Ectomycorrhiza of Populus

Forest Ecology and Management 347 (2015) 156–169

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Review

Ectomycorrhiza of Populus Agnieszka Szuba ⇑ Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland

a r t i c l e

i n f o

Article history: Received 2 November 2014 Received in revised form 28 February 2015 Accepted 5 March 2015 Available online 30 March 2015 Keywords: Poplar Ectomycorrhiza Research methods Research topics

a b s t r a c t Populus species and hybrids (poplars, aspens, cottonwoods, etc.) are important trees in forestry and landscaping, whereas Populus trichocarpa is an internationally accepted model organism for tree research. Populus roots often form symbiotic relationships with ectomycorrhizal partners, and such relationships improve the host tree’s general condition and stress tolerance. Indeed, symbioses are very important factors in poplar physiology and consequently have been a subject of frequent investigation. The aim of the presented paper is to give a background knowledge about the current status of research on poplar-specific ectomycorrhiza (ECM), to highlight the most commonly used methods as well as effects of ECM research on poplar cultivation. I focus also on predominant topics in poplar ECM research, such as diversity and specificity of ECM strains, nitrogen acquisition, transgenic poplar research and stress response. Finally I discuss the perspectives of further poplar ECM research. Ó 2015 Elsevier B.V. All rights reserved.

Contents 1. 2.

3. 4.

5.

Introduction: why poplars and their ectomycorrhizal fungi are amenable to symbiosis research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Most frequently analyzed symbiotic systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Environmental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Semi-controlled studies: from experimental field conditions to ex vitro experiments in climate chambers . . . . . . . . . . . . . . . . . . . . . . . 2.3. Sterile conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leading methods for analysis of ECM–poplar interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leading research topics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Diversity and specificity of ECM strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Fungus nitrogen assimilation and nitrogen transfer to plant partner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Research on transgenic poplar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Influence of ECM on stress response in poplars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Osmotic stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.3. Organic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.4. Biotic stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives—post-genomic research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: AM, arbuscular mycorrhiza; ECM, ectomycorrhiza; ITS, internal transcribed spacer.

157 157 157 157 161 161 163 163 164 165 165 165 166 166 166 166 167 167

⇑ Address: Laboratory of Proteomics, Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland. Tel.: +48 61 8170033; fax: +48 61 8170166. E-mail address: [email protected] http://dx.doi.org/10.1016/j.foreco.2015.03.012 0378-1127/Ó 2015 Elsevier B.V. All rights reserved.

A. Szuba / Forest Ecology and Management 347 (2015) 156–169

1. Introduction: why poplars and their ectomycorrhizal fungi are amenable to symbiosis research Members of the genus Populus (generally referred to as poplars, but including also aspens, cottonwoods, etc.) have become increasingly economically important in commercial markets, mainly in the timber, pulp, and paper industries. Poplars are also an important plant source material for energy production because they have relatively low nitrogen demand compared with other potential bioenergy crops (Somerville et al., 2010). In addition, poplars have rapid growth, simple vegetative propagation, and small genome size (450–550 Mbp; Taylor, 2002). The availability of the full genomic sequences of Populus trichocarpa (Tuskan et al., 2006) and of well-established in vitro propagation procedures have allowed poplars to be genetically engineered to create varieties with improved properties valuable for industry. Thus the combination of its biology, physiology, and genetics has established the poplar as the recognized model for tree researchers (Taylor, 2002). Poplars, like the majority of trees, interact with symbiotic fungi (Smith and Read, 2008; p. 194). Mycorrhizal fungi promote grown of poplars (Clark, 1963) by improving the tree’s ability to acquire water and nutrients, particularly under stressful environmental conditions. Ectomycorrhiza (ECM) provides poplars with phosphorous, potassium, nitrogen, and other mobilized nutrients from organic forms present in the soil that are inaccessible to trees, and in return the fungi receive photosynthetically derived carbohydrates (Baum et al., 2002; Desai et al., 2013; Gehring et al., 2006; Khasa et al., 2002; Langenfeld-Heyser et al., 2007; Quoreshi and Khasa, 2008). Mycorrhization is common in poplar and its hybrids (Table 1). For example, the mycorrhiza colonization rate of P. trichocarpa inoculated with ECM (Baum et al., 2002) was about 50% after 2 years and was as high as 100% in a second study (Danielsen et al., 2013). Poplars can form symbiotic associations with both arbuscular mycorrhizal (AM) fungi and ECM fungi (Smith and Read, 2008; p. 194). The ratio of ECM to AM varies in different poplar species and in its hybrids that are colonized by both types of fungi (Karlin´ski et al., 2010), but ECM is clearly dominant (Danielsen et al., 2012; Khasa et al., 2002). ECM fungi are usually dominant in poplar root structures even in soils in which saprophytic, pathogenic, or endophytic fungi are dominant (Danielsen et al., 2013), consequently being an important player in poplar management. In addition to poplar’s economic importance, poplar ECM fungi are also frequently studied because the genomic sequences of some such fungi are available, thereby facilitating genomic and post-genomic analyses. Complete genome sequences have been released for Laccaria bicolor (Martin et al., 2008; Martin and Nehls, 2009) and Tuber melanosporum (Martin et al., 2010). Moreover, some ECM fungi (e.g. even 50% of species associated with American aspen, Populus tremuloides; Cripps, 2001) can be easily cultivated in vitro separately from host roots as opposed to AM strains that cannot (Diop, 2003). Consequently, there are numerous multiplication techniques available for ECM cultivation including some that are under axenic conditions (for an excellent review see Müller et al., 2013).

2. Most frequently analyzed symbiotic systems Poplar ECM research may be categorized into different analysis types based on growth conditions, which vary by level of experimental control. Distinguishing between the different types of analysis is important because results differ depending on growth conditions (Dupae et al., 2014; Ma et al., 2008).

157

2.1. Environmental studies The least controlled types of studies are environmental analyses whose aim is often to determine the composition and/or the distribution of ECM strains in nature (Cripps and Miller, 1993; DeBellis et al., 2006) or in areas modified by anthropogenic activities such as planted poplar woods (Khasa et al., 2002) or various contaminated forested areas (Cripps, 2003; Krpata et al., 2008, 2009) (Table 1). These types of studies provide data on the ECM community structure and the principal species/classes of ECM (Cripps, 2001; DeBellis et al., 2006; Jakucs et al., 2005) as well as ECM population dynamics (Gryta et al., 2006; Kaldorf et al., 2004). Knowledge of the ECM species composition in nature is often the first step in more controlled analyses. For example, because native ECM species are typically the most beneficial for promoting poplar growth, i.e., as opposed to commercial inocula (Cripps, 2001; Table 1), and pose no danger of introducing dangerous, exotic species (Cripps, 2003; Meinhardt and Gehring, 2012), one must first determine the ECM species composition in nature. Environmental studies have also been performed to characterize the utility of poplar–ECM symbiosis for phytoremediation of polluted sites (Karlin´ski et al., 2010). The beneficial effect of ECMs on poplar growth in the natural restoration (Bent et al., 2011) as well as during afforestation of reclaimed and poor soils (Bois et al., 2005) are important aspects for poplar breeding potential. 2.2. Semi-controlled studies: from experimental field conditions to ex vitro experiments in climate chambers Breeding tests, which include e.g. experimental field research, should precede the establishment of large poplar plantations. In an experimental field study, it is possible to control selected growth conditions such as poplar genotype and/or released fungal genotype (Table 1) (Danielsen et al., 2012, 2013; Ma et al., 2008) as well as to limit pests/pathogens or soil nutrients using fungicides, pesticides, or fertilizers. In greenhouse studies, researchers also have the ability to alter factors such as water, humidity, nutrition, and lighting (Jentschke et al., 1999). Greenhouse studies are most frequently carried out in pot experiments, and in numerous cases they are often conducted simultaneously with field studies using soil obtained from the field analysis (Baum et al., 2002; Heslin and Douglas, 1986). Such semi-controlled field and pot greenhouse poplar experiments are often compared with each other to determine various ECM impacts, e.g. factors important for breeding, such as mycorrhization percentage or tree biomass increase (Table 1; Baum et al., 2002). Almost fully controlled, but not axenic conditions, were proposed by Jentschke et al. (1999) in the form of a sand culture. They established systems for cultivation of inoculated plants in PVC tubes filed with acid-washed quartz placed in an air-conditioned greenhouse with additional illumination and an automatic irrigation system. This arrangement allowed the researchers to control water and nutrient status and supply both at low and environmentally realistic concentrations (Jentschke et al., 1999). Even more control can be achieved in poplar ECM ex vitro experiments, which are studies performed in climate chambers that allow researchers to further manipulate experimental conditions. In these chambers, all external parameters are not only monitored but also controlled with great accuracy. Despite the lack of sterility of the entire system, most ex vitro experiments use some artificial sterilized ground medium such as sterilized sand (Ma et al., 2013) or peat and soil mixtures (Ma et al., 2008). These types of controlled conditions are also available in hydroponic cultures, which allow control of nutrient status even under greenhouse conditions (Müller et al., 2013).

