 50°01.918N 119°21.526W 724 B T 50°02.325N 119°15.994W 1318 O B 49°46.737N 119°36.203W 360 O T 49°42.792N 119°36.101W 1396 R B 50°44.477N 120°32.409W 648 R T 50°49.064N 120°42.524W 1387 111 Table 5.2: Fertility characteristics of soils collected from six sites from the Thompson-Okanagan region of British Columbia. Values are from a composite of 6 samples per site. P Organic (Bray-Site matter Organic N H 4 NO3/NO2 Total P1) Label PH (LOI) (%) C (%) (mg/kg) (mg/kg) N (%) (mg/kg) C:N BB 7.3 8.4 4.2 10 6 0.30 44.3 14 BT 6.0 11.7 5.8 9 0.1 0.15 25.9 40 OB 5.0 16.8 8.4 20 0.1 0.22 86.9 38 OT 4.7 18.2 9.1 19 0.1 0.21 51.9 44 RB 6.2 11.0 5.5 22 16.5 0.19 100.0 29 RT 5.6 9.8 4.9 9 0.5 0.14 57.7 35 11 Table 5.3: Types of statistical ana lyses (canonical cor respondence analys is [CCA] or redundancy analys is [RA]) used and signi f icance of explanatory var iables tested to explain measures of ectomycorrhizal fungal community composi t ion or seedl ing growth traits. Only those soil aspec ts of soil fertility found to be significant are reported in table. Numbers in brackets represent percentage of var iance expla ined by each significant explanatory factor. Type of analysis C C A Explanatory variable* Soi l moisture Seed l ing growth trai ts 0 0 Soi l fertility (% total N) Response variable Presence /absence of Abundance of individual morphotypes P 0.34 0.27 0.012 (31%) individual morphotypes P 0.45 0.024 (22%) > 0.0741 R A Seed l ing growth traits Soi l moisture 0 .018(32%) Soi l fertility (C:N) 0.0090 (63%) * Var iab les are categor ized as explanatory, however it should be recognized that in both ana lyses , causat ion cannot be inferred. t none of the measures of soi l fertility (pH, % organic matter, % organic C , % total N, mg kg" 1 of N H 4 , NO3/NO2 and P, and C:N) were significant in explaining variation in abundance of individual morphotypes. The minimum p-value ac ross all measu res is g iven. °°seedling growth traits include shoot height, total b iomass and root:shoot 113 Figure 5.1: Relat ionships between soil moisture (%) and seedl ing height, b iomass and root:shoot ratio. cu co c o Q. CO 8> CD CD CO CD CO > 18 16 14 12 10 8 6 4 2 0 10 20 % soil moisture 30 • Height(cm); p=0.037; r=0.53 • B iomass(g) ; p=0.027, r=0.55 • Root :shoot ; p=0.76 Figure 5.2: Relat ionships between soil C : N and seedl ing height, b iomass and root:shoot. • Height(cm); p=0.13 • B iomass(g ) ; p=0.018, r=0.89 A Root :shoot ; p=0.056, r=0.8 C:N 115 Figure 5.3: Frequency of ectomycorrhizal morphotypes observed ac ross seedl ings of Pseudotsuga menziesii war. glauca. 116 Figure 5.4: Canon ica l cor respondence analys is of ectomycorrhizal morphotypes observed on seedl ings of Pseudotsuga menziesii war. glauca ordinated along gradient of % total soil nitrogen. Amphinema-Xype • p=0.012 r2=0.31 , l/l//7cox/7ia-types Cenococcum geophilum ——^ • Total nitrogen Rhizopogon-Xype c Piloderma-Xype MRA-type - 1 . 0 +1.0 CCA axis 1 Figure 5.5: Relat ionship between percent ectomycorrhizal colonizat ion and rootshoot ratio of Pseudotsuga menziesii var. glauca seedl ings. 118 Figure 5.6: Canon ica l cor respondence analys is of ectomycorrhizal morphotypes observed on seedl ings of Pseudotsuga menziesii var. glauca ordinated a long gradient of seedl ing height. p=0.024 r2=0.22 CM CO X CO < o MRA-type Wilcoxina II Cenococcum geophilum » , j Wilcoxina mikolae Piloderma-type • Amphinema-Xype Height Rhizopogon-\ype -1.0 CCA axis 1 + 1.0 References Bidartondo M l , Baar J , Bruns T D . 2001 . Low ectomycorrhizal inoculum potential and diversity from soi ls in and near ancient forests of brist lecone pine (Pinus longaeva). Canad ian Journa l of Botany 79: 293-299 Boyle C D , Hel lenbrand K E . 1991. 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Communi ty structure of ectomycorrhizal fungi in a mature Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communit ies. Molecular Eco logy 8: 1837-1850 Ter Braak C J F , Smi laurer P. 1998. C A N O C O Reference Manua l and User 's Gu ide to C a n o c o for Windows: Software for Canon ica l Communi ty Ordinat ion (version 4), Microcomputer Power Thompson J N . 1994. The R e v o l u t i o n a r y P rocess . University of Ch i cago P r e s s Tol jander J F , Eberhardt U, Tol jander Y K , Pau l L R , Taylor A F S . 2006. S p e c i e s composi t ion of an ectomycorrhizal fungal community a long a local nutrient gradient in a boreal forest. New Phytologist 170: 873-883 Treseder K K , Al len M F . 2000. Mycorrhizal fungi have a potential role in soi l carbon storage under elevated C 0 2 and nitrogen deposi t ion. New Phytologist 147: 189-200 V a l d e s M, Asb jornsen H, G o m e z - C a r d e n a s M, Jua rez M, Vogt K A . 2006. Drought effects on fine-root and ectomycorrhizal root b iomass in managed Pinus oaxacana Mirov s tands in O a x a c a , Mex ico . Mycorrh iza 16: 117-124 Wa l lenda T, Kottke I. 1998. Nitrogen deposit ion and ectomycorrh izas. New Phytologist 139:169-187 123 6 Conclusions Ectomycorrhizal assoc ia t ions represent interactions among spec ies that are tightly l inked, both physical ly and physiological ly. W e therefore expect o rgan isms involved in ectomycorrhizal assoc ia t ions to be more sensit ive to variation within each partner than organisms involved in free-living assoc ia t ions (e.g. predator-prey relationships). In this thesis, I used meta-analysis and experimental approaches to cons ider how variation in one partner of the ectomycorrhizal symbios is affected the other. In particular, my object ives were to evaluate: i. how colonizat ion levels, regardless of ectomycorrhizal fungal taxon, correlated with host growth ii. how ectomycorrhizal fungi differentially inf luenced growth of different genera of plant hosts, and iii. how variation in growth of a single host spec ies was correlated to the composi t ion of ectomycorrhizal fungal communi t ies in var ious soil environments. B e c a u s e some of my conc lus ions relied on compar isons of inoculated and non-inoculated seedl ings, I a lso tested the eff icacy of two methods to control colonizat ion by ectomycorrhizal fungi on host plants. T h e s e results are of practical s igni f icance because prior to our experiment, no one had tested whether it was possib le to reduce ectomycorrhizal colonizat ion in unsteri l ized field soi l . My thesis object ives can be distil led into one summary quest ion: to what level of organizat ion of ectomycorrhizal fungi does the growth of host plants respond? I cons idered severa l levels of organizat ion: 1. colonizat ion levels regardless of ectomycorrhizal fungal taxon, 2. taxonomic identity of ectomycorrhizal fungi, and 3. communi t ies of ectomycorrhizal fungi. By conduct ing a meta-analys is on a large body of previously publ ished work, and by applying multivariate ana lyses to my exper imental data, I was able to evaluate the contribution of the three levels of organizat ion to variation in growth responses of host plants. Three main conc lus ions emerged . 124 The relationship between colonization level and host growth response is inconsistent W h e n consider ing individuals of a particular spec ies of host plant, variation in host growth varied with the abundances of different morphotypes of ectomycorrhizal fungi (Chapter 5). In other words, colonizat ion levels of some fungi increased with seedl ing height, and decreased for other fungal morphotypes. The relationship between total colonizat ion level and seedl ing growth differed among s e e d famil ies of a host spec ies (Chapter 4). W h e n considered ac ross many host genera, I detected no relationship between colonizat ion level and host growth response, regardless of fungal taxon (Chapter 2). These findings suggest that for the most part, host growth response to colonizat ion level is unpredictable. In order to study the effects of ectomycorrhizal fungi on plants, it is helpful to have control plants that are completely free of ectomycorrhizal contaminat ion. However , this can be chal lenging in the lab (Chapter 2), and almost impossible in non-steri le field soi ls (Chapters 3). My exper iments demonstrated that fungicides or m e s h have the potential to reduce colonizat ion, but this may be of little value if host growth responses do not consistently sca le with colonizat ion levels. If plants somet imes respond to very low levels of colonizat ion, this ra ises the quest ion of whether reductions in colonizat ion levels are meaningful treatments to a s s e s s host response to ectomycorrh izas. There is little sensitivity in growth responses of host plants to variation in the identity of ectomycorrhizal fungi Seed l ings ac ross multiple host genera increased in b iomass and shoot height when inoculated with ectomycorrhizal fungi regardless of the identity of the fungal assoc ia te (Chapter 2). W h e n ectomycorrh izas were cons idered in a multi-specif ic context (i.e. one host spec ies assoc ia ted with a community of ectomycorrhizal fungi), variation in host shoot properties did not correlate with spec ies composi t ion of the community of ectomycorrhizal fungi on their roots but rather appeared to be more tightly coupled to edaphic condit ions (Chapter 5). Thus , the variation a host plant perce ives and selects for in ectomycorrhizal fungi may be of a discrete rather than cont inuous nature, i.e., host plants respond to the presence or absence of ectomycorrhizal fungi but not to variation in their identity. A consequence of the coevolut ion among organ isms in multi-specif ic sys tems may be that reciprocal special izat ion is unlikely, therefore host 125 plants tend to be general ists in their responses to variation in the identity of ectomycorrhizal fungi. Publication bias exists in the ectomycorrhizal literature The meta-analysis investigating the mutual ism-parasit ism cont inuum in ectomycorrh izas represents a significant advance in the field of ectomycorrhizal research because it statistically evaluates and summar izes nearly four d e c a d e s of research on inoculation trials. I demonstrated that publication b ias has c louded our ability to determine general principles of host response to ectomycorrhizal inoculat ion. In the past, mycorrhizas were synonymous with mutual isms, and the tendency to publ ish results congruent with this percept ion has resulted in the under representat ion of studies reporting contrary results demonstrat ing a more parasit ic role for ectomycorrh izas. Future research directions The approaches used in my thesis represent initial tests to determine the importance of symbiotic variation to host growth. I suggest severa l avenues of further research. Whi le the effects of host genotype on colonizat ion level have been documented (Tagu et a l . 