Ectomycorrhizal community structure and fi,mction in relation to forest residue harvesting and wood ash applications

Shahid Mahmood

LUND UNIVERSITY

Dissertation 2000 A doctoral thesis at a university in Sweden is produced either as a monograph or as a collection of papers. In the latter case, the introducto~ part constitutes the formal thesis, which summarises the accompanying papers. These have either already been published or are manuscripts at various stages (in press, submitted or in ins).

ISBN9!-7105-138-8 sE-LuNBDs/NBME-oo/lo14+l10pp 02000 Shahid Mahmood Cover drawing: Peter Robemtz I DISCLAIMER

Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. Organhtion Documentname LUND UNIVERSITY I DOCTORALDISSERTATION Department of Ecology- Mtcrobial Ecology I “eda fday16,20@ Ecology Building, S-223 62 Lund, Sweden I coDfw SE-LUNBDSINBME-OOI1 OI4+I 10 pp Author(a) sponsoringOrgmiration Shahid Mahmood

T&feandsubtitls Eotomycorhkal community structure and function in relation to forest residue hawesting and wood ash applications

Ectomycorrhizal fungi form symbiotic associations with tree roots and assist in nutrient-uptake and -cycling in forest ecosystems, thereby constitutinga most significantpart of the microbial community. The aims of the studies described in this thesis were to evaluate the potential of DNA-baeed molecular methods in below-ground ectomycorrhizal community studies and to investigate changes in actomycortilzal communities on spruce roots in sites with different N deposition, and in sites subjected to harvesting of forest rasidues or application of wood ash. The ability of selected ectomycorrhizal fungi to mobilise nutriente from wood ash and to colonise root systems in the presence and absence of ash was also studied. In total 39 ectomycorrhizal species were detected in the experimental forests located in southern Sweden. At each site five to six species colonised around 60% of the root tips. The dominant species, common to the sites, were Tylospora fibrillosa, Theleptrora terreatris and Cenococcum geoptrilum. Differancas between two sites with differing levels of N deposition suggested that community structure may be influenced by N deposition, although site history, location and degree of isolation may also influence species composition. Repeated harvesting of forest residues reduced numbers of mycorrhizai roots in the humus layer to approximately 50% of that in control plots but no shift in the actomycorrhizal community could be detected. At another site, application of granulated wood ash induced a shift in ectomycorrhizal community structure and three ectomycorrhizal fungi (“ash fungi”)were found to colonise ash granules. Two “ash fung~ showed a superior ability to solubilise stabilised wood ash in laboratory experiments compared to other ectomycorrhizal isolates from the same site. In laborato~ microcosms containing intact myoorrhizal myceiia, colonisation of wood ash patches by ons “ash fungus” was good whereas colonisation by Pi/oderma croceum was poor. In a competition experiment with these two fungi, colonisation of spruce roots by the “ash fungus” increased significantly in the presence of wood ash, whereas colonisationby P. croceum decreased.

~* Ectomycorrhiza, Communiv stmcture, Sprucer pCR.RFLp, ITS region, rDNA, N deposition, Forest residue, Wood ash, Tricalcium phosphate, Solubilisation, Calcium oxalate, P uptake, “C allocation, Wocd ash coloniestkm, Spruce colonisation,Competition ctasifiition system*or index tsrms(iiolly)

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Shahid Mahmood, Department of Ecology - Microbial Ecology, Lund University, S-22362 Lund, Sweden 1,thsnadsm&d, beii &s copydgbtownerof the abstractof the abowe-mentioneddisertatio~ bueby grantto affreference ~~@lJ-~~ - e the abstmctof tbs abovHocntiOosddksertation. April 16,2000. Sii Date Ectornycorrhizal community structure and function

in relation to forest residue harvesting and wood ash applications

Shahid Makmood

LUND UNIVERSITY

Akademisk avhandling, som for avlaggande av filosofie doktorsexamen vid matematisk-naturvetenskapliga fakulteten vid Lunds Universitet, kommer att offentligen forsvaras 3 Blt?JHal/en, Ekologihuset, Solvegatan 37, Lund. Tisdagen 16 Maj 2000, kl 10W.

The thesis will be defended in English.

The cover illustration The picture illustrates the proposed model that. forest residues can be used for bioenergy production and resultant ashes can be applied back to the forest soils to compensate the lost nutrients. The bioenergy from forest residues is ‘C02-neutral’ and has been regarded as ‘environmentally friendly’. DNA-based molecular studies suggested that wood ash application might induce a shift in the ectomycorrhkzd community on the roots. The wood ash granules collected from the field were normally colonised by ectomycorrhizal mycelia. The functional aspects of these ectomycorrhizal fungi in nutrient mobilisation and acquisition fkom wood ash are also described in thk thesis. Contents

1. List of papers

2. Important results

3. Background Biofuels as an energy source Soil acidification Wood ash as a liming agent

4. Mycorrhiza Methods for studying ectomycomh~al communities

5. Effects of pollution and forest management on ectomycorrhizal corn] Effects of N deposition Effects of forest harvesting practices Effects of ash fertilisation and liming

6. Experimental approaches Field sites Community studies Laboratory studies

7. Results and Discussion Community studies Functional aspects of selected ectomycorrhizal fimgi

8. Conclusions

9. Acknowledgements

10. References

11. Papers I-V

12. Appendix

List of papers

This thesis is based on the following papers, which will be r numerals.

I Erland, S., Jonsson, T., Mahmood, S., Finlay, R ectomycomhizal community structure in two Picea abies Scandinavian Journal of Forest Research 14 (3): 209-217

II Mahrnood, S., Finlay, R.D., Erland, S. 1999. Effects of n residues on the ectomycorrhizal community in a SW Phytologist 142 (3): 577-585.

III Mahmood, S., Finlay, R.D., Wallander, H., Erland, colonisation of roots and ash granules in a spruce forest tr ash. (submitted to Forest Ecology and Management).

Iv Mahmood, S., Finlay, R.D., Erland, S., Walkmder, H colonisation of wood ash by ectomycorrhizal fungi i fertilised spruce forest. (manuscript). v Mahmood, S. 2000. Colonisation of spruce roots by two fimgi in wood ash amended substrates. (manuscript).

Thepubiishedpapers are reproduced by kindpermissionfiom th

Important results

The aims of the studies described in this thesis were to evall (ITS-typing) methods in below-ground ectomycorrhizal (E investigate changes in ectomycorrhlzal communities on spru deposition, and in sites subjected to harvesting of forest resi{ Four spruce forests in southern Sweden (Sk&ne & Halland) methods to identifi mycorrhizal fungi colonizing single r{ ectomycorrhizal fungi to mobilise nutrients from wood ash the presence and absence of ash was also studied.

In total 39 ectomycorrhizal species were detected (Papers At each site five to six species colonised around 60% of t] Dominant species, common to the sites, were Tylospora, and Cenococcuin geophilum (Papers I, II, III). Differences between two sites with differing levels c community structure may be influenced by N deposition and degree of isolation may also influence species compo Repeated harvesting of forest residues reduced numbf humus layer to approximately 50% of that in control community could be detected (Paper II). At another site, application of granulated wood ash indt structure and three ECM iimgi (“ash fi.mgi”) were found III). Two “ash fimgi” showed a superior ability to solubilise s experiments compared to other ECM isolates from the saI In laboratory microcosms containing intact mycorrhizal ash patches by one “ash fimgus” was good whereas cola was poor (Paper IV). In a competition experiment with these two fimgi, COIC “ash fimgus” increased significantly in the presence of W( Pilodei-ma croceum decreased (Paper V).