Symbiotic partnersa

158

Table 1 Ectomycorrhizal partners of symbiosis with Populus species and hybrids. Cultivation method

Genetic analysisb

ECM colonization (% of roots)

Main aspect of analysis

Effect on host biomass

Reference

Characterization and identification of ECM of white truffles from natural samples Morphological-anatomical characterization and molecular identification of Tomentella sp. ECM abundance and host relationships Root hydraulic properties and poplar growth

NA

Kovács and Jakucs (2006) Jakucs et al. (2005)

ECM

Populus alba

White truffles (e.g. Tuber rapaeodorum, Tuber puberulum, Tuber rufum)

Forest

PCR

NA

Tomentella stuposa

Forest

PCR

NA

Populus balsamifera

Hebeloma crustuliniforme

NA

22.3%

Populus deltoides

Pisolithus tinctorius, Boletus speciosus, Boletus edulis, Laccaria amethystea, Rhizopogon luteolus, Scleroderma luteus, Leccinum scabrum, Xerocomus chrysenteron, Calvatia craniiformis (analysis also made for Populus symonii) Laccaria bicolor (P. deltoides as a mother of F1)

In vitro and ex vitro (pots filled with sterile sand) Pots

NA

25–65%

Selection of ECM in respect of growth-promoting effects on poplars

Pots

Quantitative trait loci (QTL)

Laccaria bicolor

In vitro

RT-PCR

10–61%, depending on genotype; average 35% 12%

Populus euphratica

Paxillus involutus

NA

NA

Populus maximowiczii

Laccaria amethystina, Hebeloma mesophaeum, Thelephora terrestris, Tomentella sp. Tricholoma populinum, Tricholoma scalpturatum 122 ECM species; Cenococcum geophilum was most frequent 54 species, rich in Basidiomycota (43 species) and dominated by Cenococcum geophilum and fungi with corticoid basidiomes (e.g. Thelephoraceae) Hebeloma crustuliniforme, Hebeloma cylindrosporum, Laccaria laccata, Laccaria proxima, Laccaria bicolor, Paxillus involutus Numerous ECM species, e.g. Phialocephala fortinii, Tomentella atramentaria, Pezoloma ericae, Thelephora terrestris or Russula spp. Amanita muscaria v. formosa, Amanita pantherina, Inocybe lacera, Piloderma croceum Over 50 ECM species, e.g. Paxillus vernalis, Tricholoma scalpturatum, Hebeloma mesophaeum, Thelephora terrestris or Laccaria spp. found in forest;

Ex vitro (hydroponic and pots filled with sterile rooting medium) Pots

Genetic analysis of phenotypic variation for ECM formation in an interspecific F1 poplar full-sib family Metabolomic responses to colonization Mechanisms of drought stress response

NA

38.8–63.1%

Natural sites (fruiting bodies) Forest

RAPD, ISSR and RFLP PCR

Forest (smelter surroundings)

PCR

In vitro

Populus nigra Populus tremula

Populus tremuloides

NA

No significant differences in shoot height and leaf area, but stem diameter greater in inoculated poplar Significant promotion of poplar growth (effect dependent on strain used) under symbiosis

Siemens and Zwiazek (2008) Ma et al. (2008)

NA

Tagu et al. (2005)

NA

Tschaplinski et al., 2014 Luo et al. (2009)

NA

ECM colonization and mechanisms of heavy metal stress response Pattern of colonization

Stem height, length of main roots and leaf biomass unaffected but root and stem biomass lower in inoculated poplars Seedling height and biomass greater in inoculated seedlings (effects differed between ECM species) NA

72.2 ± 15.6%

Species richness

NA

Fungal diversity and species richness

NA

NA

Not given as sum but as species abundance NA

Optimization of inoculation procedure

NA

Langer et al. (2008)

Forest

PCR

NA

Different impacts of various hostfungus combinations

Bent et al. (2011)

In vitro

NA

NA

Role of ECM (common mycorrhizal networks) in natural regeneration of forest after fire ECM mantle formation

Cripps and Miller (1995)

Forest and pots (sterile medium)

NA

up to 86% (pots)

Good condition of mycorrhized seedlings (except those inoculated with I. lacera). Plant survival differed between pottested strains and native strains (except I. lacera), causing better plant survival. All strains (except I. lacera)

Species richness/diversity of ECM (in forest) and inoculum potential (pots)

Obase et al. (2009) Gryta et al. (2006) Bahram et al. (2011) Krpata et al. (2008)

Cripps (2001)

A. Szuba / Forest Ecology and Management 347 (2015) 156–169

Tree

out of them, 6 native (Amanita muscaria, Amanita pantherina, Boletus piperatus, Inocybe lacera, Paxillus vernalis, Tricholoma scalpturatum) and 3 nonnative (Cenococcum graniforme, Piloderma croceum, Pisolithus tinctorius), all well grown in vitro, were tested in pots 30 species of native fungi, e.g. Laccaria proxima, Tricholoma flavovirens, Tricholoma populinum, Scleroderma cepa or Paxillus vernalis 26 morphotypes, with dominating Cenococcum geophilum and most common Piloderma sp., Russula sp. Cortinarius sp. and Lactarius sp. Laccaria bicolor

caused a significant increase (up to 430% of control) in total plant biomass

NA

NA

Species richness/diversity of ECM (fruiting bodies)

NA

Cripps (2003)

Forest, postfire location

PCR and RLFP sorting before sequencing

Not given as a sum of ECM families.

Fungal diversity and species richness

NA

DeBellis et al. (2006)

In vitro

NA

Differences in phosphorus acquisition

Small differences in growth responses but significant in P acquisition from low P ion treatments.

Desai et al. (2013)

Laccaria bicolor

In vitro

Prediction of ectomycorrhizal metabolome

NA

Larsen et al. (2011)

Hebeloma crustuliniforme

Styroblocks containing sterile silica sand

RT-PCR: RNA deep sequencing NA

71–79% (increased with decreasing P availability) NA

22–51.6% of root length

Nitrogen assimilation

Siemens et al. (2011)

Laccaria bicolor, Paxillus involutus

Pots

NA

14–40%

Laccaria laccata

Pots and experimental field

NA

In vitro

RT-PCR

Inoculation by L. laccata caused significant increases in shoot length (but only after first year of experiment) NA

Baum et al. (2002)

Laccaria bicolor

6–53%, depending on variant 44%

Interactive effects of substrates and mycorrhization on poplar growth Poplar growth response to inoculation

In most treatments no differences in leaf DW and root DW (only under 8 mM NO3 treatment, roots of inoculated plants significantly smaller) Shoot biomass increased under ECM symbiosis.

Populus alba  glandulosa

Pisolithus tinctorius

Pots

NA

68% (initial percentage of root colonization)

Populus alba  grandidentata

50 species of ECM fungi with domination of Cortinariaceae family (symbiosis with wild and GM poplar) 186 described fungal families in soil (23% ECM)/vs 115 families in roots (87% ECM) in symbiosis with wild and GM poplars 30 ECM species, e.g. Peziza ostracoderma, Paxillus involutus, Hebeloma sp., Geopora sp., Laccaria tortilis, Tomentella ellisii (in symbiosis with wild and GM poplar)

Experimental field/plantation

PCR and RLFP

NA

Experimental field/plantation

454 pyrosequencing (target: ITS region) PCR and DGGE

Not given as a sum of ECM families

Laccaria bicolor

In vitro

Populus trichocarpa

Populus alba  tremula (Populus  canescens)

Experimental field/ Plantation

Oligoarraybased transcript profile

58–86% after first year and almost 100% after second year of growth NA

Metabolomic responses to colonization Mechanisms of heavy metal (Cd+2) stress response

Impact of an 8-year-old transgenic poplar plantation on ECM community Fungal diversity and species richness. Comparison between analysis of soil and roots

DW of leaves, stems and roots did not differ significantly between mycorrhized and non-mycorrhized plants. However, losses of total DW, observed under Cd stress, were bigger in mycorrhized poplars (17.1% vs 18.5%) NA

Baum et al. (2002)

Tschaplinski et al. (2014) Han et al., 2011

A. Szuba / Forest Ecology and Management 347 (2015) 156–169

Forest (smelter surroundings)

Stefani et al. (2009)

NA

Danielsen et al. (2012)

ECM effect on poplar nutrition status as well as ECM colonization and community composition investigation

Stem biomass positively related to degree of ECM root tip colonization

Danielsen et al. (2013)

Analysis of mechanisms of stimulated lateral root formation under mycorrhizal symbiosis

NA

Felten et al. (2009) 159

(continued on next page)

160

Table 1 (continued) Symbiotic partnersa Tree

Cultivation method

Genetic analysisb

ECM colonization (% of roots)

Main aspect of analysis

Effect on host biomass

Reference

Paxillus involutus

In vitro

NA

NA

NA

Paxillus involutus

Ex vitro (axenic medium)

NA

39–40%

Analysis of ECM formation in context of H2O2 production Analysis of effect of ECM on Na+responses of poplar

Paxillus involutus

In vitro

NA

NA

Better whole plant conditions under symbiosis

Paxillws involutus

Ex vitro (axenic medium)

RT-PCR: whole genome poplar microarray

up to 63%

Mechanisms of NaCl stress response under ECM symbiosis (K+/Na+ homeostasis) Mechanisms of salt stress response

Gafur et al. (2004) LangenfeldHeyser et al. (2007) Li et al. (2012)

Paxillus involutus

RT-PCR

61.5–65.2%

PCR and RLFP

up to 50%

NA

Not given as% values

ECM

Laccaria sp., Pezizales sp, Tuber sp.

Populus deltoides  [P. laurifolia  P. nigra]

Pisolithus tinctorius

Populus nigra  deltoids (Populus  canadensis)

Paxillus involutus

Pots

NA

5%

P. nigra  maaximowiczii

Geopora cervina, Tuber rufum, Laccaria laccata, Laccaria apethystina, Tomentella cinerascens, Tomentella subtestacea Amanita muscaria

Experimental field

PCR

6–81%

Impact of poplar rotation periods on ECM community

In vitro

RT-PCR

NA

Amanita muscaria (symbiosis with wild and GM poplar)

In vitro

NA

Not given as% values

Leccinum populinum (symbiosis with GM poplar)

In vitro

RT-PCR

23 observed ECM morphotypes; 90% of them formed by Cenococcum geophilum, Laccaria sp., Phialocephala fortinii, Thelephoraceae sp., Pezizales sp. (symbiosis with wild and GM aspen) Laccaria bicolor (with GM poplar)

Experimental field

PCR and PCRRLFP

In vitro

RT-PCR

4.2–37% depending on transgenic line Not given as a total% (but as abundance of particular morphotypes) about 45%

Activity of poplar monosaccharide transporter Documenting of mycorrhizal associations between ECM and GM poplar Role of plant hemoglobin under ECM symbiosis

Amanita muscaria

In vitro

Amanita muscaria

In vitro

RT-PCR and massive EST analysis PCR and transformation of Amanita muscaria

Populus tremula  tremuloides

Mechanisms of heavy metal (Cd2+) stress response Inoculum potential on revegetated tailing sands from Canadian oil sand industry Impact of ECM on poplar remediation of dieselcontaminated soil ECM colonization and mechanisms of heavy metal stress response

Luo et al. (2009a)