2001 , 2005, Gehr ing et a l . 2006), we are far from understanding host genotype x ectomycorrh iza interactions. Resea rch is necessary to determine the relative importance of host genet ics versus the presence, a b s e n c e and spec ies of ectomycorrhizal fungi on intraspecific variation in growth among individual host plants. Th is type of research would clarify the importance of interactions between hosts and ectomycorrhizal fungi in influencing seedl ing growth. I have contributed to this particular topic in Chapter 4 with ev idence that the relationship between colonizat ion level and host growth can be positive or negative, depending on plant genotype, within a host spec ies . However, the weakness of this experiment is that the seed l ings were not co lon ized by target fungi. Implementing other inoculation techniques, such a s submerging root sys tems of seedl ings in slurries of inoculum or use sol id inoculum, may increase the s u c c e s s of inoculation. It is a lso critical that future research explores the magnitude of specia l izat ion between host taxa and communi t ies of ectomycorrhizal fungi. Mult i-specif ic rather than pairwise interactions have been recognized to be the norm for coevo lved o rgan isms 126 (Stanton 2003 , S t rauss and Irwin 2004) and those organ isms involved in ectomycorrhizal assoc ia t ions are no except ion - individual trees frequently host communi t ies of ectomycorrhizal fungi. In other study sys tems, the role of host plant morphological variation has been shown to be important in determining the composi t ion of dependent communi t ies (Whitham et a l . 2006). Until very recently, we have known virtually nothing about how host plants influence the composi t ion of ectomycorrhizal communit ies. Two pioneering studies have finally addressed this quest ion and found that the relative growth rates (Korkama et a l . 2006) and taxonomic identity (Ishida et a l . 2007) of hosts alter the composi t ion of their ectomycorrhizal fungal communi t ies, yet much more research is required to adequately a s s e s s the sensitivity of host plants to changes in membersh ip within ectomycorrhizal fungal communi t ies and vice versa . Al though the meta-analys is in Chapter 2 is powerful in synthesiz ing d e c a d e s of research, it is limited by the features of the studies included in the analys is . In particular, pair-wise host-fungal combinat ions were the norm, thus conc lus ions on the sensitivity of hosts to variation in the identity of ectomycorrhizal fungi may change if interactions among spec ies of ectomycorrhizal fungi were to be present. Co- inoculat ion of hosts by several fungi is chal lenging and sampl ing in the field may yield more information on the specificity between hosts and communi t ies of ectomycorrhizal fungi. For example , surveying the taxonomic affinities between var ious host taxa and their ectomycorrh izal fungal communit ies, compl imented by field exper iments that manipulate host character ist ics would be a useful initial approach to address this quest ion. That hosts perceive ectomycorrhizal fungi as functionally redundant, as suggested by the meta-analys is in Chapter 2, may indicate that edaphic condit ions are more important than the presence and/or variation in composi t ion of ectomycorrhizal fungal communi t ies in determining seedl ing growth. In addit ion, results from Chapter 5 indicate that host growth is coupled to edaphic condit ions. Variat ion in edaphic condit ions takes many forms and, compared to descript ions of vegetative variation, our understanding of variation in soil character ist ics is poor (but see Bel l and Lechowicz 1991, Bel l et a l . 1993, Far ley and Fitter 1999). In particular, the role of spatial structure in the edaphic environment will be critical to understanding the role of ectomycorrhizal assoc ia t ions to host growth given the strong spatial covar iance between the composi t ion of ectomycorrhizal fungal communi t ies and soi l character ist ics. Th is feature makes it difficult to untangle the ecological importance of either to host plant growth. 127 The strength of the analytical approach taken in Chapter 5 is that it identifies and statistically parses the variation in host growth due to variation in the composi t ion of fungal communi t ies and soi l character ist ics - a conceptual ly novel framework. The w e a k n e s s e s of the experiment presented in Chapter 5 are that no causat ion c a n be inferred and the low number of soi ls sampled effectively shortens the environmental gradient that might inf luence host growth and membersh ip within ectomycorrhizal fungal communi t ies. Whi le the contribution of abiotic and symbiot ic factors in structuring ectomycorrhizal fungal communi t ies has been evaluated (Nantel and Neumann 1992, Kernaghan et a l . 2003 , Gehr ing et a l . 2006, Tol jander et a l . 2006, Hogberg et a l . 2007 , Taniguchi et a l . 2007), it has rarely been posed from a host perspect ive (but s e e Dickie et a l . 2007). A n exper imental des ign for the field that renders changes in the composi t ion of ectomycorrhizal fungal communi t ies independent from variation in soi l character ist ics remains elusive yet s tands as an important chal lenge in mycorrhizal research. Final conclusion Numerous opportunit ies now exist to investigate the distribution and abundance of plant spec ies in the context of ectomycorrhizal assoc iat ions. In particular, the degree to which host plants and ectomycorrhizal fungal communi t ies are spec ia l ized will be relevant information for forecasts of spec ies ' shifts with cl imate change. A s ranges of symbionts are unlikely to change concordantly, it will be crucial to understand the bas is and consequences of coevolut ion between hosts and fungi to predict their future distributions. 128 References Bell G , Lechowicz M J . 1991. The ecology and genet ics of f i tness in forest plants. 1. Environmental heterogeneity measured by explant trials. Journa l of Eco logy 79: 663-685 Bel l G , Lechowicz M J , Appenze l le r A , Chand le r M, DeBlo is E, J a c k s o n L, M a c K e n z i e B, Prez ios i R, Scha l lenberg M, Tinker N. 1993. The spatial structure of the environment. 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Biotic and abiotic factors affecting ectomycorrhizal diversity in boreal mixed-woods. O ikos 102: 497-504 Korkama, T., Pakkanen , A . and Pennanen , T. 2006. Ectomycorrh izal communi ty structure var ies among Norway spruce (Picea abies) c lones. N e w Phytologist 171: 815-824 Nantel P, Neumann P. 1991. Eco logy of ectomycorrh izal -basid iomycete communi t ies on a local vegetat ion gradient. Eco logy73: 99-117 Stanton M L . 2003. Interacting guilds: Moving beyond the pairwise perspect ive on mutual isms. Amer ican Naturalist 162: S 1 0 - S 2 3 St rauss S Y and Irwin R E . 2004. Ecolog ica l and evolutionary c o n s e q u e n c e s of mult ispecies plant-animal interactions. Annu . R e . Eco l . Evo l . Syst . 35: 435-466 Taniguchi T, Kanzak i N, Tama i S , Y a m a n a k a N, Futai K. 2007. D o e s ectomycorrhizal 129 fungal communi ty structure vary a long a J a p a n e s e black pine (Pinus thunbergii) to black locust (Robinia pseudoacacia) gradient? New Phytologist 173: 322-334 Tol jander J F , Eberhardt U, Tol jander Y K , Pau l L R , Taylor A F S . 2006. S p e c i e s composi t ion of an ectomycorrhizal fungal community a long a local nutrient gradient in a boreal forest. New Phytologist 170: 873-883 Whi tham T G , Bai ley J K , Schwei tzer J A , Shuster S M , Bangert R K , Leroy C J , Lonsdorf E V , Al lan G J , D iFaz io S P , Potts B M , F ischer D G , Gehr ing C A , Lindroth R L , Marks J C , Hart S C , W i m p G M , Woo ley S C . 2006. A framework for communi ty and ecosys tem genet ics: from genes to ecosys tems. Nature Rev iews Gene t i cs 7: 510-523 Appendices A. identity of host plant and fungal species pairings and effect sizes (Ln R) for seedling biomass, shoot height and shoot.root ratio for each study used in meta-analysis. Ln R Authors Host species Fungal species Biomass Height Shootroot n=459 n=329 n=235 Baum et al. 2000 Populus trichocarpa Laccaria bicolor -0.518 -0.334 -0.522 Baum et al. 2000 Populus trichocarpa Laccaria bicolor 0.856 0.529 1.627 Baum et al. 2000 Populus trichocarpa Paxillus involutus -0.319 -0.401 -0.895 Baum et al. 2000 Populus trichocarpa Paxillus involutus 0.797 0.485 1.600 Baum et al. 2002 Populus trichocarpa Laccaria laccata 0.305 Baum et al. 2002 Populus trichocarpa Laccaria laccata 0.101 Baum et al. 2002 Populus trichocarpa Laccaria laccata 0.005 Baumann et al. 2005 Pinus sylvestris Paxillus involutus 0.251 -0.198 Baumann et al. 2005 Pinus sylvestris Paxillus involutus -0.111 0.358 Baumann et al. 2005 Pinus sylvestris Paxillus involutus 0.064 -0.087 Baumann et al. 2005 Pinus sylvestris Paxillus involutus 0.066 -0.399 Baumann et al. 2005 Pinus sylvestris Paxillus involutus 0.292 0.048 Baumann et al. 2005 Pinus sylvestris Paxillus involutus Beyeler & Heyser 1997 Fagus sylvatica Lactarius subdulcis 0.272 Bougher e ta l . 