* Background

Biofuels as an energy source In Sweden there is a growing interest in the utilisation of forest derived biomass as biofuel. The main reasons for using bioenergy are the possible hazards of conventional energy sources to the environment such as production of greenhouse gases and disposal of nuclear wastes,, Bioenergy is regarded as one possible substitute for fossil fiels, which contribute substantially to the rise of carbon dioxide in the atmosphere and thus cause long-term changes in the global carbon cycle (Schkunadinger et al., 1995; Schlamadinger and Marland, 1996). There are a number of studies which indicate that forest derived biofuels have a great potential for mitigating atmospheric C02 increase in an environmentally acceptable manner (Burschel et al., 1993; Dixon et al., 1993a,b; Kursten and Burschel, 1993; Hall and House, 1994; Schlamadinger and Marland, 1999).

In a reeent report (Energilaget, 1999) on energy in Sweden it has been reported that during the year 1998, biofiels provided almost 16%(=63 Twh) of the total energy requirements (= 395 Twh). The current use of biofuels originating from forests corresponds to = 67 Twh, of which =48 Twh is derived from forest residues or by-products from the forest industry. The technical potential for energy production from forest residues has been estimated at between 22-24 Twh and it has been estimated that this figure may be increased by a fbrther 25-35 with cc)mpensatory applications of wood ash, changed quality standards within the forestry industry and an increased thhming for energy purposes.

Changed forestry management practices may fi,n-ther increase the potential for biofuel removal but the long-term sustainability and environmental acceptability of these practices is still uncertain. Intensive harvesting of biofbels from forests would result in removal of organic matter and could reduce long-term site productivity by decreasing availability of non-recalcitrant organic nitrogen or phosphorus compounds and disrupting nutrient cycling process (Zabowski et al., 1994; Jurgensen et al., 1997). Investigations have shown that clear-filling and slash removal results in the depletion of base cations and consequent soil acidification (Nykvist and Ros&, 1985). There are no reports of significant effects on the ground. flora (Olsson and Staaf, 1995), but there is a long-term effect on soil arthropods (Bengtsson et al., 1997, 1998). There is a significant lack of information on the effects of biofuel removal on soil micro-organisms, in particular ectomycorrhizas which form symbicttic associations with the tree roots and assist in nutrient uptake and cycling in forest ecosystems (Smith and Read, 1997). Continuous removal of biofuels may affect development, community structure and function of ectomycorrhiza and may thus influence nutrient uptake and tree growth with possible effects on site productivity.

Soil AcidWlcation In southern Sweden, a decline in pH of coniferous forest soils of 0.5- 1.0 units has been recorded since the 1940’s (Falkengren-Grerup, 1987). Apart from the biological acidification caused by the coniferous trees themselves (Nihlgtird, 1971; Rehfuess, 1981), 10 anthropogenic N and S deposition have been important factors contributing to this decrease in soil pH (Larsson el al., 1995; Sverdrup et al., 1995). As a consequence, there are reports on depletion of base cations by 30°A in some southern Swedish forests (Falkengren-Grerup, 1992). Recent interest in utilising forest residues for bioenergy production would further intensi~ the on-going acidification process because an increase in biomass harvesting results in depletion of base cations from the ecosystem (Nykvist and Rosen, 1985). A decrease in pH due to deposition of atmospheric pollutants can also result in an increase in the availability of heavy metals and aluminium in the ecosystem (Matzner and Prenzel, 1992; Larsson et al., 1995). For instance, at pH 3.8 in soil, the activation of the aluminiurn buffer system occurs and toxic aluminium ions are released into the soils. Elevated levels of heavy metals affect forest productivity and longevity in a number of ways (Andersson, 1988). Elevated levels of heavy metals also affect diversity of ectomycorrhizal fimgal tiit- bodies in forest soils (Riihling et al., 1984; Ohtonen et al, 1990; Riihling and Soderstr6m, 1991).

Wood ash as a liming agent One way to counteract acidification is the application of a liming agent such as wood ash to raise the soil pH. Wood ash is a by-product of combustion and is produced in large amounts at bioenergy plants. It is highly alkaline and causes arise in pH when applied to forest soils. It contains substantial amounts of base cations (Q Mg, K) and different trace elements in varying concentrations. Untreated biofuel ash contains reactive oxides in addition to significant amounts of soluble salts which may produce undesirable effects such as pH shock and burning damage to plant tissue (Steenari and Lindqvist, 1997). Technologies have therefore been developed to stabilise biofiel ashes by pelleting, granulation or self- hardening for recycling to forest soil (Steenari and Lindqvist, 1997). Application of granulated wood ash gives long-term benefits due to slow release of ions, thus avoiding large, rapid increases in pH (Clarholm, 1994). It should thereby act as a slow release fertiliser with few damaging side effects on the soil ecosystem. Recent laboratory and field investigations have shown that most of the base cations and trace elements are relatively easily leachable born the ash except phosphorus which is bound in apatite type compounds with low solubilities (Clarholm, 1994; Fransman and Nihlg5rd, 1995; Steenari and Lindqvist, 1997; Steenari, et al., 1998; Eriksson, H.M., 1998; Eriksson, J., 1998). It has been estimated that except for N and S, an application of 1-5 tons of ash per ha is sufilcient to compensate for the amount of nutrients removed through conventional harvesting (Clarholm, 1994).

Depending on the stability, type and dose of wood ash used, there is usually a decrease in acidity and aluminium concentration, and an increase in base saturation after wood ash application (Bramryd and Fransman, 1995; Jacobson and Ring, 1995). Fransman and Nihlg&d (1995) reported that wood ash affected the forest ecosystem only slowly, but after six years the runoff water indicated increased pH values as well as K, Ca concentrations and Ca/Al ratios, and decreased concentrations of Al, Fe and Mn. The beneficial effects of wood

11 ash last for several years depending on the initial acidity and atmospheric deposition on the soil.

In general, there are reports of tree growth improvements following wood ash fertilisation (Ferm et al., 1992; Jacobson and Rhg, 1995). However, Clemensson-Lindell and Persson (1995]1 found a decrease in fine-root biomass and an increase in SRL (SRL = fine root lengthlfine root dry weight) following wood ash application.

Many investigations indicate that P is the most strongly bound element in the wood ash. Clarhcdm (1994) investigated the effects of granulated wood ash on P availability to trees and micro-organisms in the humus layer 18 months after ash application. She reported an increase in acid phosphatase activity but the mean was not significantly different from the control. Furthermore, none of the P in the granulated ash was water-soluble, while around 20% was extractable with ammonium acetate pH 4.2. In the studies conducted by Clarhohn and Rosengren-Brink (1995), low P concentrations in the tree needles were associated with increased acid phosphatase activity in the humus layer. Wood ash application had no effect on P concentration as measured in soil water after five years of fertilisation (Fransman and Nihlgiird, 1995). The dissolution of P from granulated wood ash seems to be slow as indicated by very low concentrations of POAin the soil solution (Jacobson and Ring, 1995). In the same study, the initial P concentrations in tree needles from plots treated with a high dose of ash, showed markedly higher values compared to the untreated control (Jacobson and Ring, 1995).