Belowground and aboveground biomass was unaffected, but ECM colonization changed root architecture and protected fine roots against saltinduced biomass loss. ECM resulted in positive effects on poplar growth Shoot:root ratio positively correlated with ECM fungal colonization

Ma et al. (2013) Bois et al. (2005)

ECM increased significantly plant biomass in control and treated plants

Gunderson et al. (2007)

Stem biomass production highest in poplars mycorrhized with P. involutus. Root biomass not significantly greater under symbiosis NA

Sell et al. (2005)

NA NA

Hrynkiewicz et al. (2010) Grunze et al. (2004) Hampp et al. (1996)

Increased root FW under symbiosis

Jokipii et al. (2008)

ECM invasion strategies

NA

Kaldorf et al. (2004)

Role of phytohormones

NA

NA

Molecular mechanisms of nitrogen assimilation

NA

NA

Investigation of horizontal gene transfer

NA

Plett et al. (2014) Willmann et al. (2007, 2014) Zhang et al. (2005)

A. Szuba / Forest Ecology and Management 347 (2015) 156–169

Populus deltoides  nigra

Ex vitro (axenic medium) Sterilized soil cores cultivated after inoculation in controlled conditions Pots

Greater total biomass of mycorrhized poplars

Pots Hebeloma mesophaeum Populus trichocarpa  deltoides

ECM = ectomycorrhiza; GM = genetically modified poplar; NA = not analyzed; a References were used only when symbiotic partners were clearly defined. b Leading genetic methods used: DGGE = denaturing gradient gel electrophoresis; EST = expressed sequence tag; ISSR = inter-simple sequence repeat; PCR = polymerase chain reaction (in a majority of reports, PCR was made on the ITS region) and subsequent sequencing of PCR products; RAPD = random amplified polymorphic DNA; RFLP = restriction fragment length polymorphism; RT-PCR = reverse transcription polymerase chain reaction.

Pfabel et al. (2012) Inoculation had no effect on total aboveground biomass of poplars 26 vs 17% (control vs rust)

Impact of ectomycorrhizal colonization and rust infection on secondary metabolism of poplar

Plett et al. (2014) In vitro Laccaria bicolor Populus tremula  alba

Poplar transformation and RT-PCR: oligoarray – based transcript profile NA

about 60%

Role of phytohormones

NA

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The majority of semi-controlled experiments are designed to identify the impact of inoculation on poplar growth (Table 1); most such experiments have shown that, under ECM symbiosis, the development of poplar shoots (Baum et al., 2002) and roots is enhanced (Meinhardt and Gehring, 2012; Navratil and Rochon, 1981), especially in the initial phase of the experiment (Baum et al., 2002). Evaluation of host growth response is a key element of field experiments, in which the goal is to select the best symbiotic system (Baum et al., 2002; Ma et al., 2008). Poplar ECM selection is one important issue because poplars as well as ECM fungi vary in their ability to form mycorrhiza or to respond to symbiotic partner presence (Ma et al., 2008; Navratil and Rochon, 1981; Obase et al., 2009). Selection is particularly important in arable soils, which are characterized by low content of indigenous ECM inoculums and high content of soil-borne competitive pathogens (Baum et al., 2002). 2.3. Sterile conditions In vitro studies allow researchers to control almost all factors in an experimental setting and minimize variation in temperature, humidity, and isolation as well as know with certainty the composition of the symbiotic system being analyzed. The data from such tightly controlled experiments allows researchers to explore the molecular mechanisms involved in ECM–poplar symbiosis, which may contribute to the improvement of future management procedures. Despite the challenge of extrapolating the results/conclusions to natural conditions (Dupae et al., 2014), in vitro conditions allow researchers to assess transcription of a particular gene (Kemppainen et al., 2009; Kemppainen and Pardo, 2013; Selle et al., 2005; Willmann et al., 2007) or homology (Plett et al., 2011). One such axenic study was that of Cripps and Miller (1995), who described previously unreported symbioses between quaking aspen (P. tremuloides) and Inocybe lacera and Amanita pantherina. There are many established techniques for the propagation of ECMs in poplar under aseptic conditions (Cripps and Miller, 1995; Heslin and Douglas, 1986; Peterson and Chakravarty, 1991). Examples of fully axenic procedures used in poplar mycorrhizal studies include hydroponic (Luo et al., 2009) as well as sand cultures that use washed sterile silica sand in tissue culture vessels (Siemens and Zwiazek, 2008) or in square Petri dishes (Müller et al., 2013). The use of sand as a support medium results in root development that is closer to natural conditions. However, most in vitro studies that involve mycorrhized poplar propagation have been based on use of agar medium. Inoculated poplars can be grown on agar in glass jars (Heslin and Douglas, 1986), but most experiments have used a Petri dish system (Gafur et al., 2004; Hampp et al., 1996; Langer et al., 2008; Peterson and Chakravarty, 1991; Selle et al., 2005; Willmann et al., 2007). In the Petri dish method, plant roots are spread on the agar medium in the Petri plate and secured with parafilm while the stems are located outside the plate. Usually, during aseptic conditions, the hyphal mantle and Hartig net (see below, Section 3) are well developed within 3–6 weeks (Hampp et al., 1996; Szuba, unpublished data). 3. Leading methods for analysis of ECM–poplar interactions Concerning source materials, environmental ECM–poplar studies usually rely on randomly collected soil cores (Fig. 1) (Danielsen et al., 2013; Karlin´ski et al., 2010) or fruit bodies collected under poplar trees (Gryta et al., 2006; Healy et al., 2013; Krpata et al., 2009). Nevertheless, only root tip tests can guarantee that detected ECM species are indeed symbiotic partners with a particular poplar

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A Root sample

Soil cores

Root p samples Soil samples Morphotyping RNA isolaon DNA isolaon

PCR cDNA synthesis; Transcriptome analysis Sequencing

Idenficaon

B Analysis paths

References

Danielsen et al., 2012 EF;*; Stefani et al., 2009 EF

Bois et al., 2005 P; Danielsen et al., 2013 EF and 2012 EF; DeBellis et al., 2006 F Jakucs et al., 2005F; Kovács and Jakucs, 2006 F; Krapta et al., 2008 F; Stefani et al., 2008 EF Hrynkiewicz et al., 2010 EF; Bahram et al., 2010 F; Bent et al., 2011 F; Ding et al., 2011 P; Kaldorf et al., 2002 EF** and 2004EF Felten et al., 2014 IV; Jokipi et al., 2008 IV; Grunze et al., IV; Larsen et al., 2011 IV; Luo et al., 2009a EV; Ma et al., 2013 EV; Plett et al., 2014 IV; Willmann et al., 2007IV and 2014 IV; Tschaplinski et al., 2014 IV Fig. 1. (A) Leading paths of genetic analysis performed during poplar ectomycorrhiza research. Leading paths of genetic analysis for particular cultivation procedures were additionally indicated by red (for experimental field studies and forest experiments) and blue background (for in vitro and ex vitro experiments). (B) Examples of reports where the paths were utilized, with poplar cultivation methods marked as superscripts: F = forest analysis; EF = experimental field study; P = pot experiment; EV = ex vitro experiment; IV = in vitro experiment. ⁄ = report without identification of PCR products (only PCR-RLFP); ⁄⁄ = pyrosequencing.

host, and indeed root tips from soil cores are most often analyzed (Fig. 1B) (Danielsen et al., 2012; Karlin´ski et al., 2010; Krpata et al., 2009). However, there are also reports of cleaned roots analysis (Kaldorf et al., 2002) as well as direct soil analysis, showing whole fungal composition in the poplar rhizosphere (Fig. 1) (Danielsen et al., 2012, 2013; Stefani et al., 2009). ECM communities that are recovered from root tips differ substantially from those identified by soil cloning analyses, which suggests that these approaches

for collection of research material are complementary and together are sufficient to fully document ECM fungal diversity (Stefani et al., 2009). The presence of a fungal partner causes morphological changes in root architecture. Mycorrhizal roots are usually shorter and thicker, without root hairs (Müller et al., 2013), and form structures specific for particular fungal partners (http://www.deemy. de/; Peterson et al., 2004, p. 15) (Fig. 2B and D); notably, such structures may be massive (Fig. 2C). These changes in the host root are a consequence of mantle formation (Fig. 3B), a hyphal layer covering the surface of the fine roots, which are responsible for penetrating the surrounding soil and absorbing nutrients. The most characteristic feature of the ECM structure-penetrated mycelia is the Hartig net (Fig. 3B; arrowheads)—a single-layer-thick hyphal network within the plant root cortex (Peterson et al., 2004; pp. 7–42; Smith and Read, 2008; pp. 191–268) that constitutes the surface that mediates a greatly increased rate of solute exchange and is considered a marker for establishment of a functional mycorrhiza (Hampp et al., 1996). These morphological changes, which can be used to identify ECM strains (Kaldorf et al., 2002; Ma et al., 2008), are estimated with light microscopy and in some cases with additional cotton blue staining of chitin in the fungal cell wall (Karlin´ski et al., 2010). Morphotyping is usually the first step of ECM identification (Fig. 1) because mycorrhizal anatomy alone (characteristics such as mantle structure, cell wall thickness, shape and size of cystidia, or fruit body) is insufficient to distinguish ECM groups (Kovács and Jakucs, 2006). Conclusive determination of ECM groups for selected morphotypes, however, is provided by genome analysis (Fig. 1), because results of morphological estimations may differ from the genetic analysis results (DeBellis et al., 2006; Kovács and Jakucs, 2006). The use of genetic deep sequencing provides information previously unavailable, such as unexpected enzyme composition or large numbers of ECM-induced transcripts that encode unknown proteins. Moreover, use of molecular genetics tools has allowed us to better understand the patterns, ecological role and evolutionary mechanisms involved in development of symbiotic ECM–poplar associations (Martin and Nehls, 2009). Genetic analysis of ECM–poplar symbiosis has been dominated by PCR approaches. For amplification of rDNA fragments such as the partial large subunit (LSU), nuclear rDNA (Healy et al., 2013) is usually selected. In the early 1990s, however, PCR reactions of ECM fungi were directed primarily toward analysis of internal transcribed spacer (ITS) regions within rDNA (Fig. 1) (Bent and Taylor, 2010; Danielsen et al., 2013; Gardes et al., 1991; Kaldorf et al., 2004). The most frequently used primers are ITS1 or ITS1-F and ITS4 (Bent et al., 2011; Danielsen et al., 2012; Krpata et al., 2008; Stefani et al., 2009). DNA analysis of poplar ECM has also included the use of restriction fragment length polymorphisms to count the unique types of strains (DeBellis et al., 2006; Gehring et al., 2006; Gryta et al., 2006) as well as analysis of nuclear loci to establish intercontinental ECM divergence (Grubisha et al., 2012) and to evaluate the ability of poplar trees to form ECMs (Tagu et al., 2005). Under controlled conditions (Fig. 1 and Table 1), in addition to standard PCR analyses, real-time PCR or more advanced transcriptome analysis, such as oligo-array – based transcript profile research, has been used to understand the molecular mechanisms of ECM influence on poplar (Table 1), including nitrogen acquisition (Willmann et al., 2014; within the framework of a new big EST project of Populus tremula  tremuloides/Amanita muscaria), carbohydrate transport (Grunze et al., 2004), hormone cross-talk (Plett et al., 2014) or lateral root formation (Felten et al., 2009). Transcriptome-level changes were examined also for inoculated poplars growing in stress conditions, e.g. heavy metal (Ma et al., 2013) or salt stress (Luo et al., 2009a) and next-generation short-read transcriptome sequencing data from