1990 Eucalyptus diversicolor Descolea maculata 0.894 Bougher e ta l . 1990 Eucalyptus diversicolor Descolea maculata 0.368 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 0.575 Browning & Whitney 1991 Pinus banksiana Pisolithus tinctorius -0.031 0.236 Browning & Whitney 1991 Pinus banksiana Cenococcum geophilum 0.000 0.086 Browning & Whitney 1991 Pinus banksiana Laccaria proxima -0.014 0.114 Browning & Whitney 1991 Pinus banksiana Hebeloma cylindrosporum -0.030 -0.150 Browning & Whitney 1991 Pinus banksiana Tricholoma pessundatum -0.065 0.233 Authors Host species Browning & Whitney 1991 Pinus banksiana Browning & Whitney 1991 Pinus banksiana Browning & Whitney 1991 Pinus banksiana Browning & Whitney 1991 Pinus banksiana Browning & Whitney 1991 Pinus banksiana Browning & Whitney 1991 Pinus banksiana Browning & Whitney 1991 Pinus banksiana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning &• Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Browning & Whitney 1991 Picea mariana Burgess & Malajczuk 1989 Eucalyptus globulus Burgess & Malajczuk 1989 Eucalyptus globulus Burgess & Malajczuk 1989 Eucalyptus globulus Burgess et al. 1994 Eucalyptus grandis Burgess et al. 1994 Eucalyptus grandis Burgess et al. 1994 Eucalyptus grandis Burgess et al. 1994 Eucalyptus grandis Ln R Fungal species Biomass Height Shoot.root Thelephora terrestris -0.046 0.231 Suillus granulatus -0.058 0.242 Hebeloma cylindrosporum 0.126 -0.022 Pisolithus tinctorius 0.163 -0.004 Laccaria proxima 0.198 0.034 Tricholoma pessundatum 0.138 0.143 Cenococcum geophilum 0.129 -0.056 Laccaria bicolor -0.099 0.186 Laccaria proxima 0.037 0.307 Pisolithus tinctorius -0.092 0.313 Tricholoma pessundatum -0.020 0.336 Hebeloma cylindrosporum -0.007 0.275 Thelephora terrestris 0.030 0.374 Cenococcum geophilum 0.020 0.306 Suillus granulatus 0.020 0.234 Laccaria bicolor -0.104 0.153 Hebeloma cylindrosporum -0.005 0.329 Pisolithus tinctorius 0.010 0.058 Laccaria proxima 0.172 0.320 Tricholoma pessundatum -0.005 0.015 unknown 0.813 unknown 0.957 unknown 1.253 Pisolithus sp 0.511 0.303 Pisolithus sp 0.863 0.493 Pisolithus sp 1.111 0.614 Pisolithus sp 1.215 0.650 Authors Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Burgess et al. 1994 Chakravarty & Unestam 1987 Chakravarty & Unestam 1987 Chakravarty & Unestam 1987 Chakravarty & Unestam 1987 Chakravarty & Unestam 1987 Chakravarty & Unestam 1987 Chakravarty & Unestam 1987 Chakravarty & Unestam 1987 Chen et al. 2006 Chen et al. 2006 Chen et al. 2006 Host species Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Eucalyptus grandis Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Ln R Fungal species Biomass Height Pisolithus sp 1.804 1.249 Pisolithus sp 1.956 1.308 Pisolithus sp 2.408 1.434 Pisolithus sp 2.576 1.531 Pisolithus sp 2.650 1.590 Pisolithus sp 2.696 1.632 Pisolithus sp 2.802 1.646 Pisolithus sp 2.824 1.686 Pisolithus sp 3.086 1.762 Pisolithus sp 3.116 1.809 Pisolithus sp 3.173 1.843 Pisolithus sp 3.173 1.842 Pisolithus sp 3.189 1.886 Pisolithus sp 3.202 1.896 Pisolithus sp 3.305 1.928 Pisolithus sp 3.566 2.099 Laccaria laccata 0.489 0.143 Hebeloma crustulinforme 0.032 0.000 Pisolithus tinctorius 0.614 0.208 unknown 0.643 0.268 Laccaria laccata 0.489 0.143 Hebeloma crustulinforme 0.032 0.000 Pisolithus tinctorius 0.614 0.208 unknown 0.643 0.268 Scleroderma cepa 0.000 0.248 Scleroderma cepa 0.000 0.686 Scleroderma cepa 0.000 0.598 Authors Host species Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus globulus Chen et al 2006 Eucalyptus globulus Chen et al 2006 Eucalyptus globulus Chen et al . 2006 Eucalyptus globulus Chen et al . 2006 Eucalyptus globulus Chen et al . 2006 Eucalyptus globulus Chen et al . 2006 Eucalyptus globulus Chen et a . 2006 Eucalyptus globulus Chen et a . 2006 Eucalyptus globulus Chen et a . 2006 Eucalyptus globulus Fungal species Biomass Scleroderma cepa 0.000 Scleroderma cepa 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma cepa 0.000 Scleroderma cepa 0.000 Scleroderma cepa 0.000 Scleroderma cepa 0.000 Scleroderma cepa 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma citrinum 0.000 Scleroderma albidum 0.034 Scleroderma albidum 0.128 Scleroderma areolatum 0.128 Scleroderma areolatum -0.116 Scleroderma areolatum 0.476 Scleroderma cepa -0.065 Scleroderma cepa 0.049 Scleroderma cepa 0.311 Scleroderma citrinum -0.112 Scleroderma citrinum 0.171 Ln R Height Shoot:root 1.152 1.152 0.092 0.430 0.213 1.021 1.510 0.666 0.759 1.275 1.214 0.319 0.093 0.378 1.491 0.903 0.325 0.198 0.168 0.104 0.059 0.329 0.021 0.238 0.104 0.104 0.168 Authors Host species Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus globulus Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al. 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al 2006 Eucalyptus urophylla Chen et al 2006 Pinus elliottii Chen et al . 2006 Pinus elliottii Chen et al . 2006 Pinus elliottii Chen et al . 2006 Pinus elliottii Chen et al . 2006 Pinus elliottii Chen et al . 2006 Pinus elliottii Chen et a . 2006 Pinus elliottii Ln R Fungal species Biomass Height Scleroderma flavidum 0.153 0.147 Scleroderma flavidum 0.430 0.329 Scleroderma paradoxum 0.125 0.389 Scleroderma sp 0.220 0.247 Scleroderma verrucosum 0.155 0.168 Scleroderma albidum 0.714 0.294 Scleroderma albidum 0.042 0.216 Scleroderma areolatum 0.300 0.152 Scleroderma areolatum 0.005 0.184 Scleroderma areolatum 0.123 0.205 Scleroderma cepa -0.079 0.108 Scleroderma cepa 0.612 0.275 Scleroderma cepa -0.165 0.085 Scleroderma citrinum -0.214 -0.013 Scleroderma citrinum 0.327 0.426 Scleroderma flavidum 0.419 0.375 Scleroderma flavidum 0.750 0.483 Scleroderma paradoxum 0.451 0.393 Scleroderma sp 0.559 0.536 Scleroderma verrucosum 0.507 0.331 Scleroderma albidum 0.400 0.270 Scleroderma albidum 0.253 0.277 Scleroderma areolatum -0.102 0.178 Scleroderma areolatum -0.150 0.109 Scleroderma areolatum 0.302 0.034 Scleroderma cepa -0.221 0.079 Scleroderma cepa 0.270 0.134 Shoot:root co Authors Host species Chen eta l . 2006 Pinus elliottii Chen eta l . 2006 Pinus elliottii Chen et al. 2006 Pinus elliottii Chen et al. 2006 Pinus elliottii Chen et al. 2006 Pinus elliottii Chen eta l . 2006 Pinus elliottii Chen et al. 2006 Pinus elliottii Chen et al. 2006 Pinus elliottii Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen eta l . 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen eta l . 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Chen et al. 2006 Pinus radiata Choi et al. 2005 Pinus densiflora Choi et al. 2005 Pinus densiflora Conjeaud et al. 1996 Pinus pinaster Diedhiou et al. 2005 Afzelia africana Fungal species Scleroderma cepa Scleroderma citrinum Scleroderma citrinum Scleroderma flavidum Scleroderma flavidum Scleroderma paradoxum Scleroderma sp Scleroderma verrucosum Scleroderma albidum Scleroderma albidum Scleroderma areolatum Scleroderma areolatum Scleroderma areolatum Scleroderma cepa Scleroderma cepa Scleroderma cepa Scleroderma citrinum Scleroderma citrinum Scleroderma flavidum Scleroderma flavidum Scleroderma paradoxum Scleroderma sp Scleroderma verrucosum Pisolithus tinctorius Pisolithus tinctorius Hebeloma cylindrosporum Scleroderma dictyosporum Ln R Biomass Height -0.326 0.100 -0.312 -0.007 0.558 0.205 0.438 0.297 0.236 0.170 0.224 0.146 0.224 0.170 -0.262 0.021 0.959 0.342 1.260 0.302 1.134 0.164 0.895 0.146 0.794 0.312 1.080 0.165 0.830 0.255 0.959 0.176 0.391 0.070 0.717 0.213 0.935 0.435 0.717 0.266 0.830 0.400 0.747 0.422 0.623 0.198 0.303 -0.518 Authors Host species Diedhiou e ta l . 2005 Afzelia africana Diedhiou et al. 2005 Afzelia africana Diedhiou e ta l . 2005 Afzelia africana Diedhiou e ta l . 2005 Afzelia bella Diedhiou et al. 2005 Afzelia bella Diedhiou et al. 2005 Afzelia bella Diedhiou et al. 2005 Afzelia bella Diedhiou et al. 2005 Anthonotha macrophylla Diedhiou et al. 2005 Anthonotha macrophylla Diedhiou et al. 2005 Anthonotha macrophylla Diedhiou et al. 2005 Anthonotha macrophylla Diedhiou et al. 2005 Cryptosepalum tetraphylum Diedhiou et al. 2005 Cryptosepalum tetraphylum Diedhiou et al. 2005 Cryptosepalum tetraphylum Diedhiou et al. 2005 Cryptosepalum tetraphylum Diedhiou et al. 2005 Paramacrolobium coeruleum Diedhiou et al. 2005 Paramacrolobium coeruleum Diedhiou et al. 2005 Paramacrolobium coeruleum Diedhiou et al. 2005 Paramacrolobium coeruleum Diedhiou et al. 2005 Uapaca somon Diedhiou et al. 2005 Uapaca somon Diedhiou e ta l . 2005 Uapaca somon Diedhiou et al. 2005 Uapaca somon Dixon et al. 1981 Quercus velutina Dixon et al. 1981 Quercus velutina Dixon et al. 1983 Quercus velutina Dixon et al. 1983 Quercus velutina Fungal species Scleroderma verrucosum Pisolithus sp Thelephora sp Scleroderma dictyosporum Scleroderma verrucosum Pisolithus sp Thelephora sp Scleroderma dictyosporum Scleroderma verrucosum Pisolithus sp Thelephora sp Scleroderma dictyosporum Scleroderma verrucosum Pisolithus sp Thelephora sp Scleroderma dictyosporum Scleroderma verrucosum Pisolithus sp Thelephora sp Scleroderma dictyosporum Scleroderma verrucosum Pisolithus sp Thelephora sp Pisolithis tinctorius Pisolithis tinctorius Pisolithus tinctorius Pisolithus tinctorius Ln R Biomass Height Shootxoot 0.175 -0-535 0.288 -0.417 0.281 -0.461 0.571 , . -0976 0.595 -0-939 0.360 -1-182 0.358 -0-973 0.081 0.181 -0.010 0.214 0.199 -0088 0.186 0.235 0.741 -0.380 0.302 -0 510 . 0.474 -0.492 0.489 -1.063 0.456 -0.624 0.426 -0.650 0.375 -0-749 0.358 -0-744 2.209 -0726 2.035 -0-434 2.242 -0.642 1.984 -0-688 1.244 0.598 -0.850 -0.405 0.162 -0.015 Authors Host species Dixon e ta l . 1984 Quercus robur Dixon e ta l . 1984 Quercus robur Dixon et al. 1984 Quercus robur Dixon et al. 1984 Quercus robur Dixon e ta l . 