There are only a few studies which involve the effects of wood ash fertilisation on soil micro-organisms. Frosteg&d et al. (1993) using PLFA analysis, reported that an increase in pH due to wood ash caused a shift in the bacterial community to more Gram-negative and fewer Gram-positive bacteri% while the amount of fungi was unaffected in this study. By using the same method (PLFA analysis), B&ith et al. (1995) reported a significant reduction in microbial biomass (total PLFAs) and a decreased index of fungal:bacterial PLFAs indicating a larger reduction of fungi than bacteria due to the highest rate of wood ash fertilisation. However, Fritze et al. (1994), using a finnigation-extraction method, reported no change in the level of microbial biomass C or fi.mgal ergosterol in response to ash application. Wood ash fertilisation in the same experiment, however, increased the microbial respiration rate in coniferous soils which indicates faster mineralisation of organic material (Weber et al., 1985). Bi&th and Arnebrant (1994) reported higher total microbial activities and higher bacterial growth rates (measured as soil respiration rate and thymidine incorporation rate, respectively) after increasing the soil pH with ash. A higher C mineralisation rate in peat-lands (Silov~ 1988) and a high vitrification rate and N03- leaching (Ros6n et al., 1993) have been reported after wood ash fertilisation. A clear difference in species composition of microfungi in a coniferous forest both in response to liming and wood ash fertilisation, was reported by Btith and Arnebrant (1993). There are only a few investigations which involve dhect effects of ash fertilisation on ectomycorrhkas

12 and these show little or no effect on mycorrhizal abundance (Erland, 1990; Erland and Soderstr6m, 1991a).

Mycorrhiza

The term “Mycorrhiza” (Greek nzykes, fi.mgus; rhiza, root) was first used by Frank (1885) to describe an association of fungi and plant roots. In general, this type of association is mutualistic, and the fungus absorbs nutrients and water from the soil while the host plant, in return, supplies carbon to the mycorrhizal fimgus. Seven different types of mycorrhiza can be distinguished on the basis of their structure and the host plants involved viz., vesicular- arbuscular, ecto-, ectendo-, arbutoid, monotropoid, ericoid and orchid mycorrhizas (Smith and Read, 1997). The earliest land plants had rudimentary roots colonised by flmgi that formed vesicles and arbuscules very similar to present day vesicular-arbuscular mycorrhizas. The fossil record thus suggests that the colonisation of land was achieved by symbiotic organisms (Smith and Read, 1997). The majority of higher land plants are mycorrhizal with the exception of plants belonging to the families Carry ophyllaceae, Cyperaceae and Brassicaceae (Harley and Harley, 1987; Newman and Reddell, 1987).

This thesis focuses mainly on ectomycorrhiza, which are commonly formed by trees dominating temperate and boreal ecosystems (Meyer, 1973). The ectomycorrhizal flmgi are normally basidiomycetes or ascomycetes, or rarely zygomycetes belonging to the genus Endogone (Smith and Read, 1997). Anatomically, three distinct structures can be distinguished in ectomycorrhti, a) a sheath or mantle of fhngal hyphae, which encloses the root and serves as an interface between the plant and the soil; b) the Hartig net, a branched network of fungal tissue growing between the epidermal and cortical cells of the roo~ this is the interface between the symbionts where nutrients are exchanged; c) the extramatrical mycelium, a network of more or less differentiated hyphae growing from the mantle and extending into the surrounding soil to provide a large surface area for nutrient uptake (Smith and Rea~ 1997).

There are an estimated 5000-6000 species of ectomycorrhizal fungi world-wide (Molina et al., 1992) and in Sweden, 500-1000 species are reported to form mycorrhiza (Hallingb5ck, 1994). Ectomycorrhizal fungi affect host plants not only by increasing the root absorbing area (Marks and Kozlowski, 1973; Harley and Smith, 1983; Smith and Read, 1997), but also by providing protection against invasion of root pathogens (Marx, 1973; Perrin, 1985; Duchesne et al., 1988; Stenstrom et al., 1997; Morin et al., 1999; Chakravarty et al., 1999), and resistance against heavy metal toxicity and environmental stresses (Godbold et al., 1998; Smith and Read, 1997). Moreover ectomycorrhiza are important for nutrient uptake from organic matter and nutrient cycling in the forest ecosystem (Read, 1991; Leak and Read, 1997). There is also increasing evidence that ectomycomhizal fungi can weather primary minerals to mobilise nutrients essential for plant growth (Leyval and Bertheli@

13 1989, 1991; Paris et aI., 1995kb; Wallander et al., 1997; Wallander and Wickman, 1999; Wallander, 2000).

Methods for studying ectomycorrhizal communities Fruit-body irwentories Most of the earlier studies on ectomycorrhizal communities in forests have relied on identi~>ing and quanti~ing fimgal fruit-bodies. There are, however several limitations with this approach. Fruit-body production is highly dependent on season and weather conditions and data from several years fruit-body production is required to establish a reliable map of fruiting species. There are also ectomycorrhizal species which do not produce fluit-bodies (e.g. Cenococcwn), produce below-ground fruit-bodies (e.g. truffles) or produce inconspicuous fruit-bodies (resupinate ectomycorrhizal fhngi). It is also likely that there are species within the resupinate genera that are not yet known as ectomycomhiza-formers (Erlan,d and Taylor, 1999). Additionally there are studies which show that the relative abundance of the same species above- and below-ground can differ markedly. Gardes and Bruns (1996) found dominant fruit-body production by Suillus pungens and Amanita j7ancheti in a Pinus muricata stand while mycorrhiza production by these species was low. Russu,la amoenoiens, however made up nearly 30% of the mycorrhizas but produced very few fruit-bodies.

Direct studies of ectomycorrhizas It is now well established that there is little correlation between above- and below-ground ectomycomhizal communities (Mehmann et al., 1995; Gardes and Bruns, 1996; K5r& and Nylund, 1997; Dahlberg et al., 1997; Jonsson et al.; 1999). There are, however, also many difficulties in direct studies of ectomycorrhizas on the roots. Enormous numbers of fine root tips are typically found in the upper organic layer in the boreal forest with figures often approaching 2-4 million per square metre (Dahlberg et al., 1997) and only a small fraction of these can be analysed. Because of the vast heterogeneity in the soil environment representative sampling is very ditllcult. Then there is the matter of identification of the fimgi involved in the symbiosis. Two main approaches, or combinations of these have been used. a) Morphological methods This method is based on detailed macro- and microscopical features of the mantle, Hartig net and extrarnatrical mycelium. Attempts have been made to classi@ mycorrhlzas on the basis of their external morphology and relate this to the fungal genera or species involved (Agener, 1987-1997). The main disadvantages of this method are that: a) it is time consulting and requires detailed descriptions of the identified material, b) there are no available descriptions on changes in morphology of mycorrhizas during the course of development and in response to environmental stresses etc., c) there are few available details on difference in appearance depending on the host plan~ d) it demands a high degree of skill and experience. Probably due to the above reasons, only a small number of

14 mycorrhkzal fungal species have been identified by this method. The main advantages of this method are that a) it is cheap and b) relatively larger numbers of roots can be analysed than with molecular methods. b) Molecular biological methods Since the discovery of the polymerase chain reaction (PCR) (Mullis and FaloonA 1987), a number of DNA-based methods have been developed for the identification of ectomycorrhizal fungi both in pure cultures and in symbiotic association with roots (Gardes et al., 1991; Henrion et al., 1992; Gardes and Bruns, 1993; Erkmd et aI., 1994; Kreuzinger et al., 1996; Martin et al., 1998). The method which has been used extensively in this thesis involves PCR amplification of the internal transcribed spacer region (ITS) of ribosomal DNA, and its restriction fiagrnent length polymorphism (RFLP) analysis (M&mood et al, 1999). The ITS-region allows identification at the level of species because it has high inter- and low intra-specific variation at least at the regional level (K&ren el al., 1997). It is easily accessible by universal primers (e.g. ITS-1 and ITS-4, which exclusively ampli@ ITS- regions of fungal origin from Picea abies mycorrhizas). For an ITS-type to be identified to species a match with an RFLP ffom an identified fruit-body is needed. Other molecular methods which have been used are sequencing and taxon or species specific probes (Terashima and Nakai, 1996; Kreuzinger et al., 1996). If the ITS-sequence is known it is possible to identi@ matches, or at least close relationships to identified species in databases. The best resolution of below-ground communities has so far been obtained by combinations of highly skilled morphotyping and PCR/RFLP-typing, which have allowed comparatively large samples to be analysed (Taylor et al., 2000).