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Fig. 2. Various forms of ectomycorrhizal root tips formed in vitro with Populus  canescens micro-cuttings cultivated in an agar medium. (A) Control roots; (B) Hebeloma spp. ectomycorrhiza; (C) Paxillus involutus mycorrhiza; massive occurrence of mycorrhizal tips are visible; (D) Single poplar root tips colonized by P. involutus; shortened, thickened roots are seen with no visible hairs. Photo: A. Szuba, L. Karlin´ski.

E C

A M

En

H

E C En

B

M

Fig. 3. Populus  canescens root cross-sections. Microscopy images of fixed poplar control roots (A) and roots mycorrhized with Paxillus involutus (B). E = epidermis; En = endodermis; C = cortex cells; M = fungal mantle; H = Hartig net. Scale = 100 lm. Photo: J. Mucha and L. Karlin´ski.

RNA deep sequencing have been used to understand the symbiotic interactions for generation of a model of the mycorrhizal metabolome (Larsen et al., 2011). Finally, genetics has been used to produce engineered symbiotic partners (Hampp et al., 1996). Both poplar and ECM fungi have been genetically engineered, although poplar transformants are more frequent (see Section 4.3.). However, RNA silencing of fungi genes has been used in studies of nitrate reductase from L. bicolor (Kemppainen et al., 2009).

4. Leading research topics 4.1. Diversity and specificity of ECM strains In environmental studies (Table 1) or even in pot analyses, one of the leading research areas is the exploration of species composition and diversity of poplar-specific mycorrhizal fungi. Numerous ECM species are associated with those trees. Moreover, one individual poplar may potentially host hundreds

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of different ECM species (Bahram et al., 2011; Cripps and Miller, 1993; Krpata et al., 2008). Thus even non-artificially inoculated young poplar plantations, including transgenic ones, constitute a rich reservoir of symbiotic fungi (Danielsen et al., 2012), yet usually only a few of the detected ECM fungi clearly dominate all others (Kaldorf et al., 2004). Poplar ECMs may be distinguished based on whether they are early-stage colonizers such as I. lacera and L. laccata, which generally reside in nutrient-poor soil, or late-stage fungi such as A. muscaria and L. controversus (Cripps and Miller, 1993). Additionally, poplar genotype has been shown to significantly influence the colonization ability of distinct ECM, and more genetically diverse poplar populations show increased variation in the ECM community composition (Danielsen et al., 2013). ECM abundance and spatial distribution are also connected with proximity to neighboring forests (Bahram et al., 2011; Danielsen et al., 2012) because the major proportion of ECM fungi have unrestricted host ranges and can enter into symbiosis with multiple plant species (Bent et al., 2011; Hedh et al., 2009). For this reason, extensive monocultures of poplars may have decreased the potential impact of native ECM (DeBellis et al., 2006). Host preference has a substantial role in the structure of the ECM fungal community (Ding et al., 2011). Changes in host range occur frequently and are independent of other ECM strains present (Hedh et al., 2009). Variation of host choice is associated with variance in nucleotide sequences of symbiosis-regulated genes (Le Quéré et al., 2006) and with quantitative differences in fungal gene expression (as showed for birch; Le Quéré et al., 2004). Even in particular species known to form mycorrhiza with poplar, such as the frequently analyzed Paxillus involutus (Ma et al., 2013), not all isolates are competitive (Gafur et al., 2004). Moreover, gene expression varies among strains (Le Quéré et al., 2004), and metabolomic response varies in aspects such as H2O2 production (Gafur et al., 2004) following contact with poplar roots. In addition, fungal impact may vary depending on the host species and may even act in an opposite manner—for example, either promoting or inhibiting tree growth depending on the host-fungus combinations (Bent et al., 2011). Finally, in addition to host genetic factors, also management procedures, e.g. timing of rotation in Short Rotation Coppice management systems or of fertilizer application, affect mycorrhiza frequency (Hrynkiewicz et al., 2010) or ECM influence on host growth (Quoreshi and Khasa, 2008). Additionally, environmental factors, such as season (Hrynkiewicz et al. 2010), elevation, soil type (Baum et al., 2002; Cripps and Miller 1993), and especially soil moisture (Gehring et al., 2006), affect mycorrhizal colonization of poplar plants (Gehring et al., 2006). Notably, soil characteristics and depth influence the ratio of ECM and AM colonization (Gehring et al., 2006; Karlin´ski et al., 2010). Due to such a high ecological plasticity of poplar–ECM interactions (Karlin´ski et al., 2010), artificial inoculation (if planned, usually before planting of rooted seedlings) should be controlled, especially in former agricultural, abandoned and degraded areas (Khasa et al., 2002). 4.2. Fungus nitrogen assimilation and nitrogen transfer to plant partner One the most important benefits of mycorrhizae symbiosis to the poplar tree is delivery of extra nitrogen to plant cells (Willmann et al., 2007). For this reason, ECM influence on tree nitrogen nutrition is a potential important player in management of poplars growing on N-deficient substrates (Obase et al., 2009) as well as in areas with elevated nitrate/nitrite concentration (Willmann et al., 2014). Moreover, there is evidence that nitrogen utilization under ECM symbiosis may positively affect wood production (Danielsen et al., 2013). Improvement of nitrogen

acquisition by ECM fungi is more efficient then by AM fungi (Averill et al., 2014). Nitrogen is an essential component of the global terrestrial carbon cycle (Averill et al., 2014), and therefore issues related to nitrogen metabolism and translocation during ECM symbiosis have been relatively well studied in crop trees. ECM fungi associated with poplar may use different nitrogen sources such as nitrate, NH+4, urea (Guidot et al., 2005; Morel et al., 2008), di- and tripeptides (Benjdia et al., 2006), and protein (Guidot et al., 2005). Additionally, the types of EMC strains that are present differ depending on their preferred nitrogen source(s) (Guidot et al., 2005). In forested ecosystems, the types of nitrogen source provided by fungi are mainly soil protein, amino acids (Nuutinen and Timonen, 2008), amino sugars, or heterocyclic N molecules, usually in the form of recalcitrant organic matter complexes (Nannipieri and Eldor, 2009). Processes that disrupt recalcitrant organic matter complexes in poplar ECM are relatively poorly understood. However, P. involutus transcriptome profiling during the degradation of the organic matter has shown that, when protein is released from these complexes, the fungi partially degrade polysaccharides and modify polyphenols in a process involving Fenton chemistry during hydroxyl radical attack in a manner similar to that of brown-rot fungi (Rineau et al., 2012). In contrast to wood-decay fungi, P. involutus is unable to metabolize the resulting compounds because they lack the appropriate enzymes. During the disruption process, increased activity of fungal peptidases having an acidic pH optimum is detected, including activation of numerous extracellular endo- and exopeptidases (Shah et al., 2013). This process is induced by protein substrates and partially repressed by NH+4-inorganic nitrogen sources (Shah et al., 2013). Upregulation of the secreted proteases also has been demonstrated in L. bicolor but only in the presence of the poplar partner (Martin et al., 2008). The next enzyme important for nitrogen mineralization from soil amino acids during ECM symbiosis is fungus L-amino acid oxidase, which catalyzes the oxidative deamination of the alpha-amino group of L-amino acids (Nuutinen and Timonen, 2008). The breakdown and mobilization of nitrogen from organic complexes, as well as the transcription of nitrogen transporter genes, are strongly activated by carbon availability (Rineau et al., 2013). The host partner is therefore an important regulator of nutrient absorption by fungi and may control the supply of carbohydrates, e.g. if mineral supply is insufficient and sugar-flux back into root cells may occur (Grunze et al., 2004). Moreover, there is evidence that the host may be able to monitor its nitrogen-nutrition status and avoid unnecessary fungal interactions—for example during times when nitrate is readily available. Such inhibition of mycorrhization is reversed when an organic nitrogen source is accessible only to the fungus (Kemppainen et al., 2009), revealing the crucial role of the nitrogen source in establishing ECM symbiosis. Further, the silencing of LbNrt—the sole nitrate-transporter–encoding gene of the fungus—was found to substantially decrease poplar growth capacity on nitrate and thereby alter the symbiotic interaction with poplar (Kemppainen and Pardo, 2013), illustrating the important role of fungi during the establishment of poplar ECM. Indeed, symbiosis improves nitrogen assimilation ability of both partners, as measured in a 15N-labeled analysis of P. tremuloides seedlings inoculated with Hebeloma crustuliniforme (Siemens et al., 2011). This experiment showed that ectomycorrhized poplars assimilated nitrogen more efficiently than nonmycorrhized trees owing to the relatively higher efficiency of the combined enzymatic responses to NH+4 by the fungi and NO3 by the host tree (Siemens et al., 2011). Ammonium seems to be the major nitrogen source delivered by the fungus to the poplar in ECM symbiosis (Selle et al., 2005; Willmann et al., 2007), although poplars have low tolerance to NH+4 toxicity (Min et al., 1999). In the roots of P. trichocarpa, 14