1984 Quercus robur Dixon et al. 1984 Quercus robur Dixon et al. 1984 Quercus velutina Dixon et al. 1984 Quercus velutina Dixon e ta l . 1984 Quercus velutina Dixon etal . 1984 Quercus velutina Dixon et al. 1984 Quercus velutina Dixon et al. 1984 Quercus alba Dixon et al. 1984 Quercus alba Dixon e ta l . 1984 Quercus alba Dixon e ta l . 1984 Quercus alba Dixon etal . 1984 Quercus alba Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon e ta l . 1987 Pinus taeda Dixon e ta l . 1987 Pinus taeda Dixon e ta l . 1987 Pinus taeda Dixon e ta l . 1987 Pinus taeda Dixon e ta l . 1987 Pinus taeda Dixon etal . 1987 Pinus taeda Ln R Fungal species Biomass Height Shoot:root Pisolithuis tinctorius 0.711 0.397 -0.443 Pisolithus tinctorius 0.657 0.451 -0.335 Suillus granulatus 0.249 0.323 -0.074 Thelephora terrestris 0.601 0.420 -0.357 Suillis lute us 0.477 0.307 -0.206 Cenococcum geophilum 0.601 0.411 -0.267 Pisolithuis tinctorius 0.443 0.209 0.129 Pisolithus tinctorius 0.246 0.222 0.352 Suillus granulatus 0.413 0.265 0.163 Thelephora terrestris 0.443 0.162 -0.042 Suillis lute us 0.282 0.241 0.311 Pisolithuis tinctorius 0.288 0.211 0.208 Pisolithus tinctorius 0.201 0.305 0.154 Suillus granulatus 0.065 0.141 0.312 Thelephora terrestris 0.105 0.148 0.087 Suillis lute us 0.201 0.205 0.154 Pisolithus tinctorius 0.095 0.136 Pisolithus tinctorius 0.000 0.109 Pisolithus tinctorius 0.501 0.331 Pisolithus tinctorius 0.071 0.134 Pisolithus tinctorius -0.025 0.080 Pisolithus tinctorius 0.476 0.305 Pisolithus tinctorius 0.048 0.152 Pisolithus tinctorius 0.024 0.138 Pisolithus tinctorius 0.491 0.352 Pisolithus tinctorius 0.000 0.088 Pisolithus tinctorius 0.000 0.111 Authors Host species Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al. 1987 Pinus taeda Dixon et al 1987 Pinus taeda Dixon et al 1987 Pinus taeda Dixon et al 1987 Pinus taeda Dixon et al 1987 Pinus taeda Dixon et al 1987 Pinus taeda Dixon et al 1987 Pinus taeda Dixon et al . 1987 Pinus taeda Dixon et al . 1987 Pinus taeda Dixon et al . 1987 Pinus taeda Dunabeitia et al. 2004 Pinus radiata Dunabeitia et al. 2004 Pinus radiata Dunabeitia et al. 2004 Pinus radiata Dunabeitia et al. 2004 Pinus radiata Dunabeitia et al. 2004 Pinus radiata Dunabeitia et al. 2004 Pinus radiata Duponnois et al. 2000 Acacia holosericea Garbayeet al. 1988 Eucalyptus urophylla x E. kirtoniana Garbaye et al. 1988 Eucalyptus urophylla x E. kirtoniana Garbaye et al. 1988 Eucalyptus urophylla x E. kirtoniana Garbaye et al. 1988 Eucalyptus urophylla x E. kirtoniana Fungal species Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Pisolithus tinctorius Rhizopogon luteolus Rhizopogon roseolus Scleroderma citrinum Rhizopogon luteolus Rhizopogon roseolus Scleroderma citrinum Pisolithus tinctorius Pisolithus tinctorius Scleroderma texense Scleroderma aurantium Hebeloma cylindrosporum Ln R Biomass Height 0.243 0.211 0.023 0.080 0.190 0.095 0.171 0.203 0.023 0.088 0.151 0.095 0.190 0.196 0.223 0.010 0.377 -0.095 0.583 0.310 0.298 0.049 0.391 -0.106 0.649 0.303 0.189 0.010 0.318 -0.062 0.606 0.307 0.075 0.206 0.079 0.023 0.143 0.175 0.621 0.715 0.215 0.148 0.000 -0.041 Ln R Authors Host species Fungal species Biomass Height Shootxoot Garbaye et al. 1988 Eucalyptus urophylla x E. kirtoniana Scleroderma dictysporum -0.083 Garbayeet al. 1988 Eucalyptus urophylla x E. kirtoniana Pisolithis tinctorius -0.083 Grandcourt et al. 2004 Dicorynia guianensis unknown 0.342 Grandcourteta l . 2004 Eperua falcata unknown -0.209 Heinrich et al. 1988 Eucalyptus pilularis Pisolithus tinctorius 1.188 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.352 0.154 0.158 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.388 0.373 0.412 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.385 0.270 0.312 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.384 0.194 0.297 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.277 0.223 0.260 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.390 0.305 0.420 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.298 0.134 0.283 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata -0.039 0.046 0.162 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.104 0.097 0.121 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata -0.137 0.000 0.119 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata -0.136 -0.074 0.001 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata -0.193 -0.133 0.539 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata -0.266 -0.166 0.163 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata -0.029 -0.017 -0.511 Hung & Molina 1986 Pseudotsuga menziesii Laccaria laccata 0.021 -0.082 -0.106 Ivory & Munga 1983 Pinus caribaea Pisolithis tinctorius -0.130 Ivory & Munga 1983 Pinus caribaea Rhizopogon nigrescens -0.130 Ivory & Munga 1983 Pinus caribaea Scleroderma bovista -0.109 Ivory & Munga 1983 Pinus caribaea Scleroderma texense 0.048 Ivory & Munga 1983 Pinus caribaea Thelephora terrestris 0.065 Lamhamedi et al. 1990 Pinus pinaster Pisolithus arhizus -0.261 -0.129 Lamhamedi et al. 1990 Pinus pinaster Pisolithus arhizus -0.061 -0.294 Authors Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Host species Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Ln R Fungal species Biomass Height ShooLroot Pisolithus arhizus -0.361 -0.294 Pisolithus arhizus -0.319 0.133 Pisolithus arhizus -0.334 -0.217 Pisolithus arhizus 0.013 -0.224 Pisolithus arhizus -0.410 0.096 Pisolithus arhizus -0.022 -0.121 Pisolithus arhizus -0.280 -0.197 Pisolithus arhizus -0.335 -0.370 Pisolithus arhizus -0.242 0.072 Pisolithus arhizus -0.277 -0,198 Pisolithus arhizus 0.076 0.035 Pisolithus arhizus -0.300 -0.254 Pisolithus arhizus -0.247 -0.229 Pisolithus arhizus 0.061 0.033 Pisolithus arhizus -0.230 -0.167 Pisolithus arhizus -0.270 -0.121 Pisolithus arhizus -0.337 -0.166 Pisolithus arhizus 0.141 0.079 Pisolithus arhizus -0.220 0.084 Pisolithus arhizus -0.329 -0.143 Pisolithus arhizus -0.149 -0.279 Pisolithus arhizus -0.166 0.025 Pisolithus arhizus -0.172 0.026 Pisolithus arhizus 0.053 0.139 Pisolithus arhizus -0.364 -0.066 Pisolithus arhizus -0.346 0.095 Pisolithus arhizus -0.194 0.043 Authors Host species Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster. Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et a l , 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Lamhamedi et al. 1990 Pinus pinaster Ln R Fungal species Biomass Height Shoot.root Pisolithus arhizus -0.337 0.093 Pisolithus arhizus -0.276 -0.142 Pisolithus arhizus -0.321 0.010 Pisolithus arhizus -0.119 0.004 Pisolithus arhizus -0.222 0.120 Pisoiithus arhizus 0.098 0.006 Pisolithus arhizus -0.136 -0.045 Pisolithus arhizus -0.170 0.011 Pisolithus arhizus -0.232 0.021 Pisolithus arhizus 0.058 0.017 Pisolithus arhizus -0.018 -0.009 Pisolithus arhizus -0.268 0.059 Pisolithus arhizus -0.332 0.067 Pisolithus arhizus -0.312 0.032 Pisolithus arhizus -0.067 0.108 Pisolithus arhizus -0.029 0.072 Pisolithus arhizus -0.130 0.057 Pisolithus arhizus 0.002 0.238 Pisolithus arhizus -0.269 0.073 Pisolithus arhizus -0.131 -0.040 Pisolithus arhizus -0.039 -0.213 Pisolithus arhizus 0.051 0.051 Pisolithus arhizus -0.050 0.054 Pisolithus arhizus 0.010 0.103 Pisolithus arhizus -0.144 0.058 Pisolithus arhizus -0.370 -0.199 Pisolithus arhizus -0.235 0.008 Authors Lamhamedi et al. 1 990 Lamhamedi et al. ' 990 Lamhamedi et al. ' 990 Lamhamedi et al. ' 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamedi et al. 1990 Lamhamed et al. 1990 Lamhamed et al. 1990 Lamhamed et al. 1990 Lamhamed et al. 1990 Lamhamed et al. 1990 Lamhamed et al. 1990 Lamhamed et al. 1990 Lamhamed et al. 1990 Lamhamed i e al. 1990 Lamhamed i e al. 1990 Lamhamed i e ta l . 1990 Lamhamec i e ta l . 1990 Host species Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Pinus pinaster Ln R Fungal species Biomass Height Shoot: root Pisolithus arhizus -0.337 0.093 Pisolithus arhizus -0.276 -0.142 Pisolithus arhizus -0.321 0.010 Pisolithus arhizus -0.119 0.004 Pisolithus arhizus -0.222 0.120 Pisolithus arhizus 0.098 0.006 Pisolithus arhizus -0.136 -0.045 Pisolithus arhizus -0.170 0.011 Pisolithus arhizus -0.232 0.021 Pisolithus arhizus 0.058 0.017 Pisolithus arhizus -0.018 -0.009 Pisolithus arhizus -0.268 0.059 Pisolithus arhizus -0.332 0.067 Pisolithus arhizus -0.312 0.032 Pisolithus arhizus -0.067 0.108 Pisolithus arhizus -0.029 0.072 Pisolithus arhizus -0.130 0.057 Pisolithus arhizus 0.002 0.238 Pisolithus arhizus -0.269 0.073 Pisolithus arhizus -0.131 -0.040 Pisolithus arhizus -0.039 -0.213 Pisolithus arhizus 0.051 0.051 Pisolithus arhizus -0.050 0.054 Pisolithus arhizus 0.010 0.103 Pisolithus arhizus -0.144 0.058 Pisolithus arhizus -0.370 -0.199 Pisolithus arhizus -0.235 0.008 Ln R Authors Host species Fungal species Biomass Height Lu et al. 1998 Eucalyptus globulus Hydnangium submellatum -0.026 Lu et al. 1998 Eucalyptus globulus Hdynotrya sp -0.042 Lu et al. 1998 Eucalyptus globulus Hydnum repandum -0.030 Lu et al. 1998 Eucalyptus globulus Laccaria lateritia -0.002 Lu et al. 1998 Eucalyptus globulus Laccaria lateritia 0.000 Lu et al. 1998 Eucalyptus globulus Laccaria laccata -0.007 Lu et al. 1998 Eucalyptus globulus Laccaria sp 0.021 Lu et al. 1998 Eucalyptus globulus Leucopaxillus lilacinus -0.023 Lu et al. 1998 Eucalyptus globulus Mesophellia -0.144 Lu et al. 1998 Eucalyptus globulus Mesophellia -0.023 Lu et al. 1998 Eucalyptus globulus Paxillus muelleri -0.014 Lu et al. 1998 Eucalyptus globulus Paxillus sp -0.062 Lu et al. 1998 Eucalyptus globulus Pisolithus albus -0.055 Lu et al. 1998 Eucalyptus globulus Pisolithus microcarpus -0.035 Lu et al. 1998 Eucalyptus globulus Pisolithus tinctorius -0.199 Lu et al. 1998 Eucalyptus globulus Pisolithus sp -0.007 Lu et al. 1998 Eucalyptus globulus Pisolithus sp -0.030 Lu et al. 1998 Eucalyptus globulus Scleroderma cepa -0.026 Lu et al. 1998 Eucalyptus globulus Scleroderma cepa 0.016 Lu et al. 1998 Eucalyptus globulus Scleroderma cepa -0.112 