Effects of pollution and forest management on ecto- mycorrhizal community structure

Effects of N deposition Most of the available information on the effects of N deposition on ectomycorrhizal communities comes fkom fruit-body inventories. The dynamics of ectomycorrhizal fmit- body production have been used extensively as a bio-indicator of environmental or pollution-induced changes (Wtisterlund, 1982; Terrnorshuizen and Schaffers, 1987; Markkola and Ohtonen, 1988; Amolds, 1988, 1991), mainly because it is much easier to observe changes in above-ground fi-uit-body production than below-ground mycorrhlzas. However, recent investigations have shown that the species composition based on above- -ground fruh-body production has little or no correlation with the below-ground mycorrhizal community structure (Mehmann et al., 1995; Gardes and Bruns, 1996; K5r& and Nylund, 1997; Dahlberg et al., 1997; Jonsson et al., 1999).

In general, a number of investigators have reported a clear decline in the diversity of ectomycorrhizal sporocarps in response to N-deposition/N-fertilisation (Wiisterlund, 1982;

15 Ruhling and Tyler, 1990; Arnolds, 1991; Termorshuizen and Schaffers, 1991; Brandrud, 1995; IBaar, 1996). The genera most sensitive to N deposition appear to be Cortinarizis and Russuia, whereas the genus Lactarius shows a small response, and a few species such as Paxillus involutus and Lactarius rujixs show an increase in tluit-body production (Brandrud, 1995). Termorshuizen (1993) reported a reduction in above-ground production of fruit- bodies due to higher levels of N fertilisers but the below-ground mycorrhizal frequency and the number of mycorrhizas per unit of soil vohune were not affected. Similarly, Brandrud and Ti]mmermann (1998) reported a lack of effect on the below-ground mycorrhiza and fine- root density and diversity, however there was a rapid and substantial decrease in species diversity and fruit-body production of most ectomycomhizal species. In contrast to these below-ground studies, Baxter et al. (1999) reported a decrease in ectomycorrhizal morphotypes from 26 to 16 on mature oak in rural (low N deposition and heavy metal) and urban (high N deposition and heavy metal) soils respectively. In the same study, nine ectom!ycorrhlzal types were distinguished on Quercus rubi-a seedlings grown in rural soils versus seven in urban soils. Despite fewer ectomycorrhizal types in urban soils, the richness of ectc}mycorrhizal types per centimetre fine root of mature oak or Q. rubra seedlings did not differ between urban and rural soils. Ectomycorrhizal colonisation of mature trees (ectomycorrhizal tips/m fine root) was lower in urban than in rural soils but higher on Q. rubra seedlings grown in pots containing urban soils than those with rural soils. Fine root length per unit soil volume was higher in urban than rural stands. In another investigation, Woellecke et al. (1999) reported a strong reduction in ectomycorrhizal morphotypes on Scots pine at a high N site (only nine types) compared with a control (low N) site (eighteen types), At the low N site the highest number of mycorrhizal root tips was found in the organic layer. At the high N site the amount of mycorrhizas per unit soil volume was similar in both organic and mineral soil layers, and also significantly lower compared to the amount at the low N site.

In the study by K5r6n and Nylund (1997), the nitrogen treatment reduced the fine-root biomass (to 49% of the control) but did not decrease the mycorrhizal frequency (close to 100%)I or concentration of ergosterol in fine roots. By using PCR-RPLP methods, they reported 21 restriction profiles on roots and only four of them matched the restriction patterns of dominant sporocarps on the site. They suggested that nitrogen deposition will primarily change the community structure of ectomycorrhizal fimgi, whereas the number of species may be less affected than has been previously inferred tlom sporocarp inventories. Wallenda and Kottke (1998) reviewed the available data from long-term N deposition studies and concluded that the most prominent negative effects might be on the formation of hit-bodies. According to them, “generalist” species, which form mycorrhizas with a wide range of tree species, are less affected by increased N availability than “specialist” species, especially those which establish symbiosis with conifers. Recently Taylor et al. (2000) examined the fimgal diversity in ectomycorrhizal communities of spruce and beech forests along north-south transects in Europe. They collected information on below-ground mycorrhizas and above-ground fruh-bodies, and concluded that in spruce forests the decline

16 in diversity towards the south (highly N polluted) of the transect is accompanied by an increase in single species dominance and is paralleled by changes in the nitrogen nutrition of the fhngi involved. Characteristic fimgai species of northern sites (less N polluted), which are able to utilise organic N, decreased in number as the availability of mineral N increased towards the south.

Most of the above mentioned studies of N deposition involve either fruit-body surveys or morphotyping of mycorrhizas. The study mentioned in Paper I, was carried out by using a DNA-based analysis of ectomycorrhizal community structure on roots in two geographically separated 60-yr-old spruce forests with different levels of atmospheric N deposition - to further expand our knowledge on the factors which may induce changes in below-ground ectomycorrhizal communities.

Effects of forest harvesting practices Removal of residues from forests results in changes in the soil physical and chemical environment and may disrupt nutrient recycling processes in the ecosystem. Ectomycomhizal fungi depend on living trees for their carbon supply and are deprived of thk by tree harvesting.

Hagerrnan et al. (1999) investigated the effects of ciear-cut logging on the diversity and persistence of ectomycomhiza in a subalpine forest in British Columbia. Over the course of the study, they reported a total of 39 distinct mycorrhizal types. The dominant types matched descriptions of E-strain mycorrhlzae and of mycorrhiza formed by Cenococcum spp., two types of Lactarius spp., Piloderma spp., Hebeloma spp., Amphinema spp., and Cortinarius spp. There was no effect on the diversi~ of ectomycorrhiza with treatment after one growing season. However, after two and three growing seasons the numbers of active fine roots as well as the diversity of ectomycorrhiza in clear-cuts was significantly reduced with distance from the forest edge. Changes in nursery and indigenous mycorrhka of Scots pine seedlings planted out in forests and clear-cuts were examined by Dahlberg and Stenstrom (1991). They reported that in general clear-cutting had a minor effect, both qualitatively and quantitatively. Nineteen different mycorrhizal types were recorded in their study. Piloderma croceum colonised seedlings significantly more ilequently in forests than in clear-cuts, whereas the reverse was found for Cenococcum geophilum, and two other mycorrhizai types. Clear-cut areas of different sizes in the Interior Cedar-Hemlock forests of British Columbia decreased ectomycorrh~al fungal richness (based on epigeous sporocarps) exponentially as gap size increased (Durall et al., 1999). In the same study, ectomycorrhlzal richness on seedlings decreased slightly with increasing distance from the edge of the intact forest. The decrease in richness with distance horn the forest was associated with an increase in the proportion of I%elephora mycorrhkas in the samples. Numbers of ectomycomhiza were assessed 3 yr after harvesting approximately 50% of the overstory in 2 Douglas-fir-larch stands in western Montan% one was subjected to intensive

17 residue removal, the other broadcast burned 1 yr after harvest. Numbers of active ectomlycorrhizal root tips were significantly reduced in the broadcast burned stand compared to either the intensively utilised stand or to an adjacent, undisturbed stand (Harvey et al., 1980). Page-Dumroese et al. (1998) analysed the impact of soil compaction and tree stump removal on out planted seedlings in northern Idaho, and reported that these treatments reduced the numbers and morphological types of ectomycorrhizas and non-my corrhizal short roots on Douglas-fir. Western white pine seedlings had reduced numbers of non- mycorrhlzal short roots in the same treatments. Waters et al. (1994) investigated the effects of commercial thinning on sporocarp production of hypogeous ectomycorrhizal fungi at two sites in north-eastern California. Total relative frequency and biomass of sporocarps did not differ significantly among thinning levels at either site. There was, however, a significant association between thhing level and the frequencies of the most common genera at one site (Jennie Springs), suggesting that thinning significantly affected the composition of hypogeous ectomlycorrhlzal fungi. The study mentioned in Paper II was carried out to find whether harvesting of clear-cutting and thinning residues would affect below-ground ectom ycorrhlzal community structure in a 35-yr-old spruce forest.