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putative NH+4 transporters have been identified (Couturier et al., 2007), considerably more than in Arabidopsis thaliana, a plant that does not form mycorrhizal symbiosis partnerships. It has been speculated that ECM may cause physiological changes in aspen NH+4 tolerance by altering plant enzymatic processes (Siemens et al., 2011). Mycorrhiza formation has also been shown to strongly increase expression of fungal high-affinity NH+4 transporters (Couturier et al., 2007), and genes encoding the putative NH+4 importers have been identified in fungi (Javelle et al., 2001, 2003; Küster et al., 2007; Willmann et al., 2007). Moreover, genes that are typically induced by nitrogen starvation are suppressed by high nitrogen availability (Javelle et al., 2001, 2003). 4.3. Research on transgenic poplar Low levels of lignin in wood are advantageous when the wood is used for industrial purposes (Danielsen et al., 2013; Pilate et al., 2002), and therefore it is common for poplars to be genetically modified to alter the activities of enzymes of the lignin biosynthetic pathway such as downregulation of cinnamoyl coenzyme A reductase, caffeic acid O-methyl transferase, or cinnamoyl alcohol dehydrogenase (Danielsen et al., 2013). Transgenic poplars are utilized mainly for the mass planting of trees for ultimate conversion to biomass, and changes in the composition of phenolic compounds may affect root interactions with microbes (Danielsen et al., 2013). Hence, the potential environmental impact of genetically modified trees is a valid, longstanding issue for poplar management, especially because in other tree species it has been recorded that genetically modified trees can trigger unforeseen, pleiotropic effects on the local ECM community (Hoenicka and Fladung, 2006). Consequently, one of the most frequently analyzed issues is the impact of transgenic manipulation on ECM composition in experimental fields (Danielsen et al., 2013; Stefani et al., 2009). The majority of such studies have shown no major differences in ECM colonization between wild-type and transgenic poplars in both short-term (Kaldorf et al., 2002) and long-term experiments (8-year-long investigation; Stefani et al., 2009). However, some differences have been reported between wild-type and transgenic poplars, especially after longer periods, such as differences in ECM community composition or abundance and development of ECM morphotypes. Those differences were similar to those observed between different hybrid poplars (Danielsen et al., 2013) and were explained by the authors as being caused probably by circumstances such as the clone-specific effect (Kaldorf et al., 2002) or secondary effect of draftism caused by the construct (35S::rolC) used for manipulation (Hoenicka and Fladung, 2006) and not a direct result of genetic manipulations. Effects of poplar gene modification on mycorrhiza formation have also been examined in axenic conditions (Hampp et al., 1996). In these studies, no differences were detected in the formation or morphology of the symbiotic interaction between A. muscaria and transgenic aspens that had been modified to have an altered indoleacetic acid balance. The danger of horizontal gene transfer has been analyzed in transgenic poplars (reviewed in: Nehls et al., 2006) and such knowledge is especially important for poplar breeding politics. DNA stability and risk of transgenic DNA uptake from decomposed leaves in a natural forest environment was examined over a decade ago and showed a rather low danger of engineered DNA contamination in non-transformed species (Hay et al., 2002). However, ECM symbiotic fungi are in close contact with host root cells, and consequently when these cells degenerate and host DNA is released, the fungus potentially can take up the DNA. Studies of possible horizontal DNA transfer to the ECM partner are motivated by documented horizontal transfers between other fungi and plants. For example, the phytopathogenic fungus Plasmodiophora

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brassicae (Bryngelsson et al., 1988) and saprophyte Aspergillus niger (Hoffmann et al., 1994) may take up host DNA from dead plant material. Studies to date have been mixed, but overall they have confirmed that the risk of horizontal DNA transfer during ECM–poplar symbiosis is low. For example, poplars carrying the gene rolC from Agrobacterium rhizogenes were co-cultivated in vitro with ECM ascomycete Phialocephala fortinii for 12 weeks, after which the fungal hyphae (exclusive of host material) were examined and found to contain no rolC signals (Kaldorf et al., 2004). Another example involved transgenic aspen poplar that expressed the herbicide resistance gene, bar, from Streptococcus hygroscopicus, that were mycorrhized with wild A. muscaria in an axenic Petri dish system and co-cultivated. In the 35,000 ectomycorrhized aspen samples that were tested, researchers found no evidence of horizontal gene transfer (Zhang et al., 2005). In poplar species, genetic-engineering-related work has been carried out on also the prokaryotic hemoglobin gene, VHb (heterologous Vitreoscilla hemoglobin gene), from the Gram-negative bacterium Vitreoscilla sp. In this experiment, VHb transgenic hybrid aspen were used to investigate the role of plant hemoglobin during symbiotic interactions (Jokipii et al., 2008). The study found that endogenous plant hemoglobins may be involved in early growth responses caused by ECM fungi and that VHb may compensate for the function(s) of the endogenous hemoglobins (Jokipii et al., 2008). Poplar genes have also been used to genetically modify other ECM trees. For example poplar’s caffeate/5-hydroxyferulate Omethyltransferase genes (PtCOMT; causes a decrease in the lignin syringyl/guaiacyl composition ratio; Sutela et al., 2009) were used to genetically alter silver birch (Betula pendula Roth). This study investigated whether potential changes in the repertoire of phenolic compounds could influence the formation of ECM with P. involutus. Similar to the findings for poplar trees, no significant effect was seen with respect to the composition or quantity of phenolic compounds, and thus the researchers concluded that genetic modification did not affect the fungi-host interaction. 4.4. Influence of ECM on stress response in poplars One of the leading topics of current poplar ECM research concerns our understanding of the physiological and molecular mechanisms responsible for the commonly observed enhanced poplar tolerance to various stress conditions in the presence of mycorrhiza (Li et al., 2012). 4.4.1. Osmotic stress Lack of water is one of the biggest problems in many parts of the world. Mycorrhizal poplar roots are characterized by increased water transport capacity (Marjanovic´ et al., 2005) and higher root hydraulic conductivity (Siemens and Zwiazek, 2008) compared with non-inoculated poplar roots. When water is scarce, symbiosis may facilitate water uptake by increasing the expression of aquaporins (Marjanovic´ et al., 2005a). Langenfeld-Heyser et al. (2007) showed that, although mycorrhization does not suppress salt-induced oxidative stress, but the mycelia growth is not affected by 150 mM NaCl; moreover, the ECM has positive effects on the host by increasing plant biomass and decreasing Na+ accumulation in poplar leaves. Furthermore, in roots under salt stress, ECMmediated remodeling of ion flux helps to maintain K+/Na+ homeostasis by increasing the release of Ca2+ (Li et al., 2012). ECM also activates genes related to abiotic and biotic stress responses as well as genes involved in vesicle trafficking (Luo et al., 2009a). Finally, ECM changes poplar phytohormone balance during salt stress. The presence of P. involutus increases the level of abscisic

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acid and salicylic acid and decreases the level of jasmonic acid and auxin in poplar roots compared with non-mycorrhizal roots (Luo et al., 2009a). 4.4.2. Heavy metals ECM improves the condition of stressed plants by improving nutrient status (Ma et al., 2013). It is well documented that ECM increases a tree’s defense processes via production of extracellular exudates as well as by binding heavy metal ions to the fungal cell wall or sequestering the ions in the fungal cytosol and/or vacuoles (Luo et al., 2014; Smith and Read, 2008 pp. 375–378). Mycorrhiza are thus an important player in phytoremediation of heavy metals, an environment-friendly and low-cost procedure of metal detoxification (Sell et al., 2005). Moreover, poplars are important for the forestation of smelter sites because they are fast-growing early successional tree species (Cripps, 2003; Sell et al., 2005). However, the impact of a heavy metal on ECM development (Harris and Jurgensen, 1977) and action (Krpata et al., 2009) may depend on the particular experimental conditions. For example, ECMs enhance poplar tolerance to Cd2+ (Ma et al., 2013), but Krpata et al. (2009) showed that the ECM does influence the level of cadmium accumulation in poplar trees; in fact, ECMs may facilitate even higher Cd2+ uptake in roots (Han et al., 2011; Ma et al., 2013) and leaves (Sell et al., 2005) compared with non-mycorrhizal plants. Ma et al. (2013) explained this as a consequence of enlarged ECM roots, but Han et al. (2011) showed that ECM trees under Cd2+ stress are characterized by decreased root dry weight. Effects of ECM on oxidative response again differ between various reports; according to Ma et al. (2013), symbiosis alleviates oxidative stress in the presence of Cd2+, whereas Han et al. (2011) showed that superoxide dismutase activity and thiol content are not changed in ECM plants compared with non-ECM plants. One proposed reason for these differing results is that the strains from contaminated sites are adapted to be more efficient for phytoremediation (Colpaert et al., 2011). However, other studies have found no beneficial influence of strains from contaminated areas (Jean-Philippe et al., 2011). Heavy metals also usually cause ECM reduction. Soil contamination with some heavy metals, such as copper, lead to reduction of poplar root tip abundance, mycorrhizal colonization ratio, and soil fungi biomass and an increase in non-mycorrhizal fungal endophyte levels compared with non-polluted soils (Karlin´ski et al., 2010). According to some reports, soils polluted by copper mines have no mycorrhiza, and addition of an extract of natural forest soil to such copper-contaminated soils fails to initiate symbiosis. Interestingly, however, iron mine tailings have been shown to develop extensive ECM (Harris and Jurgensen, 1977). 4.4.3. Organic compounds ECM has been shown to be ineffective at removal of total petroleum hydrocarbons from soil (Gunderson et al., 2007). Infection with ECM fungi may, however, benefit biomass as evidenced by increased fine root production as well as nitrogen and phosphorus uptake by hybrid poplars growing in diesel-contaminated soil (Gunderson et al., 2007). In contrast, soils recently contaminated with oil sands in Canada were found to be devoid of active mycorrhizal propagules in comparison with other reclamation areas (Bois et al., 2005). Some artificial inoculation is possible, however, because ECM have the ability to inoculate poplar stands growing in denuded areas such as volcanic debris (Obase et al., 2009). Attempts have been made also to use mycorrhizal fungi to improve phytoremediation of organic compounds, such as