Lu et al. 1998 Eucalyptus globulus Scleroderma sp -0.123 Lu et al. 1998 Eucalyptus globulus Scleroderma sp -0.125 Lu et al. 1998 Eucalyptus globulus Tricholoma sp 0.016 MacFal l& Slack 1991 Pinus resinosa Thelephora terrestris MacFall & Slack 1991 Pinus resinosa Pisolithus tinctorius MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.398 0.070 Shootroot -0.425 Ln R Authors Host species Fungal species Biomass Height Shoot:root MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.171 0.050 -0.386 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.398 0.050 0.086 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.023 0.017 0.038 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.134 -0.013 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.266 0.042 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.294 0.045 . MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.132 0.058 MacFall & Slack 1991 Pinus resinosa Pisolithus tinctorius 0.149 -0.112 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.069 -0.072 MacFall & Slack 1991 Pinus resinosa Pisolithus tinctorius 0.233 -0.024 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.285 -0.017 MacFall & Slack 1991 Pinus resinosa Pisolithus tinctorius 0.304 0.163 MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa 0.318 0.199 MacFall & Slack 1991 Pinus resinosa Pisolithus tinctorius MacFall & Slack 1991 Pinus resinosa Hebeloma arenosa MacFall et al. 1991 Pinus resinosa Hebeloma arenosa 2.187 -0.802 Marx et al. 1976 Pinus clausa Pisolithus tinctorius 0.284 Marx et al. 1976 Pinus clausa Pisolithus tinctorius 0.464 Marx e ta l . 1976 Pinus taeda Pisolithus tinctorius 0.035 Marx e ta l . 1976 Pinus taeda Pisolithus tinctorius 0.063 Marx e ta l . 1976 Pinus elliottii Pisolithus tinctorius 0.144 Marx e ta l . 1976 Pinus elliottii Pisolithus tinctorius 0.165 Marx e ta l . 1976 Pinus strobus Pisolithus tinctorius 0.693 0.261 0.000 Marx e ta l . 1976 Pinus strobus Pisolithus tinctorius 0.118 0.044 0.028 Marx e ta l . 1976 Pinus taeda Pisolithus tinctorius 0.877 0.348 -0.208 Marx e ta l . 1976 Pinus taeda Pisolithus tinctorius 0.603 0.206 -0.321 Marx e ta l . 1976 Pinus virginiana Pisolithus tinctorius 0.732 0.231 -0.455 4^ Authors Host species Marx et al. 1976 Pinus virginiana Mason et al. 2000 Eucalyptus globulus Mason et al. 2000 Eucalyptus globulus Mason et al. 2000 Eucalyptus globulus Morte et al. 2001 Pinus halapensis Morte e ta l . 2001 Pinus halapensis Muhsin & Zwiazek 2002 Picea glauca Nylund & Wallander 1989 Pinus sylvestris Nylund & Wallander 1989 Pinus sylvestris Osonubi et al. 1991 Acacia auriculiformis Osonubi et al. 1991 Albizia lebbeck Osonubi et al. 1991 Leucaena leucocephala Osonubi et al. 1991 Gliricidia sepium Repac1996 Picea abies Repac1996 Picea abies Repac 1996 Picea abies Repac1996 Picea abies Repac1996 Picea abies Repac1996 Picea abies Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle ST inus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus sylvestris Ln R Fungal species Biomass Height ShooLroot Pisolithus tinctorius 0.177 , 0.022 -0.245 Laccaria fraterna Laccaria fraterna Pisolithus tinctorius Suillus mediterraneansis 0.105 -0.006 Suillus mediterraneansis 0.103 -0.006 Hebeloma crustuliniforme 0.125 0.017 Hebeloma crustuliniforme -0.515 Laccaria laccata -0.599 Boletus suillus 0.726 0.303 Boletus suillus -1.526 -1.282 Boletus suillus 1.127 -0.062 Boletus suillus 0.683 -0.404 Suillus bovinus 0.000 -0.032 -0.238 Suillus bovinus 0.112 0.023 -0.136 Suillus bovinus -0.070 -0.040 -0.107 Suillus bovinus 0.050 0.012 -0.076 Inocybe lacera 0.157 -0.018 -0.153 Inocybe lacera 0.136 0.021 -0.037 Rhizopogon roseolus 0.936 0.095 Suillis granulatus 0.771 0.013 Thelephora terrestris 0.736 -0.076 Pisolithus tinctorius 0.805 0.020 Cenococcum geophilum 0.906 0.204 unknown 0.794 0.052 unknown 0.724 0.026 Suillus cothurnatus 0.039 -0.055 0.147 Authors Host species Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle ST inus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus ponderosa Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Riffle &Tinus 1982 Pinus sylvestris Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Fungal species Rhizopogon roseolus Suillus granulatus Thelephora terrestris Pisolithus tinctorius Cenococcum geophilum unknown unknown Rhizopogon roseolus Suillis granulatus Thelephora terrestris Pisolithus tinctorius Cenococcum geophilum unknown unknown Suillus cothurnatus Rhizopogon roseolus Suillus granulatus Thelephora terrestris Pisolithus tinctorius Cenococcum geophilum unknown unknown Hebeloma crustulinforme Hebeloma crustulinforme Hebeloma crustulinforme Hebeloma crustulinforme Hebeloma crustulinforme Ln R Biomass Height Shootroot 0.136 0.026 0.256 0.122 0.037 0.088 -0.028 -0.124 0.146 -0.082 -0.083 0.060 0.146 -0.011 0.196 0.039 -0.055 0.190 0.230 0.016 0.283 -0.125 -0.008 0.000 -0.058 -0.092 -0.071 -0.030 0.039 -0.061 -0.049 Authors Host species Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Rincon et al. 2001 Pinus pinea Ln R Fungal species Biomass Height Laccaria laccata 0.000 0.166 Laccaria laccata 0.000 -0.070 Laccaria laccata 0.027 0.119 Laccaria laccata 0.154 0.005 Laccaria laccata 0.027 0.027 Pisolithus tinctorius -0.121 -0.020 Pisolithus tinctorius -0.154 0.044 Melanogaster ambiguus -0.208 -0.311 Melanogaster ambiguus -0.043 -0.100 Rhizopogon luteolus -0.262 0.134 Rhizopogon luteolus -0.230 0.064 Rhizopogon luteolus -0.108 -0.012 Rhizopogon luteolus -0.080 -0.012 Rhizopogon luteolus -0.080 0.160 Rhizopogon luteolus -0.026 0.248 Rhizopogon roseolus -0.241 0.180 Rhizopogon roseolus -0.241 0.222 Rhizopogon roseolus -0.241 0.155 Rhizopogon roseolus -0.304 0.166 Rhizopogon roseolus -0.182 0.158 Rhizopogon roseolus -0.304 0.121 Pisolithus tinctorius -0.211 -0.120 Pisolithus tinctorius -0.182 -0.159 Pisolithus tinctorius -0.049 -0.209 Scleroderma verrucosum -0.267 -0.046 Scleroderma verrucosum -0.384 0.080 Scleroderma verrucosum -0.187 0.126 Authors Rincon et al. 2001 Rincon et al. 2005 Rincon et al. 2005 Rincon et al. 2005 Rouhier& Read 1998 Rouhier & Read 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Host species Pinus pinea Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pinus sylvestris Pinus sylvestris Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pinus contorta Pinus contorta Pinus contorta Pinus contorta Pinus contorta Pinus contorta Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pseudotsuga menziesii Pinus ponderosa Pinus ponderosa Pinus ponderosa Fungal species Scleroderma verrucosum Rhizopogon luteolus Rhizopogon roseolus Scleroderma verrucosum Paxillus involutus Suillus bovinus Laccaria laccata Laccaria laccata Laccaria laccata Rhizopogon vinicolor Rhizopogon vinicolor Rhizopogon vinicolor Laccaria laccata Laccaria laccata Laccaria laccata Laccaria laccata Laccaria laccata Laccaria laccata Laccaria laccata Laccaria laccata Laccaria laccata Rhizopogon vinicolor Rhizopogon vinicolor Rhizopogon vinicolor Laccaria laccata Laccaria laccata Laccaria laccata Ln R Biomass Height ShooLroot -0.239 0.028 -0.363 -0.038 -0.363 0.000 -0.245 -0.099 -0.067 0.320 -0.120 0.380 0.236 0.167 0.051 0.217 0.131 0.138 0.090 0.200 0.182 0.612 0.382 -0.305 0.390 0.305 -0.428 0.242 0.243 -0.234 0.084 0.111 -0.277 0.032 - 0.093 -0.447 0.136 0.048 -0.756 0.215 0.327 -0.373 0.370 0.367 0.102 0.218 0.283 -0.155 0.530 0.296 -0.931 0.485 0.216 -0.573 0.007 0.244 -0.019 0.599 0.132 -0.377 0.457 0.210 -0.809 0.319 0.263 -0.630 0.167 0.262 0.042 0.192 0.225 -0.070 0.245 -0.016 -0.227 Authors Scagel & Linderman 1998 Scagel & Linderman 1998 Scagel & Linderman 1998 Schier & McQuattie 1995 Schier & McQuattie 1996 Schier & McQuattie 1996 Tarn & Griffiths 1994 Tarn & Griffiths 1994 Tarn & Griffiths 1994 Tarn & Griffiths 1994 Tarn & Griffiths 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Host species Pinus ponderosa Pinus ponderosa Pinus ponderosa Pinus strobis Pinus rigida Pinus rigida Castanopsis fissa Castanopsis fissa Castanopsis fissa Castanopsis fissa Castanopsis fissa Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Ln R Fungal species Biomass Height ShooLroot Laccaria laccata 0.240 0.260 0.212 Laccaria laccata 0.194 0.172 0.198 Laccaria laccata -0.037 0.050 -0.082 Pisolithus tinctorius 0.304 0.109 0.700 Pisolithus tinctorius 0.654 0.193 Pisolithus tinctorius 0.270 0.171 Pisolithis tinctorius 0.117 -0.480 Cenococcum geophilum 0.256 -0.256 Thelephora terrestris -0.094 -0.561 Hymenogaster 0.033 -0.623 Sclerodema sp -0.158 -0.674 Protubera -0.223 unknown 0.148 Chondrogaster 0.336 Cortinarius -0.174 Cortinarius Cortinarius Cortinarius Cortinarius Cortinarius Cortinarius Hysterangium 0.039 Hysterangium Hysterangium Hysterangium Amanita sp -1.022 Amanita sp Authors Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Host species Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Ln R Fungal species Biomass Height Shoot.root Amanita sp Amanita sp Hydnangium 0.336 Hydnangium Hydnangium Zelleromyces 0.307 Zelleromyces Zelleromyces Hymenogaster 0.278 Hymenogaster Hymenogaster Hymenogaster Thaxterogaster sp 0.542 Thaxterogaster sp Scleroderma sp 0.000 Scleroderma sp Scleroderma sp Scleroderma sp Scleroderma sp Setchelliogaster sp 0.732 Pisolithus sp 0.307 Pisolithus sp Pisolithus sp Pisolithus sp Laccaria 0.365 cn o Laccaria Laccaria Authors Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Thomson et al. 1994 Turjaman et al. 2005 Turjaman et al. 2005 Wallander 2000 Wallander 2000 Wallander 2000 Wallander 2000 Wallander 2000 Wallander 2000 Wallander et al. 1997 Wallander et al. 1997 Wallander et al. 1997 Wallander e ta l . 