Effects of ash fertilisation and liming Both wood ash fertilisation and liming increase soil pH and base saturation and reduce the avaiklbility of toxic heavy metals in soils. They have therefore been proposed as countermeasures to on-going acidification. To date there are only a few studies which report a direct effect of ash fertilisation on ectomycorrhiza.

According to Riihling (1993), wood ash fertilisation had no significant effect on t?uit-body production. There are several reports of reductions in ectomycorrhizal fruit-bodies after liming (Kuyper and De-Vries, 1990; Andersson and Soderstrom, 1995; Kraepelin and Michaelis, 1997), however, De-Vries et al. (1995) found no significant effects of liming on ectom,ycorrhizal fkuit-bodies. Similarly, Agerer et al. ( 1998) reported no adverse effects of acid rain and ameliorator liming on either the diversity or productivity of ectomycorrhizal fimgi in a spruce stand. In their study, a combination of acid irrigation and liming resulted in thirty times more fruit-bodies than in the control.

Erlaml and S5derstr6m (199 la) reported six different ectomycorrhizal morphotypes on Pinus sylvestris seedlings planted in a pine forest treated with lime and ash. Despite a large pH difference (control plots pH 3.8, limed plots pH 5.2 & ash plots pH 6.4), no marked changes in the ectomycorrhizal community colonizing roots were observed. An unidentified pink type of mycorrhiza was more than twice as common in the limed plots than in the ash treated and control plots. Similarly, Piloderma croceum mycorrhizas were significantly more abundant in the lime treatment than in the ash treatment although they were infrequent

18 in both (3% and 1YOrespectively). In a laboratory assay, Erland and Soderstrom (199 lb) found five different mycorrhizal morphotypes on seedlings grown in forest humus treated with different amounts of lime to produce a soil pH gradient. These types were present throughout the entire pH range, except for 1? croceum which was not found at values higher than 6.2.

Ectomycorrhizal colonisation of Picea abie.s seedlings planted in a limed spruce forest, was investigated by Andersson and Soderstrom (1995). They reported six ectomycorrhizal morphotypes: three types (Paxdlus invoiutus, a white type and Pinirhiza roses) decreased after liming, two types (the brown type and Piceirhiza nigra) increased and one type (Cenococcum geophilum) was not affected consistently by the liming.

In a study by Antibus and Linkins (1992), liming did not appear to affect diversity of ectomycorrhizal morphotypes, however, lime did increase the relative fkequency of certain ectomycorrhizal morphotypes. Recently, Jonsson et al. ( 1999) using PCR-RFLPS, reported 16 ectomycorrhizal ITS-types on roots in a limed spruce forest. A similarity assessment suggested a shMt in ectomycorrhizal community structure as a result of the treatment. But variation within treatments was large and there were no statistically significant differences between treatments with respect to specific taxa. To date there are no reports on the effects of granulated wood ash on below-ground ectomycorrhkal community structure on spruce trees. Paper III describes the colonisation of roots and ash granules in a spruce forest treated with granulated wood ash.

19 Experimental approaches

A major objective of the studies described in this thesis was to integrate field studies of mycolThizal community structure with laboratory based studies of function. Three community studies were performed to examine the effects of a) N deposition (Paper I), b) removal of forest residues (Paper II) and c) application of granulated wood ash (Paper III). Where possible mycorrhizal fungi identified in field studies were isolated and used to synthesize mycorrhizal associations which were used in subsequent laboratory experiments (Papers IV, V, Appendix).

Field !sites Vedby & SkrylIe Two similar forests (Skrylle and Vedby) with contrasting patterns of N deposition were chosen for the initial community study (I). The forests were similar with respect to stand age, soil type and previous vegetation. Vedby is located fi.u-thestto the north (lat. 56°10’ N, long. 13°10’ E, alt. 50 m) and was planted with Norway spruce (f’ices abies (L) Kant.) in 1935. Skrylle (lat. 55°40’ N, 13°20’ E, alt. 90 m) was planted with Norway spruce in 1934. Both forests are first-generation spruce plantations and were used as pasture prior to planting. The soil type is brown podzol at both sites (humus pH (H20) 3.6 in Vedby, 4.1 in Skrylle). The average yearly N throughfall was 14-15 kg N ha-l in Vedby and 24-29 kg N ha-l in Skrylle. The N03 content of percolated soil water was <0.01 mg din-3 in Vedby and >2 mg din-3 in Skrylle. Average rainfall 1988-95 was 395 mm and 345 mm for summer and winter respectively at Vedby and 329 mm and 356 mm for summer and winter respectively at Skrdle. There were five blocks (20 x 20 m2) in each forest arranged within a 1 ha area.

Tonnersjoheden Tonnersjoheden Experimental Forest located in the south-west (56° 41’ N, 4°57’ E) of Sweden was chosen for the study of forest residue removal (II). The annual mean temperature is 6.5 ‘C and the annual precipitation 1000 mm. The soil type and texture have been classified as podzol and sandy-silty till respectively. The experiment was started in

1961 ifter clear-cutting and plantation of a second generation Norway spruce (Picea abies (L.) Karst.) forest. Residues have been removed fkom treated plots on four occasions, after the clear-cut in 1961, and during subsequent thinning in 1975, 1986 and 1992. Control plots were thhmed at the same time but residues were left on the forest floor. All plots were of the same size (15 m x 10 m) and arranged in a randomised block design.

Torup The study of effects of ash application (III) was performed at an experimental forest at Torup in the south-west of Sweden (56° 55’ N, 13°05’ E). The region has an amual precipitation of 800-1000 mm and a mean temperature of 6-7”C. The soil type and texture have been classified as podzol and sandy-till respectively. The present forest was planted in 1957 with a second generation Norway spruce (Picea abies (L.) Karst) after clear-cutting of

20 Scots pine (Pinus sylvestris L.). Three treatments were applied in 1990, unfertilised (control), and two treatments of granulated wood ash, 3 ton ha-l (low ash) and 6 ton ha-l (high ash). All plots were of the same size (30 m x 30 m) and arranged in a randomised block design.

Community studies Different approaches were followed in the three community studies as we gained more experience of handling root material.

In Vedby and Skrylle (I) sampling of ectomycorrhizal roots was done by taking five cores (1 O x 10 cm2) from the humus layer of each block in May 1995. Root tips were morphotyped and counted on a per m root length basis. For molecular identification, DNA was extracted from single mycorrhizas, ITS-region of the ribosomal DNA was PCR- amplified and then digested with Hirzf I, Mbo I and Tag I endonucleases to produce RFLPs. For identification of ectomycorrhizal species, these restriction fragments were compared to a reference library containing RFLPs of regionally collected, identified ectomycorrhizal fkuit-bodies and axenic cultures.

At Tonnersjoheden ectomycorrhizal roots were sampled on two different occasions (autumn 1995 and spring 1996), by taking five cores randomly tlom the humus layer of each plot (II). In addition, 10 cores from the humus layer of each plot were taken to measure the thickness of the humus layer in each core before extracting all the roots. Both the total numbers of mycorrhlzas in each core and the numbers of mycorrhlzas per m root length were counted. No prior morphotyping was done before extracting DNA from single mycorrhizas. ITS-RFLP patterns were compared with identified fruit-bodies from Tonnersj6heden and other forests in the region.