hexahydro-1,3,5-trinitro-1,3,5-triazine (Thompson and Polebitski, 2010), but have yielded a low phytoremediation efficiency. 4.4.4. Biotic stress Poplar ECM exhibit cross-talk with pathogenic organisms, which are often problematic during poplar breeding. For example, ECM remodel their metabolic defense response to Melampsora, a rust fungi, by changing their composition of flavonoids and lipids (Pfabel et al., 2012). In addition, invasion of exotic plant species may reduce ECM composition and the colonization ratio of mycorrhized poplar roots (Meinhardt and Gehring, 2012), resulting in antagonistic effects on both organisms. Alternatively, analysis of quantitative trait loci revealed numerous shared quantitative trait loci characteristics for plant responses to ECM and to the pathogenic fungus Melampsora laricipopulina (Tagu et al., 2005), pointing to similarities in responses to symbiotic and pathogenic fungi. 5. Perspectives—post-genomic research Much remains to be explored with regard to ECM–poplar interactions and dynamics. For example, one research area involves complex co-infections of ECM with symbiotic bacteria in whole rhizosphere analyses, and a second area involves exploring the effects of ECM response to expected climate changes (Cairney, 2012). Perhaps the most conspicuous area needing exploration involves a technical issue—the almost complete lack of complex, cutting-edge post-genomic analyses of not only poplar-associated ECM but also ECM associated with other plant species. Despite abundant genetic data for ECM and poplar species, the disciplines of proteomics and metabolomics have not been applied sufficiently to elucidate the biochemistry underlying ECM-related symbioses. There is only one report of two-dimensional electrophoresis for ECM-inoculated birch (Simoneau et al., 1993), and few proteomic studies have been published concerning ECM fungi. Liang et al. (2007) analyzed Boletus edulis under salt shock and identified 22 affected proteins involved in methionine biosynthesis, glycolysis, DNA repair, and cell cycle control. Boletus spore proteins were also identified (Mao et al., 2011). It is also known that L. bicolor free-living mycelia can release 224 proteins into a medium, including numerous cell wall modeling enzymes or extracellular proteases (Vincent et al., 2012). Information is also limited regarding metabolic changes. Despite this deficiency, it is known that colonization causes massive reprogramming of the metabolic pathway manifested, e.g. by shifts in the metabolism of aromatics, lowmolecular-weight organics, and fatty acids (Tschaplinski et al., 2014). There have been also some cutting-edge spectrometric analyses of inoculated poplar, e.g. focused on tannins, phenolics, and lipids (Pfabel et al., 2012; Sutela et al., 2009). All these changes probably aimed at ECM manipulation of host defense processes (Pfabel et al., 2012; Tschaplinski et al., 2014). Poplar varieties differ in their response to ECM colonization, especially in defense-related and immune-response-related metabolites or effectors such as small signaling proteins (Plett and Martin, 2012; Tschaplinski et al., 2014). Thus the application of post-genomic technologies is the most promising area to explore in the coming decade. Knowledge about metabolome changes may be very important also for future poplar management procedures. Some attempts at metabolome prediction, aimed to investigate the complex molecular ECM–poplar interactions, have already been undertaken (Larsen et al., 2011). One can speculate that in the future this may result in the creation of genetically modified poplars characterized by an increased production of desirable secondary metabolites.

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Acknowledgements This work was supported by grant No. DEC-2011/03/D/NZ9/ 05500 from the National Science Centre (NCN), Poland. I gratefully acknowledge Dr L. Karlin´ski, Dr hab. T. Leski, Dr M. Pietras, Dr J. Mucha and Prof. M. Rudawska (Institute of Dendrology, Polish Academy of Sciences) for valuable discussions and help. References Averill, C., Turner, B.L., Finzi, A.C., 2014. Mycorrhiza-mediated competition between plants and decomposers drives soil carbon storage. Nature 505, 543–545. Bahram, M., Põlme, S., Kõljalg, U., Tedersoo, L., 2011. A single European aspen (Populus tremula) tree individual may potentially harbour dozens of Cenococcum geophilum ITS genotypes and hundreds of species of ectomycorrhizal fungi. FEMS Microbiol. Ecol. 75, 313–320. Baum, C., Stetter, U., Makeschin, F., 2002. Growth response of Populus trichocarpa to inoculation by the ectomycorrhizal fungus Laccaria laccata in a pot and a field experiment. For. Ecol. Manage. 163, 1–8. Benjdia, M., Rikirsch, E., Müller, T., Morel, M., Corratgé, C., Zimmermann, S., Chalot, M., Frommer, W.B., Wipf, D., 2006. Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum: characterization of two di- and tripeptide transporters (HcPTR2A and B). New Phytol. 170, 401–410. Bent, E., Taylor, D.L., 2010. Direct amplification of DNA from fresh and preserved ectomycorrhizal root tips. J. Microbiol. Meth. 80, 206–208. Bent, E., Kiekel, P., Brenton, R., Taylor, D.L., 2011. Root-associated ectomycorrhizal fungi shared by various boreal forest seedlings naturally regenerating after a fire in interior alaska and correlation of different fungi with host growth responses. Appl. Environ. Microbiol. 77, 3351–3359. Bois, G., Piché, Y., Fung, M.Y., Khasa, D.P., 2005. Mycorrhizal inoculum potentials of pure reclamation materials and revegetated tailing sands from the Canadian oil sand industry. Mycorrhiza 15, 149–158. Bryngelsson, T., Gustafsson, M., Gréen, B., Lind, C., 1988. Uptake of host DNA by the parasitic fungus Plasmodiophora brassicae. Physiol. Mol. Plant Pathol. 33, 2163– 2171. Cairney, J., 2012. Extramatrical mycelia of ectomycorrhizal fungi as moderators of carbon dynamics in forest soil. Soil Biol. Biochem. 47, 198–208. Clark, F.B., 1963. Endotrophic mycorrhizae influence yellow poplar seedling growth. Science 140, 1220–1221. Colpaert, J.V., Wevers, J.H.L., Krznaric, E., Adriaensen, K., 2011. How metal-tolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution. Ann. For. Sci. 68, 17–24. Couturier, J., Montanini, B., Martin, F., Brun, A., Blaudez, D., Chalot, M., 2007. The expanded family of ammonium transporters in the perennial poplar plant. New Phytol. 174, 137–150. Cripps, C. L., 2001. Mycorrhizae of Aspen Forests: ecology and potential application. In: Sustaining Aspen in Western Landscapes: Proceedings of the Symposium on Western Aspen Forests, Grand Junction, CO, June 2000, pp. 285–298. Cripps, C.L., 2003. Native mycorrhizal fungi with aspen on smelter-impacted sites in the Northern Rocky Mountains: occurrence and potential use in reclamation. In: National Meeting of the American Society of Mining and Reclamation and the 9th Billings Land Reclamation Symposium, Billings, Montana, pp. 193–208. Cripps, C.L., Miller Jr., O.K., 1993. Ectomycorrhizal fungi associated with aspen on three sites in the north-central Rocky Mountains. Can. J. Botany 71, 1414– 1420. Cripps, C.L., Miller, O.K., 1995. Ectomycorrhizae formed in vitro by quaking aspen: including Inocybe lacera and Amanita pantherina. Mycorrhiza 5, 357–370. Danielsen, L., Thürmer, A., Meinicke, P., Buée, M., Morin, E., Martin, F., Pilate, G., Daniel, R., Polle, A., Reich, M., 2012. Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities. Ecol Evol. 2, 1935–1948. Danielsen, L., Lohaus, G., Sirrenberg, A., Karlovsky, P., Bastien, C., Pilate, G., Polle, A., 2013. Ectomycorrhizal colonization and diversity in relation to tree biomass and nutrition in a plantation of transgenic poplars with modified lignin biosynthesis. PLoS ONE 8, e59207. DeBellis, T., Kernaghan, G., Bradley, R., Widden, P., 2006. Relationships between stand composition and ectomycorrhizal community structure in boreal mixedwood forests. Microb Ecol. 52, 114–126. Desai, S., Naik, D., Cumming, J.R., 2013. The influence of phosphorus availability and Laccaria bicolor symbiosis on phosphate acquisition, antioxidant enzyme activity, and rhizospheric carbon flux in Populus tremuloides. Mycorrhiza 24, 369–382. Ding, Q., Liang, Y., Legendre, P., He, X.H., Pei, K.Q., Du, X.J., Ma, K.P., 2011. Diversity and composition of ectomycorrhizal community on seedling roots: the role of host preference and soil origin. Mycorrhiza 21, 669–680. Diop, T.A., 2003. In vitro culture of arbuscular mycorrhizal fungi: advances and future prospects. Afr. J. Biotechnol. 2, 692–697. Dupae, J., Bohler, S., Noben, J.P., Carpentier, S., Vangronsveld, J., Cuypers, A., 2014. Problems inherent to a meta-analysis of proteomics data: a case study on the plants’ response to Cd in different cultivation conditions. J. Proteomics 108, 30–54. Felten, J., Kohler, A., Morin, E., Bhalerao, R.P., Palme, K., Martin, F., Ditengou, F.A., Legué, V., 2009. The ectomycorrhizal fungus Laccaria bicolor stimulates lateral