1997 Yazid et al. 1994 Yazid et al. 1994 Host species Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Shorea pinanga Shorea pinanga Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Pinus sylvestris Hopea odorata Hopea belter! Fungal species Laccaria Hebeloma Descolea Descolea Pisolithus arhizus Scleroderma sp Suillus variegatus Suillus variegatus Suillus variegatus Suillus variegatus unknown unknown Piloderma croceum Paxillus involutus Suillus variegatus Suillus variegatus Pisolithus tinctorius Pisolithus tinctorius Ln R Biomass Height Shootxoot 1.138 1.151 1.099 0.619 0.930 0.533 0.288 0.431 0.693 0.526 1.386 0.932 2.122 0.617 0.155 1.215 0.496 0.239 Full citation of each study used in meta-analysis (excluding studies involving manipulations of nutrients) Study B a u m et a l . 2000 B a u m et a l . 2002 B a u m a n n et al . 2005 Full citation B a u m C , Schmid K, Makesch in F. 2000. Interactive effects of substrates and ectomycorrhizal colonizat ion on growth of a poplar clone. Journal of Plant Nutrition and Soi l Sc ience 163: 221-226 Baum C , Stetter U, Makesch in F. 2002. Growth response of Populus trichocarpa to inoculation by the ectomycorrhizal fungus Laccaria laccata in a pot and a field experiment. Forest Eco logy and Management 163: 1-8 Baumann K, Schne ider B U , Marschner P, Huttl R F . 2005. Root distribution and nutrient status of mycorrhizal and non-mycorrhizal Pinus sylvestris L. seedl ings growing in a sandy substrate withj ignite fragments. Plant and Soi l 276: 347-357 Beye ler & Beyeler M , Heyser W . 1997. The influence of mycorrhizal colonizat ion on growth in the greenhouse and Heyser 1997 on catechin, epicatechin and procyanidin in roots of Fagus sylvatica L. Mycorrhiza 7: 171-177 Bougher et al . 1990 Bougher NL , Grove T S , Malajczuk N. 1990. Growth and phosphorus acquisit ion of Karri [Eucalyptus diversicolor F-Muell) seedl ings inoculated with ectomycorrhizal fungi in relation to phosphorus supply. New Phytologist 114: 77-85 Study Browning & Whitney 1991 Full citation Browning M H R , Whi tney R D . 1991. R e s p o n s e s of jack pine and black spruce seedl ings to inoculation with selected spec ies of ectomycorrhiza fungi. Canad ian Journal of Forest Research 21 : 701-706 Burgess & Burgess T, Mala jczuk N. 1989. The effects of ectomycorrhizal fungi on reducing the variation of seedl ing Mala jczuk 1989 growth of Eucalytpus globulus. Agriculture Ecosys tems and the Environment 28: 41-46 Burgess et a l . 1994 Burgess , T, Dell B, Malajczuk N. 1994. Variat ion in mycorrhizal development and growth stimulation by 20 Pisolithus isolates inoculated on to Eucalyptus grandis W Hill Ex Maiden. New Phytologist 127: 731 -739 Chakravar ty & Chakravarty P, Unes tam T. 1987. Differential influence of ectomycorrhizae on plant-growth and d isease Unes tam 1987 resistance in Pinus sylvestris seedl ings. Journal of Phytopathology 120: 104-120 C h e n et a l . C h e n Y L , Dell B, Malajczuk N. 2006. Effect of Scleroderma spore density and age on mycorrhiza 2006 formation and growth of containerized Eucalyptus globulus and E-Urophyl la seedl ings. New Forests 31 : 453-467 Cho i et a l . 2005 Cho i D S , Quoresh i A M , Maruyama Y , J in H O , Koike T. 2005. Effect of ectomycorrhizal infection on growth and photosynthetic characterist ics of Pinus densiflora seedl ings grown under elevated C 0 2 concentrat ions. Photosynthet ica 43: 223-229 Study Full citation Conjeaud et al . Con jeaud C , Sche romm P, Mousain D. 1996. Effects of phosphorus and ectomycorrhiza on maritime 1996 pine seedl ings (Pinus pinaster). New Phytologist 133: 345-351 Diedhiou et a l . Diedhiou A G , G u e y e O, Diabate M, Prin Y , Duponnois R, Dreyfus B, B a A M . 2005. Contrast ing 2005 responses to ectomycorrhizal inoculation in seedl ings of six tropical Afr ican tree spec ies . Mycorrh iza 16: 11-17 Dixon et a l . Dixon R K , Wright G M , Garrett H E , C o x G S , Johnson P S , Sande r IL. 1981. Container-grown and 1981 nursery-grown black oak seedl ings inoculated with Pisolithis tincortius- growth and ectomycorrhizal development during seedl ing production period. Canad ian Journal of Forest Research 11: 487-491 Dixon et a l . Dixon R K , Pal lardy S G , Garrett H E , Cox G S , Sander IL. 1983. Comparat ive water relations of container-1983 grown and bare-root ectomycorrhizal and nonmycorrhizal Quercus velutina seedl ings. Canad ian Journal of Botany 61 : 1559-1565 Dixon et a l . Dixon R K , Garrett H E , C o x G S , Marx D H , Sander IL. 1984. Inoculation of 3 Quercus spec ies with 11 1984 isolates of ectomycorrhizal fungi. 1. Inoculation success and seedl ing growth relationships. Forest Sc ience 30: 364-372 Dixon et a l . Dixon R K , Garrett H E , Ste lzer H E . 1987. Growth and ectomycorrhizal development of Loblolly pine 1987 progenies inoculated with 3 isolates of Pisolithus tinctorius. S i lvae Genet ica 36: 240-245 1987 cn 4^ Study Full citation Dunabetia et al. Dunabeitia MK, Hormilla S, Garcia-Plazaoia J l , Txarterina K, Arteche U, Becerril JM . 2004. Differential 2004 Duponnois et al. 2000 responses of three fungal species to environmental factors and their role in the mycorrhization of Pinus radiata D. Don. Mycorrhiza 14: 11-18 Duponnois R, Founoune H, Ba A, Pienchette C, E! Jaafari S, Neyra M, Ducousso M. 2000. Ectomycorrhization of Acacia holosericea A. Cunn. ex G. Don by Pisolithus spp. in Senegal: Effect on plant growth and on the root-knot nematode Meloidogyne javanica. Annals of Forest Science 57: 345-350 Garbaye et al. Garbaye J , Delwaulle J C , Diangana D. 1988. Growth response of Eucalyptus in the Congo to 1988 •. ectomycorrhizal inoculation. Forest Ecology and Management 24: 151-157 Grandcourt et de Grandcourt A, Epron D, Montpied P, Louisanna E, Bereau M, Garbaye J , Guehl JM . 2004. al. 2004 Contrasting responses to mycorrhizal inoculation and phosphorus availability in seedlings of two tropical rainforest tree species. New Phytologist 161: 865-875 Heinrich et al. 1988 Heinrich PA, Mulligan DR, Patrick JW. 1988. The effect of ectomycorrhizas on the phosphorus and dry-weight acquisition of Eucalyptus seedlings. Plant and Soil 109: 147-149 Hung & Molina Hung LLL, Molina R. 1986. Use of the ectomycorrhizal fungus Laccaria laccata in forestry. 3. Effects of 1986 commercially produced inoculum on container-grown Douglas-fir and ponderosa pine seedlings. Canadian Journal of Forest Research 16: 802-806 cn cn Study Full citation Ivory & Munga Ivory M H , Munga F M . 1983. Growth and survival of container grown Pinus caribaea infected with. 1983 various ectomycorrhizal fungi. Plant and Soi l 71 :339-344 Lamhamed i et Lamhamed i M S , Fo r th J A , Kope H H , Kropp B R . 1990. Genet ic variation in ectomycorrhiza formation by al. 1990 Pisolithus arhizus on Pinus pinaster and Pinus banksiana. N e w Phytologist l 15: 689-697 Lu et ai: 1998 Lu X H , Mala jczuk N, Dell B. 1998. Mycorrhiza formation and growth of Eucalyptus globulus seedl ings inoculated with spores of various ectomycorrhizal fungi. Mycorrhiza 8: 81-86 MacFa l l & S lack 1991 MacFa l l et a l . 1991 Marx et a l . 1976 MacFa l l J S , S lack S A . 1991. Effects of Hebeloma arenosa on growth and survival of container-grown red pine seedl ings {Pinus resinosa). Canad ian Journal of Forrest Research 21: 1459-1465 Macfal l J , S lack S A , Iyer J . 1991. Effects of Hebeloma arenosa and phosphorus fertility on growth of red pine (Pinus resinosa) seedl ings. Canad ian Journal of Botany 69: 372-379 Marx D H , Brysan W C , Cordel l C E . 1976. Growth and ectomycorrhizal development of P ine seedl ings in nursery soi ls infested with fungal symbiont Pisolithus tinctorius. Forest Sc ience 22: 91-100 Mason et al . 2000 M a s o n P A , Ibrahim K, Ingleby K, Munro R C , Wi lson J . 2000. Mycorrhizal development and growth of inoculated Eucalyptus globulus (Labill.) seedl ings in wet and dry condit ions in the g lasshouse . Forest Eco logy and Management 128: 269-277 Study Full citation Morte et a l . Morte A , D iaz G , Rodr iguez P, A larcon J J , Sanchez-B ianco M J . 2001. Growth and water relations in 2001 mycorrhizal and nonmycorrhizal Pinus halepensis plants in response to drought. Bio logia Plantarum 44: 263-267 2001 Muhs in & Muhs in T M , Zwiazek J J . 2002. Colonizat ion with Hebeloma crustuliniforme increases water Zw iazek 2002 conductance and limits shoot sod ium uptake in white spruce {Picea glauca) seedl ings. Plant and Soi l 2 3 8 : 2 1 7 - 2 2 5 Nylund & Nylund J E , Wal lander H. 1989. Effects of ectomycorrhiza on host growth and carbon balance in a semi -Wal lander 1989 hydroponic cultivation system. New Phytologist 112: 389-398 Osonub i et al . Osonub i O , Mulongoy K, Awotoye O O , Atayese M O , Okal i D U U . 1991. Effects of ectomycorrhizal and 1991 vesicular-arbuscular mycorrhizal fungi on drought tolerance of 4 leguminous woody seedl ings. Plant and Soi l 136: 131-141 R e p a c 1996 R e p a c I. 1996. Inoculation of Picea abies (L) Karst seedl ings with vegetative inocula of ectomycorrhizal fungi Suillus bovinus (L: Fr) O. Kuntze and Inocybe lacera (Fr) Kumm. New Forests 12: 41-54 Riffle & T inus 1982 Riffle J W , T inus R W . 1982. Ectomycorrhizal characterist ics, growth, and survival of artificially inoculated ponderosa pine and Sco ts pine in a greenhouse and plantation. Forest Sc ience 28: 646-660 Study R incon et a l . 2001 Fuli citation R incon A , A lvarez IF, P e r a J . 2001. Inoculation of containerized Pinus pinea L. seedl ings with seven ectomycorrhizal fungi. Mycorrh iza 11: 265-271 R incon et a l . R incon A , Par lade J , P e r a J . 2005. Effects of ectomycorrhizal inoculation and the type of substrate on 2005 mycorrhizat ion, growth and nutrition of containerised Pinus pinea L. seedl ings produced in a commercia l nursery. Anna ls of Forest Sc ience 62: 817-822 R o u h i e r & R o u h i e r H , R e a d D J . 1998. Plant and fungal responses to elevated atmospheric carbon dioxide in R e a d 1998 mycorrhizal seedl ings of Pinus sylvestris. Environmental and Experimental Botany 40: 237-246 S c a g e l & S c a g e l C F , L inderman R G . 