At Torup ectomycorrhizal roots were sampled in May 1997 by randomly taldng five cores from the humus layer of each plot (III). No attempts were made to morphotype mycorrhizas before extracting DNA from single mycorrhizas. Ash granules were collected from the treated plots for scanning electron microscopy. Mycelial layers were sampled from the surface of well colonised ash granules for molecular fungal identification. ITS-RFLP patterns of mycorrhizas and mycelia from ash granules were compared with identified fkuit- bodies from Torup and other forests in the region.

Laboratory studies Isolation offungiji-om roots Ectomycorrhizal fungi were isolated from mycorrhizas collected fkom a granulated wood ash fertilised spruce forest located at Torup in southern Sweden (IV). The molecular identities of these isolates were established by ITS-typing using the PCR-RFLP method and

21 by comparing the restriction patterns with a reference library consisting of RFLPs of identified fruit-bodies and ectomycorrhizal fimgi found in our previous community structure studies on tree roots at Torup and other forests located in southern Sweden.

In vitro testing of wood ash solubilisation capacity Thirty eight fimgal isolates of Cenococcum geophilum, Piloderma croceum, Thelephora terrestris, Tylosporaj7brillosa, and five unidentified species (designated as Ha-96- 1, Ha-96- 3, Tor-97-4, To-95-3, Ve-95- 1 and Ve-95-3), were tested for their ability to solubilise tricalcium phosphate (TCP) or hardened wood ash (HWA) in vitro (IV). The fungi were grown on cellophane membranes spread on a modified Melin-Norkrans medium containing alanine, ammonium or nitrate as a nitrogen source with a surface layer of agar containing TCP. ‘Visual assessment of solubilisation after 120 days was made by observing the clear zones in the agar around and under colonies after removing the cellophane membrane. Crystal formation in the agar was observed in this experiment and a second experiment was performed using the same isolates to monitor crystal formation in TCP and HWA plates containing alanine as the sole nitrogen source. An inverted microscope was used for direct and ncm-destructive visualization of the medium in the Petri dishes. The purified crystals either horn TCP or HWA plates were subjected to scanning electron microscopy and X-ray microprobe analysis.

Analysis for P content in the mycelium Selected ectomycorrhizal fimgi were examined for P content in the mycelium when grown on cellophane membranes spread on HWA-containing plates (IV). Mycelia were collected carefi,dly avoiding any contamination and freeze-dried. Dried mycelia were digested in concentrated nitric acid prior to analysis of P by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).

Myceliial growth and carbon allocation in microcosms containing intact mycorrhizal associations and wood ash amended substrates Mycorrhizal associations were synthesised between spruce seedlings and different isolates (P. crocez.un, Tor-97-4 and C. geophilum) and well-colonised seedlings were transferred to microcosms containing peat (IV). After two months, when the mycelia started growing actively from the roots, small peat filled plastic cups were embedded carefully around the root system, close to the margin of the extending mycorrhizal mycelium (IV, Fig. 5 & 6). Within each chamber five cups contained only peah while the remaining five cups also contained a surface layer of ash, and mycelial colonisation of the cups was subsequently examined. Possible differences between the fingi in patterns of carbon allocation were investigated by labelling seedlings with 14COZand analysing the distribution of labelled carbon, using sample oxidation and liquid scintillation counting.

22 Mycorrhizal competition and root colonisation in wood ash amended sand-peat A pot experiment was performed to examine the dynamics of colonisation of spruce roots by two ectomycorrhizal fungi in sand-peat with and without wood ash (V). Non- mycorrhlzal spruce seedlings (bait seedlings) were planted between two spruce seedlings pre-colonised with P. croceuin and Tor-97-4 respectively. The growth-substrate was a sand- peat mixture amended with wood ash (or left unamended) and supplied with two levels of N, so that four substrate combinations (LN +A, HN +A, LN –A and HN –A) were obtained. The plants were grown for 120 d before harvesting. Ectomycorrhizal colonisation status of bait seedlings and pre-colonised seedlings was determined by counting mycorrhizal and NM root tips on a per m root length basis. Plant materials were oven dried for weight determinations.

Nutrient uptake jiom hardened wood ash – by mycorrhizal and non-mycorrhizal spruce seedlings An additional experiment (not fully reported in this thesis) (Appendix) was conducted to examine the role of mycorrhizal mycelium in mobilizing nutrients horn hardened wood ash and the possible effects thk may have on plant growth and nutrient uptake. Mycorrhizal spruce seedlings colonised by five fi.mgal isolates (Tor-97-4 (Tor-35), Tor-97-4 (Tor-67), P. crocewn (Tor-02), P. croceum (Tor-24) and Ha-96-3 (Tor-78)) were grown in sand-peat substrate amended with hardened wood ash (or left unamended) and two levels of a slow release N fertiliser (treatment combinations were: LN +A, HN +A, LN –A and HN –A). Non-mycorrhizal seedlings were used as controls. The plants were grown in pots for 120 d before harvesting. The central portion of each pot containing the root system of each seedling and sand-peat substrate was enclosed in a nylon mesh bag (Appendix, Fig. 1). The ash was mixed into the soil in the outer compartment of each pot. Plants and growth substrates were subjected to following analyses: dry weight of shoot and root, elemental analysis of shoot and root (by ICP-AES), soil solution (from mycelial compartment) analysis for N03, POJ and oxalate (by ion chromatography). Further analyses to illustrate bacterial activity in the mycelial compartment (as determined by thymidine and leucine incorporation rates), changes in soil microbial community structure (as determined by phospholipid fatty acid analysis (PLFA)) and fimgal biomass on roots (as determined by ergosterol analysis) are in progress.

23 Results and Discussion

Community studies Vedby & Skrylle (I) The level of mycorrhizal colonisation was almost 100% at both sites, but the total number of mycorrhizal roots was 30-42°/0 higher at the northern site. Six morphotypes were distinguished at the northern site with the lower N deposition (Vedby), and four at the southern site (Skrylle). Some morphotypes consisted of several ITS-types and identical ITS- types were also found in roots assigned to different morphotypes. The RFLP data revealed a total of 16 ITS-types. Eleven of these were identified at least to genus by comparison with local or regional reference material. Five ectomycorrhizal taxa, Cenococcwn geophilunz, Thele$)hora rerrestris, Tylospora jlbrillosa, Tylopiius felleus and Ve-95-3 were common to the two sites but the total number of taxa recorded at Vedby was twice as high as at Skrylle.

Tonnersjoheden (II) Harvesting of forest residues significantly decreased the thickness of the humus layer, as well as decreasing the numbers of ectomycorrhizal root tips both per metre root length and per urlit humus volume. In total, 19 different ITS-types were found on two different sampling occasions (autumn and spring); 11 of these were common to both samplings. Nine of the ITS-types were identified to at least the genus level by comparison with RFLP patterns of identified fruit-bodies or axenic cultures. Five species, Cortirzarius sp. 2, I%elephora terrestris, Lactarius theiogalus, Tylosporafibrillosa and Tti-96-12, occurred on over YZO of the total sampled root tips. Together these species colonised 63°A of the mycorrhizas screened. A similarity index assessment showed no shift in mycorrhizal community structure as a result of harvesting.