167

root formation in poplar and Arabidopsis through auxin transport and signaling. Plant Physiol. 151, 1991–2005. Gafur, A., Schützendübel, A., Langenfeld-Heyser, R., Fritz, E., Polle, A., 2004. Compatible and incompetent Paxillus involutus isolates for ectomycorrhiza formation in vitro with poplar (Populus  canescens) differ in H2O2 production. Plant Biol. (Stuttg) 6, 91–99. Gardes, M., White, T.J., Fortin, J.A., Bruns, T.D., Taylor, J.W., 1991. Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Can. J. Bot. 69, 180–190. Gehring, C.A., Mueller, R.C., Whitham, T.G., 2006. Environmental and genetic effects on the formation of ectomycorrhizal and arbuscular mycorrhizal associations in cottonwoods. Oecologia 149, 158–164. Grubisha, L.C., Levsen, N., Olson, M.S., Taylor, D.L., 2012. Intercontinental divergence in the Populus-associated ectomycorrhizal fungus, Tricholoma populinum. New Phytol. 194, 548–560. Grunze, N., Willmann, M., Nehls, U., 2004. The impact of ectomycorrhizal formation on monosaccharide transporter gene expression in poplar roots. New Phytol. 164, 147–155. Gryta, H., Carriconde, F., Charcosset, J.Y., Jargeat, P., Gardes, M., 2006. Population dynamics of the ectomycorrhizal fungal species Tricholoma populinum and Tricholoma scalpturatum associated with black poplar under differing environmental conditions. Environ. Microbiol. 8, 773–786. Guidot, A., Verner, M.C., Debaud, J.C., Marmeisse, R., 2005. Intraspecific variation in use of different organic nitrogen sources by the ectomycorrhizal fungus Hebeloma cylindrosporum. Mycorrhiza 15, 167–177. Gunderson, J.J., Knight, J.D., Van Rees, K.C., 2007. Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil. J. Environ. Qual. 36, 927–934. Hampp, R., Ecke, M., Schaeffer, C., Wallenda, T., Wingler, A., Kottke, I., Sundberg, B., 1996. Axenic mycorrhization of wild type and transgenic hybrid aspen expressing T-DNA indoleacetic acid-biosynthetic genes. Trees 11, 59–64. Han, S.H., Kim, D.H., Lee, J.Ch., 2011. Effects of the ectomycorrhizal fungus Pisolithus tinctorius and Cd on physiological properties and Cd uptake by hybrid poplar Populus alba  glandulosa. J. Ecol. Field Biol. 34, 393–400. Harris, M.M., Jurgensen, M.F., 1977. Development of salix and populus mycorrhizae in metallic mine tailings. Plant Soil 47, 509–517. Hay, I., Morency, M.-J., Séguin, A., 2002. Assessing the persistence of DNA in decomposing leaves of genetically modified poplar trees. Can. J. For. Res. 32, 977–982. Healy, R.A., Smith, M.E., Bonito, G.M., Pfister, D.H., Ge, Z.W., Guevara, G.G., Williams, G., Stafford, K., Kumar, L., Lee, T., Hobart, C., Trappe, J., Vilgalys, R., McLaughlin, D.J., 2013. High diversity and widespread occurrence of mitotic spore mats in ectomycorrhizal Pezizales. Mol. Ecol. 22, 1717–1732. Hedh, J., Johansson, T., Tunlid, A., 2009. Variation in host specificity and gene content in strains from genetically isolated lineages of the ectomycorrhizal fungus Paxillus involutus s. Mycorrhiza 19, 549–558. Heslin, M.C., Douglas, G.C., 1986. Effects of ectomycorrhizal fungi on growth and development of poplar plants derived from tissue culture. Sci. Hortic. 30, 143– 149. Hoenicka, H., Fladung, M., 2006. Biosafety in Populus spp. and other forest trees: from non-native species to taxa derived from traditional breeding and genetic engineering. Trees 20, 131–144. Hoffmann, T., Golz, C., Schieder, O., 1994. Foreign DNA sequences are received by a wild-type strain of Aspergillus niger after co-culture with transgenic higher plants. Curr. Genet. 27, 70–76. Hrynkiewicz, K., Baum, Ch., Leinweber, P., Weih, M., Dimitriou, I., 2010. The significance of rotation periods for mycorrhiza formation in short rotation coppice. For. Ecol. Manage. 260, 1943–1949. Jakucs, E., Kovács, G.M., Agerer, R., Romsics, C., Eros-Honti, Z., 2005. Morphologicalanatomical characterization and molecular identification of Tomentella stuposa ectomycorrhizae and related anatomotypes. Mycorrhiza 15, 247–258. Javelle, A., Rodriguez-Pastrana, B.R., Jacob, C., Botton, B., Brun, A., Andre, B., Marini, A.M., Chalot, M., 2001. Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum. FEBS Lett. 505, 393–398. Javelle, A., Morel, M., Rodriguez-Pastrana, B.R., Botton, B., Andre, B., Marini, A.M., Brun, A., Chalot, M., 2003. Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADPGDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum. Mol. Microbiol. 47, 411–430. Jean-Philippe, S.R., Franklin, J.A., Buckley, D.S., Hughes, K., 2011. The effect of mercury on trees and their mycorrhizal fungi. Environ. Pollut. 159, 2733– 2739. Jentschke, G., Brandes, B., Heinzemann, J., Marschner, P., Godbold, D.L., 1999. Sand culture of mycorrhizal plants. J. Plant Nutr. Soil Sci. 162, 107–112. Jokipii, S., Häggman, H., Brader, G., Kallio, P.T., Niemi, K., 2008. Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction. J. Exp. Bot. 59, 2449–2459. Kaldorf, M., Fladung, M., Muhs, H.J., Buscot, F., 2002. Mycorrhizal colonization of transgenic aspen in a field trial. Planta 214, 653–660. Kaldorf, M., Renker, C., Fladung, M., Buscot, F., 2004. Characterization and spatial distribution of ectomycorrhizas colonizing aspen clones released in an experimental field. Mycorrhiza 14, 295–306. Karlin´ski, L., Rudawska, M., Kieliszewska-Rokicka, B., Leski, T., 2010. Relationship between genotype and soil environment during colonization of poplar roots by mycorrhizal and endophytic fungi. Mycorrhiza 20, 315–324.

168

A. Szuba / Forest Ecology and Management 347 (2015) 156–169

Kemppainen, M.J., Pardo, A.G., 2013. LbNrt RNA silencing in the mycorrhizal symbiont Laccaria bicolor reveals a nitrate-independent regulatory role for a eukaryotic NRT2-type nitrate transporter. Environ. Microbiol. Rep. 5, 353–366. Kemppainen, M., Duplessis, S., Martin, F., Pardo, A.G., 2009. RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus. Environ. Microbiol. 11, 1878–1896. Khasa, P.D., Chakravarty, P., Robertson, A., Thomas, B.R., Dancik, B.P., 2002. The mycorrhizal status of selected poplar clones introduced in Alberta. Biomass Bioenergy 22, 99–104. Kovács, G.M., Jakucs, E., 2006. Morphological and molecular comparison of white truffle ectomycorrhizae. Mycorrhiza 16, 567–574. Krpata, D., Peintner, U., Langer, I., Fitz, W.J., Schweiger, P., 2008. Ectomycorrhizal communities associated with Populus tremula growing on a heavy metal contaminated site. Mycol. Res. 112, 1069–1079. Krpata, D., Fitz, W., Peintner, U., Langer, I., Schweiger, P., 2009. Bioconcentration of zinc and cadmium in ectomycorrhizal fungi and associated aspen trees as affected by level of pollution. Environ. Pollut. 157, 280–286. Küster, H., Becker, A., Firnhaber, C., Hohnjec, N., Manthey, K., Perlick, A.M., Bekel, T., Dondrup, M., Henckel, K., Goesmann, A., Meyer, F., Wipf, D., Requena, N., Hildebrandt, U., Hampp, R., Nehls, U., Krajinski, F., Franken, P., Pühler, A., 2007. Development of bioinformatic tools to support expressed sequence tag (EST) sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses. Phytochemistry 68, 19–32. Langenfeld-Heyser, R., Gao, J., Ducic, T., Tachd, P., Lu, C.F., et al., 2007. Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar Populus  canescens. Mycorrhiza 17, 121–131. Langer, I., Krpata, D., Peintner, U., Wenzel, W.W., Schweiger, P., 2008. Media formulation influences in vitro ectomycorrhizal synthesis on the European aspen Populus tremula L. Mycorrhiza 18, 297–307. Larsen, P.E., Sreedasyam, A., Trivedi, G., Podila, G.K., Cseke, L.J., Collart, F.R., 2011. Using next generation transcriptome sequencing to predict an ectomycorrhizal metabolome. BMC Syst. Biol. 5, 70. Le Quéré, A., Schützendübel, A., Rajashekar, B., Canbäck, B., Hedh, J., Erland, S., Johansson, T., Tunlid, A., 2004. Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus. Mol. Ecol. 13, 3809– 3819. Le Quéré, A., Eriksen, K.A., Rajashekar, B., Schützendübel, A., Canbäck, B., Johansson, T., Tunlid, A., 2006. Screening for rapidly evolving genes in the ectomycorrhizal fungus Paxillus involutus using cDNA microarrays. Mol. Ecol. 15, 535–550. Li, J., Bao, S., Zhang, Y., Ma, X., Mishra-Knyrim, M., Sun, J., Sa, G., Shen, X., Polle, A., Chen, S., 2012. Paxillus involutus strains MAJ and NAU mediate K(+)/Na(+) homeostasis in ectomycorrhizal Populus x canescens under sodium chloride stress. Plant Physiol. 159, 1771–1786. Liang, Y., Chen, H., Tang, M., Shen, S., 2007. Proteome analysis of an ectomycorrhizal fungus Boletus edulis under salt shock. Mycoll. Res. 111, 939–946. Luo, Z.B., Li, K., Jiang, X., Polle, A., 2009. Ectomycorrhizal fungus (Paxillus involutus) and hydrogels affect performance of Populus euphratica exposed to drought stress. Ann. For. Sci. 66, 106. Luo, Z.B., Janz, D., Jiang, X., Göbel, C., Wildhagen, H., Tan, Y., Rennenberg, H., Feussner, I., Polle, A., 2009a. Upgrading root physiology for stress tolerance by ectomycorrhizas: insights from metabolite and transcriptional profiling into reprogramming for stress anticipation. Plant Physiol. 151, 1902–1917. Luo, Z.B., Wua, Ch., Zhang, Ch., Lic, H., Lipkad, U., Polleda, College A., 2014. The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ. Exp. Bot. 108, 47–62. Ma, L., Wu, X., Zheng, L., 2008. Mycorrhizal formation of nine ectomycorrhizal fungi on poplar cuttings. Front. For. China 3, 475–479. Ma, Y., He, J., Ma, C., Luo, J., Li, H., Liu, T., Polle, A., Peng, C., Luo, Z.B., 2013. Ectomycorrhizas with Paxillus involutus enhance cadmium uptake and tolerance in Populus  canescens. Plant Cell. Environ. 37, 627–642. Mao, L., Jiang, S., Wang, B., Chen, L., Yao, Q., Chen, K., 2011. Protein Profile of Bacillus subtilis Spore. Curr. Microbiol. 63, 198–205. Marjanovic´, Z., Uwe, N., Hampp, R., 2005. Mycorrhiza formation enhances adaptive response of hybrid poplar to drought. Ann. N. Y. Acad. Sci. 1048, 496–499. Marjanovic´, Z., Uehlein, N., Kaldenhoff, R., Zwiazek, J.J., Weiss, M., Hampp, R., Nehls, U., 2005a. Aquaporins in poplar: what a difference a symbiont makes! Planta 222, 258–268. Martin, F., Nehls, U., 2009. Harnessing ectomycorrhizal genomics for ecological insights. Curr. Opin. Plant. Biol. 12, 508–515. Martin, F., Aerts, A., Ahrén, D., Brun, A., Danchin, E.G., Duchaussoy, F., et al., 2008. The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452, 88–92. Martin, F., Kohler, A., Murat, C., Balestrini, R., Coutinho, P.M., Jaillon, O., et al., 2010. Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature 464, 1033–1038. Meinhardt, K.A., Gehring, C.A., 2012. Disrupting mycorrhizal mutualisms: a potential mechanism by which exotic tamarisk outcompetes native cottonwoods. Ecol. Appl. 22, 532–549. Min, X.J., Siddiqi, M.Y., Glass, A.D.M., Guy, R.D., Kronzucker, H.J., 1999. A comparative study of fluxes and compartmentation of nitrate and ammonium in early-successional tree species. Plant Cell. Environ. 22, 821–830. Morel, M., Jacob, C., Fitz, M., Wipf, D., Chalot, M., Brun, A., 2008. Characterization and regulation of PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus involutus. Fungal Genet. Biol. 45, 912–921.