1998. Influence of ectomycorrhizal fungal inoculation on growth and root L inderman IAA concentrat ions of transplanted conifers. Tree Physio logy 18: 739-747 1998 Sch ie r & Sch ier G A , McQuatt ie C J . 1995. Effect of aluminum on the growth, anatomy, and nutrient content of McQuat t ie ectomycorrhizal and nonectomycorrhizai eastern white pine seedl ings. Canad ian Journal of Forest 1995 Resea rch 25: 1252-1262 Sch ie r & Sch ier G A , McQuatt ie C J . 1996. Response of ectomycorrhizal and nonmycorrhizal pitch pine {Pinus McQuat t ie rigida) seedl ings to nutrient supply and aluminum: Growth and mineral nutrition. Canad ian Journal of 1996 Forest R e s e a r c h 26: 2145-2152 cn co Study Full citation Tarn & Griffiths Tarn P C F , Griffiths DA. 1994. Mycorrhizal associat ions in Hong Kong Fagaceae. 6. Growth and nutrient uptake by Gastanopsis fissa seedl ings inoculated with ectomycorrhizal fungi. Mycorrhiza 4: 169-172 1994 Thomson et al . 1994 Turjaman et a l . 2005- -T h o m s o n B D , Grove T S , Malajczuk N, Hardy G E S J . 1994. The effectiveness of ectomycorrhizal fungi in increasing the growth of Eucalyptus globulus Labill in relation to root colonization and hyphal development in soi l . New Phytologist 126: 517-524 Turjaman M, Tamai Y , S e g a h H, Limin S H , C h a J Y , Osak i M, Tawaraya K. 2005. Inoculation with the ectomycorrhizal fungi Pisolithus arhizus and Scleroderma sp improves early growth of Shorea pinanga nursery seedl ings. New Forests 30: 67-73 Wal lander 2000 Wal lander H. 2000. Uptake of P from apatite by Pinus sylvestris seedl ings co lonised by different ectomycorrhizal fungi. Plant and Soi l 218: 249-256 Wal lander et a l . Wal lander H, W ickman T, J a c k s G . 1997. Apatite as a P source in mycorrhizal and non-mycorrhizal 1997 Pinus sylvestris seedl ings. Plant and Soi l 196: 123-131 Yaz id et a l . Yaz id S M , Lee S S , Lapeyr ie F. 1994. Growth stimulation of Hopea spp (Dipterocarpaceae) seedl ings 1994 following ectomycorrhizal inoculation with an exotic strain of Pisolithus tinctorius. Forest Eco logy and Management 67: 339-343 CD B. Identity of host plant and fungal species pairings with associated effect sizes (Ln R) seedling biomass. Authors Host species Fungal species P addition Ln R mg kg -1 Biomass Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 0 0.894 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 0 0.368 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 0 0.575 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 2 1.170 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 2 1.170 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 2 2.335 Bougher e ta l . 1990 Eucalyptus diversicolor Descolea maculata 4 2.197 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 4 2.147 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 4 3.050 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 8 1.603 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 8 1.518 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 8 1.937 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 12 0.360 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 12 0.754 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 12 0.873 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 16 0.182 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 16 0.416 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 16 0.341 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 20 -0.259 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 20 -0.288 Bougher et al. 1990 Eucalyptus diversicolor Laccaria laccata 20 -0.386 Bougher e ta l . 1990 Eucalyptus diversicolor Descolea maculata 28 -0.062 Bougher et al. 1990 Eucalyptus diversicolor Descolea maculata 28 -0.023 Authors Host species Bougher e ta l . 1990 Bougher et al. 1990-Bougher et al. 1990 Bougher e ta l . 1990 Bougher et al. 1990 Bougher et al. 1990 . Bougher et al. 1990 Browning & Whitney 1992 Browning & Whitney 1992 Browning & Whitney 1992. Browning & Whitney 1992 Browning & Whitney 1992 Browning & Whitney 1992 Browning & Whitney 1992 Browning & Whitney 1992 Burgess et al. 1993 Burgess et al. 1994 Burgess et al. 1995 Burgess et al. 1996 Burgess et al. 1997 Burgess et al. 1998 Burgess et al. 1999 Burgess et al 2000 Burgess et al 2001 Burgess et al 2002 Burgess et al 2003 Burgess et al 2004 Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Picea mariana Picea mariana Pinus banksiana Pinus banksiana Picea mariana Picea mariana Pinus banksiana Pinus banksiana Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Fungal species P addition Ln R mg kg -1 Biomass Laccaria laccata 28 -0.150 Descolea maculata 36 -0.168 Descolea maculata 36 -0.002 Laccaria laccata 36 0.013 Descolea maculata 48 -0.056 Descolea maculata 48 0.047 Laccaria laccata 48 -0.064 Laccaria bicolor 1.5 0.275 Laccaria bicolor 1.5 0.389 Laccaria bicolor 1.5 0.512 Laccaria bicolor 1.5 0.144 Laccaria bicolor 7.2 -0.089 Laccaria bicolor 7.2 -0.131 Laccaria bicolor 7.2 0.331 Laccaria bicolor 7.2 0.134 Cortinarius globuliformis 4 0.560 Paxillus muelleri 4 0.560 Hysterangium inflatum 4 0.118 Hysterangium inflatum 4 0.629 Thaxterogaster sp 4 0.694 Amanita xanthocephala 4 0.755 Hymenogaster zeylanicus 4 0.694 Hymenogaster viscidus 4 1.057 Hymenogaster zeylanicus 4 1.355 Setchelliogaster sp 4 1.099 Descolea maculata 4 1.771 Hydnangium carneum 4 1.682 Authors Host species Burgess et al. 2005 Burgess et al. 2006 Burgess et al. 2007 Burgess et al. 2008 Burgess et al. 2009 Burgess et al. 2010 . Burgess etal. '2011 Burgess et al. 2012 Burgess et al. 2013 Burgess et al. 2014 Burgess et al. 2015 Burgess et al. 2016 Burgess et al. 2017 Burgess et al. 2018 Burgess et al. 2019 Burgess et al. 2020 Burgess et al. 2021 Burgess et al. 2022 Burgess et al. 2023 Burgess et al. 2024 Burgess et al. 2025 Burgess et al. 2026 Burgess et al. 2027 Burgess et al. 2028 Burgess et al. 2029 Burgess et al. 2030 Burgess et al. 2031 Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus diversicolor Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Eucalyptus globulus Fungal species Laccaria laccata Laccaria laccata Sclerodema verrucosum Pisolithis tinctorius Cortinarius globuliformis Paxillus muelleri Hysterangium inflatum Hysterangium inflatum Thaxterogaster sp Amanita xanthocephala Hymenogaster zeylanicus Hymenogaster viscidus Hymenogaster zeylanicus . Setchelliogaster sp Descolea maculata Hydnangium carneum Laccaria laccata Laccaria laccata Sclerodema verrucosum Pisolithis tinctorius Cortinarius globuliformis Paxillus muelleri Hysterangium inflatum Hysterangium inflatum Thaxterogaster sp Amanita xanthocephala Hymenogaster zeylanicus P addition Ln R mg kg -1 Biomass 4 1.505 4 1.853 4 1.771 4 2.421 4 0.516 4 0.921 4 0.307 4 0.429 4 0.997 4 0.544 4 1.119 4 0.806 4 1.444 4 1.365 4 1.805 4 1.371 4 1.959 4 2.208 4 1.914 4 2.714 12 0.055 12 0.042 12 0.002 12 0.017 12 0.045 12 0.005 12 -0.008 CD Authors Host species Burgess 3tS al. 2032 Eucalyptus globulus Burgess et al. 2033 Eucalyptus globulus Burgess et al. 2034 Eucalyptus globulus Burgess et al. 2035 Eucalyptus globulus Burgess et al. 2036 Eucalyptus globulus Burgess et al. 2037 Eucalyptus globulus Burgess et al. 2038 Eucalyptus globulus Burgess et al. 2039 Eucalyptus globulus Burgess et al. 2040 Eucalyptus globulus Burgess et al. 2041 Eucalyptus diversicolor Burgess et al. 2042 Eucalyptus diversicolor Burgess et al. 2043 Eucalyptus diversicolor Burgess et al. 2044 Eucalyptus diversicolor Burgess et al. 2045 Eucalyptus diversicolor Burgess et al 2046 Eucalyptus diversicolor Burgess et al 2047 Eucalyptus diversicolor Burgess et al 2048 Eucalyptus diversicolor Burgess et al 2049 Eucalyptus diversicolor Burgess et al 2050 Eucalyptus diversicolor Burgess el al . 2051 Eucalyptus diversicolor Burgess el al . 2052 Eucalyptus diversicolor Burgess , el al . 2053 Eucalyptus diversicolor Burgess >e tal . 2054 Eucalyptus diversicolor Burgess ; e t a . 2055 Eucalyptus diversicolor Burgess 5 e t a . 2056 Eucalyptus diversicolor Chen e al . 2000 Eucalyptus globulus Chen e t a . 2000 Eucalyptus urophylla Fungal species P addition Ln R mg kg -1 Biomass Hymenogaster viscidus 12 0.034 Hymenogaster zeylanicus 12 -0.007 Setchelliogaster sp 12 -0.034 Descolea maculata 12 0.023 Hydnangium carneum 12 0.089 Laccaria laccata 12 -0.061 Laccaria laccata 12 0.065 Sclerodema verrucosum 12 0.012 Pisolithis tinctorius 12 0.040 Cortinarius globuliformis 12 -0.317 Paxillus muelleri 12 -0.380 Hysterangium inflatum 12 -0.379 Hysterangium inflatum 12 -0.178 Thaxterogaster sp 12 -0.124 Amanita xanthocephala 12 -0.106 Hymenogaster zeylanicus 12 -0.541 Hymenogaster viscidus 12 -0.156 Hymenogaster zeylanicus 12 -0.007 Setchelliogaster sp 12 -0.119 Descolea maculata 12 -0.101 Hydnangium carneum 12 0.097 Laccaria laccata 12 -0.205 Laccaria laccata 12 -0.065 Sclerodema verrucosum 12 -0.081 Pisolithis tinctorius 12 -0.001 Laccaria lateritia 5 0.368 Laccaria lateritia 5 2.048 Authors Host species Fungal species P addition Ln R mg kg -1 Biomass Conjeaud et al. 1996 Pinus pinaster Hebeloma cylindrosporum 0 Grandcourt et ai. 2004 Dicorynia guianensis unknown 0 0.342 Grandcourt et al. 2004 Eperua falcata unknown 0 -0.209 Grandcourt et al. 2004 Dicorynia guianensis unknown 8 1.228 Grandcourt et al. 2004 Eperua falcata unknown 8 0.312 Grandcourt et al. 2004 Dicorynia guianensis unknown 40 1.226 Grandcourt et al. 2004 Eperua falcata unknown 40 0.344 Khasa eta l . 2001 .-.