Torup (III) In total 20 different ectomycorrhizal ITS-types were recognised on roots in the soil organic horizon. Five of these were identified to species and one to genus. Six species, Tyiospora j?brillosa, Cortinarius sp. 3 and four unidentified ITS-types (Tor-97-4, Ve-95-1, To-95-3 and Ve-95-9) each occurred on over 5’%0 of the total root tips analysed. Together these comprised 55°/0 of the ectomycorrhizal community on the screened roots. A similarity index assessment suggested a shift in mycorrhizal community structure as a result of the ash application. Statistically significant differences in occurrence of single species could not be shown. Some trends could, however, be detected and fewer Cortinarius sp. 3 mycorrhlzas were hmd in plots treated with higher levels of ash while the numbers of Tor-97-4 and Ha- 96-3 mycorrhizas increased. Ash granules collected horn the fertilised plots were normally colonised by fi.mgal mycelia (III, Fig. 3). PCR-RFLP analysis of these mycelia revealed the presence of four ITS-types. Three of these (Tor-97-4, Ha-96-3 and Tor-97- 1) were also present on the mycorrhizal roots and two of them corresponded to taxa which had a tendency to increase in ash treated plots.

24 Methodological problems and overview of community studies The PCR-RFLP methodology provided a valuable tool with which to study ectomycorrhizal communities. The resolution of taxa was much higher than our coarse morphotyping and allowed us to match individual ITS-types colonizing roots and ash granules. The extremely high root density and small numbers of roots which can be examined using this method make it less usefil in detecting overall changes in community structure with a high degree of statistical certainty. However we detected similar numbers of taxa to studies using morphotyping despite having much smaller root samples. With the exception of one polluted site (Skrjdle) we found 13-20 ectomycomhizal species colonizing roots in each of the studied spruce forests. This is in general agreement with other community structure studies in Swedish coniferous forests suggesting a species range between 12-32 (llihlberg et al., 1997; K&en and Nylund 1997; Jonsso% L. et al., 1999kb; JonssoL T. et al., 1999). Similarly Bruns (1995), reported 13-35 ectomycorrhizal species in monoculture forests along the Californian coast. In each of our experimental forests, only 5-6 species were found colonizing around 60°/0of the examined root tips.

The species which could be identified by comparison to the reference material were; Amanita fulva, Cenococcum geophiluin, Lactarius necator, L. theiogalus, Piloderma croceum, Thelephora terrestris, Tylopilus felleus, and Tylospora f?brillosa (I-III). Attempts to identi~ more of the important and dominant ITS-types to species by sequencing are currently in progress. In the second and thkd communi~ studies we used a non-stratified sampling strategy (no morphotyping of root tips prior DNA-analysis). Thk allowed us to identi~ the dominant/abundant species in the community and to detect changes in overall community structure in response to the treatments (II, III). However, thk method is not sufficient for determining changes in single species with a high degree of statistical certainty or for describing total species richness. To obtain information not only on the most abundant species in the community but also on the occurrence of “rare” species, analysis of much larger samples would be necessary. The high root densities in these soils mean that even “rare” species occurring at very low frequencies may colonise large numbers of roots and thus be functionally important. Sampling of roots on different occasions during the year helps to take account of any temporal changes in the community structure. In paper II, sampling of the roots was done on two different occasions (autumn and spring), we found a high degree of similarity in the abundance of dominant species in both samplings. Four of the relatively less evenly distributed or the rare species disappeared in the spring harvest and the two new species found also belonged to the ‘tail’ of the ranked abundance distribution. This indicates that with additional samplings only the ‘tail’ of the ranked abundance gets longer but the relative abundance of dominant species remains fairly constant. Patchiness in the distribution of species was another potential problem which we encountered in our studies. We were unable to show any significant effects of treatments on single species basis because of large spatial variation in the distribution. Spatial variation is a natural phenomenon due to heterogeneous distribution of nutrient resources in soil. This variation

25 can be tackled to some extent by taking a large number of small sub-samples and then pooling them or by more intensive sampling of roots from plots.

E#ects of Ndeposition and site history A reduction in ectomycorrhizal sporocarps and diversity on roots in response to increased N availability has been reported in a number of earlier investigations (Wasterlund, 1982; Ruhling and Tyler, 1990; Arnolds, 1991; Brandrud, 1995; Wallenda and Kottke, 1998; Baxter et cd., 1999; Woellecke et al., 1999). Fruit-body production in some ectomycorrhizal species belonging to the genera Cortinarius and Russzda is very sensitive to N fertilisation (lhnclrud, 1995). Although the effects on fruh-bodies are generally more pronounced than on roots (Wallenda and Kottke, 1998), increased levels of N can reduce mycelial growth and root colonisation potential (Arnebrant, 1996; Arnebrant and Soderstrom, 1992; Wallander and Nylund, 1992). Recently, a decline in the diversity of ectomycorrhizal species in spruce forests was reported along north-south (from less to high N polluted) transects in Europe (Taylor et al., 2000). Thk decline in species was accompanied by an increase in single species dominance and changes in the N nutrition of the fingi involved. In our study (I) atmospheric N deposition may have had a strong adverse effect on mycorrhizal community structure. With a similar sampling effort only 7 ectomycorrhizal species were found in the forest with high N deposition (24-29 kg N ha-l yr-l) at Slu-ylle compared to 13 at Vedby (14-15 kg N ha-l yr-l). Moreover, there were significantly more mycorrhizas per m root length at Vedby than at Skrylle. In our investigation no Cortinarius spp. were found at Skrylle. A close examination of the geography and history of the Skrylle forest revealed that it is situated below the limit of natural occurrence of Norway spruce while the Vedby site lies on[the boundary and may have contained spruce prior to its use as pasture. Vedby is also located close to other forests while Skrylle is isolated and surrounded by farmland. Thk difference in history and location of forests complicates our interpretation of the lower number of species being due to high N deposition alone. The higher number of species at Vedby could thus also be a result of higher inoculation potential from other forests situated close by rather than being due to lower N deposition alone. Factors such as anthropogenic stress, site history and degree of isolation may thus all play a role in determining the ectomycomhizd community structure of roots (I).

Possible methods to reduce N accumulation include removal of forest residues (Lundborg 1997, 1998). The critical load for N deposition also becomes much higher if residues are removled, in comparison with when they are left on the site. The harvested biomass can be used for combustion to produce energy and resuhant ashes can thus be applied to forest soils to supplement the lost nutrients. The model proposed by Lundborg (1997, 1998), may have implications in some coniferous forests with severely high N deposition in the south- western parts of Sweden, whereas the deposition levels decrease towards the north (Grennfelt and Hultberg, 1986).

26 E#ects offorest residue harvesting In our investigation of the effects of repeated harvesting of forest residues on the ectomycorrhizal community structure (II), we could not find any significant changes in the species composition on the roots. However, harvesting of biomass significantly decreased the tlickness of the humus layer as well as decreasing the numbers of ectomycorrhizal root tips both per metre root length and per unit humus volume. This decline in the number of roots may be due to changes in the physical and chemical environment in the soil caused by intensive harvesting. A thh layer of humus responds more rapidly to temperature changes in the air and is also more sensitive to drought than a thicker layer of humus. Fine roots are sensitive to changes in temperature and moisture in the soil (Lindstrom, 1986; Tabbush, 1986; Husted and Law%der, 1989; Vapaavuori et al., 1992; Persson et al., 1995). The main explanation for the smaller numbers of mycorrhizas in the humus layer is likely to be a decrease in the numbers of fine roots available for colonisation. Analysis of roots from the treated plots showed a marked reduction in Ca/Al ratios compared with roots in the control plots (H. Erlandsson and H. Lundkvist, pers. corn.). Lower Ca/Al ratios have an adverse affect on growth of Norway spruce roots (Godbold et al., 1988; VogeIei and Rothe, 1988). Similarly, Finlay (1995) reported that long-term exposure to high concentrations of Al resulted in negative effects on ectomycorrhlzal colonisation of Fagus sylvatica and Pinus contorts seedlings by Paxilius invoiutus and suggested that this effect seemed to be related to dkect effects on the plant roots and a decrease in the number of root tips available for fimgal colonisation rather than any direct effect of Al on the fimgus. Baar & De-Vries (1995) investigated the effects of complete removal or doubling of the humus layer in severely nitrogen and sulphur polluted Pinus syivestris forests. They found a negative relationship between humus thickness and numbers of ectomycorrhlzal species colonizing seedling roots, but these results are probably explained by the high N pollution and competition from herbaceous species such as Deschainpsia fkxuosa. In our study removal of organic matter, resulting in a thinner humus layer, had a negative overall effect on the number of mycorrhbal roots but no significant effect on the number or composition of species. Chemical analysis of organic material from our biofiel harvested plots indicates a relatively higher exchangeable acidhy, lower base saturation and exchangeable base cations (J. Bengtssom H. Lundkvist and M. Olsson, pers. corn.). Moreover, a negative effect on tree growth was recorded in the treated plots (BjtMcroth, 1984).