Müller, A., Volmer, K., Mishra-Knyrim, M., Polle, A., 2013. Growing poplars for research with and without mycorrhizas. Front. Plant Sci. 4, 332. Nannipieri, P., Eldor, P., 2009. The chemical and functional characterization of soil N and its biotic components. Soil Biol. Biochem. 41, 2357–2369. Navratil, S., Rochon, G.C., 1981. Enhanced root and shoot development of poplar cuttings induced by Pisolithus inoculum. Can. J. For. Res. 11, 844–848. Nehls, U., Zhang, C., Tarkka, M., Hampp, R., Fladung, M., 2006. Investigation of horizontal gene transfer from transgenic aspen to ectomyccorhizal fungi. In: Fladung, M., Ewald, D. (Eds.), ‘‘Tree transgenesis – Recent Developments. Springer-Verlag, Berlin, Heidelberg, New York, pp. 323–333. Nuutinen, J.T., Timonen, S., 2008. Identification of nitrogen mineralization enzymes, L-amino acid oxidases, from the ectomycorrhizal fungi Hebeloma spp. and Laccaria bicolor. Myco. Res. 112, 1453–1464. Obase, K., Tamai, Y., Yajima, T., Miyamoto, T., 2009. Mycorrhizal synthesis of four ectomycorrhizal fungi in potted Populus maximowiczii seedlings. Mycoscience 50, 143–145. Peterson, R.L., Chakravarty, P., 1991. Techniques in synthesizing ectomycorrhiza. In: Norris, J., Read, D., Varma, A. (Eds.), Methods in Microbiology. Academic, U.S., pp. 75–107. Peterson, R.L., Massicotte, H.B., Melville, L.H., 2004. Mycorrhizas: Anatomy and Cell Biology NRC Research Press Ottawa. Pfabel, C., Eckhardt, K.U., Baum, C., Struck, C., Frey, P., Weih, M., 2012. Impact of ectomycorrhizal colonization and rust infection on the secondary metabolism of poplar (Populus trichocarpa  deltoides). Tree Physiol. 32, 1357–1364. Pilate, G., Guiney, E., Holt, K., Petit-Conil, M., Lapierre, C., et al., 2002. Field and pulping performances of transgenic trees with altered lignification. Nat. Biotechnol. 20, 607–612. Plett, J.M., Martin, F., 2012. Poplar root exudates contain compounds that induce the expression of MiSSP7 in Laccaria bicolor. Plant Signal Behav. 7, 12–15. Plett, J.M., Montanini, B., Kohler, A., Ottonello, S., Martin, F., 2011. Tapping genomics to unravel ectomycorrhizal symbiosis. Methods Mol. Biol. 722, 249–281. Plett, J.M., Khachane, A., Ouassou, M., Sundberg, B., Kohler, A., Martin, F., 2014. Ethylene and jasmonic acid act as negative modulators during mutualistic symbiosis between Laccaria bicolor and Populus roots. New Phytol. 202, 270– 286. Quoreshi, A.M., Khasa, D.P., 2008. Effectiveness of mycorrhizal inoculation in the nursery on root colonization, growth, and nutrient uptake of aspen and balsam poplar. Biomass Bioenergy 32, 381–391. Rineau, F., Roth, D., Shah, F., Smits, M., Johansson, T., Canbäck, B., Olsen, P.B., Persson, P., Grell, M.N., Lindquist, E., Grigoriev, I.V., Lange, L., Tunlid, A., 2012. The ectomycorrhizal fungus Paxillus involutus converts organic matter in plant litter using a trimmed brown-rot mechanism involving Fenton chemistry. Environ. Microbiol. 14, 1477–1487. Rineau, F., Shah, F., Smits, M.M., Persson, P., Johansson, T., Carleer, R., Troein, C., Tunlid, A., 2013. Carbon availability triggers the decomposition of plant litter and assimilation of nitrogen by an ectomycorrhizal fungus. ISME J. 10, 2010– 2022. Sell, J., Kayser, A., Schulin, R., Brunner, I., 2005. Contribution of ectomycorrhizal fungi to cadmium uptake of poplars and willows from a heavily polluted soil. Plant Soil. 277, 245–253. Selle, A., Willmann, M., Grunze, N., Gessler, A., Weiss, M., Nehls, U., 2005. The highaffinity poplar ammonium importer PttAMT1.2 and its role in ectomycorrhizal symbiosis. New Phytol. 168, 697–706. Shah, F., Rineau, F., Canbäck, B., Johansson, T., Tunlid, A., 2013. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus. New Phytol. 200, 875–887. Siemens, J.A., Zwiazek, J.J., 2008. Root hydraulic properties and growth of balsam poplar (Populus balsamifera) mycorrhizal with Hebeloma crustuliniforme and Wilcoxina mikolae var. mikolae. Mycorrhiza 18, 393–401. Siemens, J.A., Calvo-Polanco, M., Zwiazek, J.J., 2011. Hebeloma crustuliniforme facilitates ammonium and nitrate assimilation in trembling aspen (Populus tremuloides) seedlings. Tree Physiol. 31, 1238–1250. Simoneau, P., Viemont, J.D., Moreau, J.C., Strullu, D.G., 1993. Symbiosis-related polypeptides associated with the early stages of ectomycorrhiza organogenesis in birch (Betula pendula Roth). New Phytol. 124, 495–504. Smith, S.E., Read, D., 2008. Mycorrhizal Symbiosis. Academic Press, London, UK. Somerville, C., Youngs, H., Taylor, C., Davis, S.C., Long, S.P., 2010. Feedstocks for lignocellulosic biofuels. Science 329, 790–792. Stefani, F.O., Moncalvo, J.M., Séguin, A., Bérubé, J.A., Hamelin, R.C., 2009. Impact of an 8-year-old transgenic poplar plantation on the ectomycorrhizal fungal community. Appl. Environ. Microbiol. 75, 7527–7536. Sutela, S., Niemi, K., Edesi, J., Laakso, T., Saranpää, P., Vuosku, J., Mäkelä, R., Tiimonen, H., Chiang, V.L., Koskimäki, J., Suorsa, M., Julkunen-Tiitto, R., Häggman, H., 2009. Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birch. BMC Plant Biol. 9, 124. Tagu, D., Bastien, C., Faivre-Rampant, P., Garbaye, J., Vion, P., Villar, M., Martin, F., 2005. Genetic analysis of phenotypic variation for ectomycorrhiza formation in an interspecific F1 poplar full-sib family. Mycorrhiza 15, 87–91. Taylor, G., 2002. Populus: arabidopsis for forestry. Do we need a model tree? Ann. Bot. 90, 681–689. Thompson, P.L., Polebitski, A.S., 2010. Effect of mycorrhizal fungi on the phytoremediation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Environ. Sci. Technol. 44, 1112–1115. Tschaplinski, T.J., Plett, J.M., Engle, N.L., Deveau, A., Cushman, K.C., Martin, M.Z., Doktycz, M.J., Tuskan, G.A., Brun, A., Kohler, A., Martin, F., 2014. Populus trichocarpa and Populus deltoides exhibit different metabolomic responses to

A. Szuba / Forest Ecology and Management 347 (2015) 156–169 colonization by the symbiotic fungus Laccaria bicolor. Mol. Plant. Microbe Interact. 27, 546–556. Tuskan, G.A., Difazio, S., Jansson, S., Bohlmann, J., Grigoriev, I., Hellsten, U., et al., 2006. The genome of black cottonwood, Populus trichocarpa (Torr. and Gray). Science 313, 1596–1604. Vincent, D., Kohler, A., Claverol, S., Solier, E., Joets, J., Gibon, J., Lebrun, M.H., Plomion, C., Martin, F., 2012. Secretome of the free-living mycelium from the ectomycorrhizal basidiomycete Laccaria bicolor. J. Proteome Res. 11, 157–171.

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Willmann, A., Weiss, M., Nehls, U., 2007. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria. Curr. Genet. 51, 71–78. Willmann, A., Thomfohrde, S., Haensch, R., Nehls, 2014. The poplar NRT2 gene family of high affinity nitrate importers: Impact of nitrogen nutrition and ectomycorrhiza formation, vol. 108, pp. 79–88. Zhang, C., Hampp, R., Nehls, U., 2005. Investigation of horizontal gene transfer in poplar/Amanita muscaria ectomycorrhizas. Environ. Biosafety Res. 4, 235–242.