- • Pinus contorta Hebeloma longicaudum 18 0.902 Khasa et al. 2001 Pinus contorta Laccaria bicolor 18 0.853 Khasa et al. 2001 Pinus contorta Paxillus involutus 18 0.936 Khasa et al. 2001 Pinus contorta Pisolithis tinctorius 18 0.964 Khasa et al. 2001 Pinus contorta Rhizopogon vinicolor 18 -0.015 Khasa et al. 2001 Pinus contorta Suillis tomentosus 18 0.014 Khasa et al. 2001 Picea glauca Hebeloma longicaudum 18 0.764< Khasa et al. 2001 Picea glauca Laccaria bicolor 18 0.852 Khasa et al. 2001 Picea glauca Paxillus involutus 18 1.246 Khasa et al. 2001 Picea glauca Pisolithis tinctorius 18 1.222 Khasa et al. 2001 Picea glauca Rhizopogon vinicolor 18 0.109 Khasa et al. 2001 Picea glauca Suillis tomentosus 18 0.016 Khasa et al. 2001 Picea mariana Hebeloma longicaudum 18 0.976 Khasa et al. 2001 Picea mariana Laccaria bicolor 18 1.152 Khasa et al. 2001 Picea mariana Paxillus involutus 18 1.677 Khasa et al. 2001 Picea mariana Pisolithis tinctorius 18 1.366 Khasa et al. 2001 Picea mariana Rhizopogon vinicolor 18 0.298 Khasa et al. 2001 Picea mariana Suillis tomentosus 18 0.328 Khasa et al. 2001 Pinus sylvestris Hebeloma longicaudum 18 0.066 Khasa et al. 2001 Pinus sylvestris Laccaria bicolor 18 -0.040 Authors Host species Fungal species P addition Ln R mg kg -1 Biomass Khasa et al. 2001 Pinus sylvestris Paxillus involutus 18 0.176 Khasa et al. 2001 Pinus sylvestris Pisolithis tinctorius 18 0.152 Khasa et al. 2001 Pinus sylvestris Rhizopogon vinicolor 18 -0.034 Khasa et al. 2001 Pinus sylvestris Suillis tomentosus 18 1.615 Khasa et al. 2001 Larix-sibirica Hebeloma longicaudum 18 0.074 Khasa et al. 2001 Larix sibirica Laccaria bicolor 18 0.148 Khasa eta l . 2001 Larix sibirica Paxillus involutus 18 0.099 Khasa eta l . 2001 Larix sibirica Pisolithis tinctorius 18 0.084 Khasa et al. 2001 Larix sibirica Rhizopogon vinicolor 18 0.027 Khasa et al. 2001 Larix sibirica Suillis tomentosus 18 0.043 Khasa et al. 2001 Pinus contorta Hebeloma longicaudum 37 0.781 Khasa eta l . 2001 Pinus contorta Laccaria bicolor 37 0.755 Khasa et al. 2001 Pinus contorta Paxillus involutus 37 0.908 Khasa et al. 2001 Pinus contorta Pisolithis tinctorius 37 0.862 Khasa et al. 2001 Pinus contorta Rhizopogon vinicolor 37 0.016 Khasa et al. 2001 Pinus contorta Suillis tomentosus 37 0.128 Khasa et al. 2001 Picea glauca Hebeloma longicaudum 37 0.982 Khasa et al. 2001 Picea glauca Laccaria bicolor 37 1.061 Khasa et al. 2001 Picea glauca Paxillus involutus 37 1.281 Khasa et al. 2001 Picea glauca Pisolithis tinctorius 37 1.135 Khasa et al. 2001 Picea glauca Rhizopogon vinicolor 37 0.140 Khasa et al. 2001 Picea glauca Suillis tomentosus 37 0.131 Khasa et al. 2001 Picea mariana Hebeloma longicaudum 37 0.688 Khasa et al. 2001 Picea mariana Laccaria bicolor 37 1.025 Khasa et al. 2001 Picea mariana Paxillus involutus 37 1.217 Khasa et al. 2001 Picea mariana Pisolithis tinctorius 37 1.134 Khasa et al. 2001 Picea mariana Rhizopogon vinicolor 37 0.057 Authors Host species Fungal species P addition Ln R mg kg -1 Biomass Khasa et al. 2001 Picea mariana Suillis tomentosus 37 0.075 Khasa et al. 2001 Pinus sylvestris Hebeloma longicaudum 37 -0.010 Khasa eta l . 2001 Pinus sylvestris Laccaria bicolor 37 0.044 Khasa eta l . 2001 Pinus sylvestris Paxillus involutus 37 0.334 Khasa et al. 2001 Pinus sylvestris Pisolithis tinctorius 37 0.334 Khasa et al. 2001 Pinus sylvestris Rhizopogon vinicolor 37 0.039 Khasa et al. 2001 Pinus sylvestris Suillis tomentosus 37 0.034 Khasa et al. 2001 Larix sibirica Hebeloma longicaudum 37 -0.585 Khasa et al. 2001 Larix sibirica Laccaria bicolor 37 -0.522 Khasa eta l . 2001 Larix sibirica Paxillus involutus 37 -0.531 Khasa et al. 2001 Larix sibirica Pisolithis tinctorius 37 -0.531 Khasa et al. 2001 Larix sibirica Rhizopogon vinicolor 37 -0.648 Khasa et al. 2001 Larix sibirica Suillis tomentosus 37 -0.618 Khasa et al. 2001 Pinus contorta Hebeloma longicaudum 55 0.505 Khasa et al. 2001 Pinus contorta Laccaria bicolor 55 0.485 Khasa et al. 2001 Pinus contorta Paxillus involutus 55 0.729 Khasa et al. 2001 Pinus contorta Pisolithis tinctorius 55 0.662 Khasa et al. 2001 Pinus contorta Rhizopogon vinicolor 55 0.076 Khasa et al. 2001 Pinus contorta Suillis tomentosus 55 0.086 Khasa et al. 2001 Picea glauca Hebeloma longicaudum 55 0.670 Khasa et al. 2001 Picea glauca Laccaria laccata 55 0.716 Khasa e ta l . 2001 Picea glauca Paxillus involutus 55 0.849 Khasa e ta l . 2001 Picea glauca Pisolithis tinctorius 55 0.911 Khasa e ta l . 2001 Picea glauca Rhizopogon vinicolor 55 0.105 Khasa et al. 2001 Picea glauca Suillis tomentosus 55 0.156 Khasa et al. 2001 Picea mariana Hebeloma longicaudum 55 0.579 Khasa et al. 2001 Picea mariana Laccaria bicolor 55 0.608 Authors Host species Khasa et al. 2001 Picea mariana Khasa et al. 2001 Picea mariana Khasa et al. 2001 Picea mariana Khasa e ta l . 2001 Picea mariana Khasa et al. 2001 Pinus sylvestris Khasa et al. 2001 Pinus sylvestris Khasa et al. 2001 Pinus sylvestris Khasa et al. 2001 Pinus sylvestris Khasa et al. 2001 Pinus sylvestris Khasa et al. 2001 Pinus sylvestris Khasa et al. 2001 Larix sibirica Khasa et al. 2001 Larix sibirica Khasa et al. 2001 Larix sibirica Khasa e ta l . 2001 Larix sibirica Khasa et al. 2001 Larix sibirica Khasa et al. 2001 Larix sibirica MacFall et al. 1991 Pinus resinosa MacFall et al. 1991 Pinus resinosa MacFall et al. 1991 Pinus resinosa MacFall et al. 1991 Pinus resinosa MacFall et al. 1991 Pinus resinosa Tyminski et al. 1986 Pinus sylvestris Tyminski et al. 1986 Pinus sylvestris Tyminski et al. 1986 Pinus sylvestris Tyminski et al. 1986 Pinus sylvestris Tyminski et al. 1986 Pinus sylvestris Tyminski et al. 1986 Pinus sylvestris Fungal species P addition Ln R mg kg -1 Biomass Paxillus involutus 55 0.795 Pisolithis tinctorius 55 0.793 Rhizopogon vinicolor 55 0.034 Suillis tomentosus 55 0.045 Hebeloma longicaudum 55 0.066 Laccaria bicolor 55 0.086 Paxillus involutus 55 0.395 Pisolithis tinctorius 55 0.398 Rhizopogon vinicolor 55 0.021 Suillis tomentosus 55 0.031 Hebeloma longicaudum 55 0.045 Laccaria bicolor 55 0.058 Paxillus involutus 55 0.120 Pisolithis tinctorius 55 0.058 Rhizopogon vinicolor 55 0.036 Suillis tomentosus 55 0.031 Hebeloma arenosa 0 2.187 Hebeloma arenosa 17 1.222 Hebeloma arenosa 34 0.488 Hebeloma arenosa 68 0.175 Hebeloma arenosa 136 0.105 Laccaria laccata 1 -0.566 Hebeloma crustliniforme 1 -0.714 Laccaria laccata 3.1 -0.392 Hebeloma crustliniforme 3.1 -0.287 Laccaria laccata 10 -0.349 Hebeloma crustliniforme 10 -0.392 Authors Tyminski et al. 1986 Tyminski et al. 1986 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Walker 2001 Host species Pinus sylvestris Pinus sylvestris Pinus lambertiana Pinus jeffreyi Pinus lambertiana Pinus jeffreyi Pinus lambertiana Pinus jeffreyi Pinus lambertiana Pinus jeffreyi Pinus lambertiana Pinus jeffreyi Pinus lambertiana Pinus lambertiana Pinus jeffreyi Pinus jeffreyi Pinus lambertiana Pinus jeffreyi Pinus lambertiana Pinus jeffreyi Pinus lambertiana Pinus jeffreyi Fungal species P addition Ln R mg kg -1 Biomass Laccaria laccata 31 -0.128 Hebeloma crustliniforme 31 -0.566 Pisolithis tinctorius 0 0.053 Pisolithis tinctorius 0 0.063 Pisolithis tinctorius 8 -0.075 Pisolithis tinctorius 8 -0.328 Pisolithis tinctorius 12 -0.038 Pisolithis tinctorius 12 0.167 Pisolithis tinctorius 16 -0.413 Pisolithis tinctorius 16 -0.064 Pisolithis tinctorius 20 -0.145 Pisolithis tinctorius 20 0.321 Pisolithis tinctorius 24 -0.037 Pisolithis tinctorius 24 -0.147 Pisolithis tinctorius 24 -0.019 Pisolithis tinctorius 24 0.000 Pisolithis tinctorius 36 -0.197 Pisolithis tinctorius 36 0.071 Pisolithis tinctorius 40 -0.103 Pisolithis tinctorius 40 0.174 Pisolithis tinctorius 60 0.012 Pisolithis tinctorius 60 0.629 Full citation of each study used in the meta-analysis examining the effects of phosphorus addition on the outcome of ectomycorrhizal associations. Study Ful l citation Bougher et al . 1990 Bougher NL , Grove T S , Malajczuk N. 1990. Growth and phosphorus acquisit ion of Karri {Eucalyptus diversicolor F-Muell) seedl ings inoculated with ectomycorrhizal fungi in relation to phosphorus supply. New Phytologist 114: 77-85 Browning & Whi tney Browning M H R , Whitney R D . 1991. Responses of jack pine and black spruce seedl ings to 1992 inoculation with selected spec ies of ectomycorrhiza fungi. Canadian Journal of Forest Resea rch 2 1 : 7 0 1 - 7 0 6 Burgess et a l . 1993 Burgess T l , Malajczuk N, Grove T S . 1993. The ability of 16 ectomycorrhizal fungi to increase growth and phosphorus uptake of Eucalyptus globulus Labill and E. diversicolor F. Muel l . Plant and Soi l 153: 155-164 C h e n et a l . 2000 C h e n Y L , Brundrett M C , Dell B. 2000. Effects of ectomycorrhizas and vesicular-arbuscular mycorrhizas, a lone or in competit ion, on root colonization and growth of Eucalyptus globulus and E. urophylla. New Phytologist 146: 545-556 Conjeaud et al . 1996 Con jeaud C , Sche romm P, Mousa in D. 1996. Effects of phosphorus and ectomycorrhiza on maritime pine seedl ings (Pinus pinaster). New Phytologist 133: 345-351 Study Full citation Grandcourt et al. 2004 de Grandcourt A, Epron D, Montpied P, Louisanna E, Bereau M, Garbaye J , Guehl JM . 2004. Contrasting responses to mycorrhizal inoculation and phosphorus availability in seedlings of two tropical rainforest tree species. New Phytologist 161: 865-875 Khasa et al. 2001 Khasa PD, Sigler L Chakravarty P, Dancik BP, Erikson L, McCurdy D. 2001. Effect of fertilization on growth and ectomycorrhizal development of container-grown and bare-root nursery conifer seedlings. New Forests 221: 179-197 MacFall etal. 1991 Macfali J , Slack SA, Iyer J . 1991. Effects of Hebeloma arenosa and phosphorus fertility on growth of red pine {Pinus resinosa) seedlings. Canadian Journal of Botany 69: 372-379 Tyminski et al. 1986 Tyminski A, leTacon F, Chadoeuf J. 1986. Effect of three ectomycorrhizal fungi on growth and . phosphorus uptake of Pinus silvestris seedlings at increasing phosphorus levels. Canadian Journal of Botany 64: 2753-2757 Walker 2001 Walker RF. 2001. Growth and nutritional responses of containerized sugar and Jeffrey pine seedlings to controlled release fertilization and induced mycorrhization. Forest Ecology and Management 149: 163-179 

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