Effects of ash application One way to ameliorate acidification, caused either by high N and S deposition or increased removal of forest residues, is to apply wood ash to such forests. In paper III, we examined the effects of granulated wood ash on the ectomycorrhizal community structure of roots. A shh?tin the ectomycorrhizal community was noticed 7 yr after fertilisation but no significant differences in the occurrence of single speeies could be detected. This is in conformity with earlier findings of Erland and Soderstrom (1991a), who reported no significant effect of ash on the ectomycorrhlzal community, despite a much higher pH difference, with control spruce seedlings growing at pH 3.8 compared to pH 6.4 in treated plots. However, we

27 noticeci some trends in species occurrence, for instance, Cortinarius sp. 3 mycorrhkas were quite abundant in control plots but declined in low ash treatment and could not be detected in high ash applied plots. The sensitivity of some Cortinarius species to liming and N fertilisation has been reported previously in the literature (WWerlund, 1982; Brandrud, 1995; Jacobsson, 1993). Two unidentified mycorrhizal ITS-types designated as Ha-96-3 and Tor-97-4, showed a tendency to increase in abundance in wood ash fertilised plots. The most interesting finding was that the mycelia of these two types were found colonizing 74 and 7% of the examined ash granules (III, Fig. 3). Since most of the base cations and trace elements are easily leachable from ash granules except P which is bound in apatite compounds with low solubilities (Steenari and Lindqvist, 1997), it is likely that mycorrhizal mycelia colonizing the ash granules are involved in direct uptake of P. This speculation is further supported by the fact that the tree needles from ash treated plots showed significantly higher concentrations of P compared to the untreated controls, whereas concentrations of P04 in soil solution did not vary markedly between the treatments, indicating that P solubilisation was very slow (Jacobson and Ring, 1995). Ectomycorrhiz.al fungi were isolated from the roots to test their nutrient mobilisation abilities in laboratory experiments and to re-synthesize mycorrhizal associations for use in microcosm experiments.

Functional aspects of selected mycorrhizal fungi Mycei,ial growth and carbon allocation in microcosms containing intact mycorrhizal associations and wood ash amended substrates Tor-97’-4 and P. croceum isolated from the roots were tested for their ability to colonise peat amended with wood ash in microcosms containing intact mycorrhizal associations (IV). After seven months Tor-97-4 colonised ash amended patches completely with a thick mat like rnycelium, whereas control patches were colonised more slowly and incompletely by a less dense mycelium (IV, Fig. 5Zb). P. croceum mycelia avoided ash patches and distinct hollow zones appeared in the mycelium at the sites of the ash patches, whereas control patches were colonised (IV, Fig. 6Ab). In a carbon allocation assay, Tor-97-4 mycelia allocated significantly more 14C to ash patches than P. croceum. P. croceum allocated relatively more 14Cto control patches than to the ash patches.

Possible reasons for colonisation of ash patches by Tor-97-4 mycelia are; a) the fungus is having a high pH growth optimum and is growing well at pH 6.6 in the ash patch, b) the fungus is responding to certain nutrients in the ash (like P) which were not otherwise available, or a combination of these factors. Erland (1990) reported more extensive external mycelial growth of an unidentified pink ectomycorrhizal isolate (Pink LMT 85:2) at pH 7 than at pH 3.8. P. croceum mycelia avoided ash patches, whereas control patches were colonised. This may be due to sensitivity of P. croceum to high pH in the ash patches. P. croceurn showed a narrow pH tolerance interval compared to other mycorrhizal fungi in studies conducted by Erland and Soderstrom (1990). These authors grew pine seedlings in

28 limed humus (pH 4 – 7.5) and noticed that P. crocewn did not colonise at pH values above 6.2, and that the optimal pH for root colonisation was around 5.

In the field study, Tor-97-4 showed a tendency to increase in abundance with increasing doses of wood ash and was the most abundant species in plots treated with the highest dose of ash (III). In the next experiment we studied the influence of wood ash on the colonisation potential of Tor-97-4 in competition with P. croceum (V).

Mycori%izal competition and root colonisation in wood ash amended sand-peat P. croceum and Tor-97-4 were selected for a competition experiment (in intact symbiotic systems) to evaluate their comparative abilities to colonise roots of non-my corrhizal spruce seedlings treated with factorial combinations of wood ash and N fertiliser (V). Tor-97-4 mycelia colonised around 60% of the fine roots of bait seedlings in ash treatments regardless of nitrogen level and around 20-26°/0 in treatments not receiving ash. P. croceum colonised only around 8’XOof the root tips in the presence of ash but 56°/0 of the root tips in the low nitrogen treatment without ash. However, in the high nitrogen treatment without ash the colonisation level was reduced to around 30% (V, Fig. 1). Total numbers of root tips per seedling did not vary significantly between the treatments.

This study demonstrates that Tor-97-4 mycelia have a competitive advantage over P. croceum when grown in a wood ash amended substrate. Reduced colonisation of P. croceum in the ash treated substrate could be due to high pH as discussed earlier. Moreover, P. croceum mycorrhizas declined drastically at the high level of N. This might be due to sensitivity of P. croceum to high N concentration in the substrate. In a similar study of competition between Hebeloma crustulinl~orme and P. croceum in N amended substrates, a marked reduction in P. croceum mycorrhizas was noticed with increased N concentrations (Arnebrant, 1996). However, Jonsson et al. (1999c) reported a significant increase in the abundance of P. croceum mycorrhlzas after experimental nitrogen additions to a Norway spruce forest in south-western Sweden. Many investigators report a reduced mycelial growth and colonisation potential for a majority of ectomycorrhizal fungi in high N treatments (Wallander and Nyhmd, 1992; ArnebranL 1994; Arnebran6 1996; Wallenda and Kottke, 1998). Wood ash may have stimulated the growth of Tor-97-4 mycelia either through increased pH or by nutritional effects and thereby increased the inoculum potential for colonisation of new roots.

In vitro testing of wood ash solubilisation capacip We managed to isolate 9 out of 20 species colonizing spruce roots in the Torup community structure study, including Ha-96-3 and Tor-97-4 (which also colonise ash granules in the wood ash treated plots) (III). Thirty eight isolates of these species were used in laboratory studies of nutrient mobilisation (IV). In these studies Tor-97-4 mycelia had a great potential to solubilise both TCP and HWA in vitro and to take up P from HWA. Ha-96-3 and P. croceum had some ability to solubilise TCP or HWA and to take up P from HWA. All of

29 the other fungi tested had a generally poor ability to solubilise these substrates. Solubilisation of the HWA by Tor-97-4 was accompanied by abundant production of crystals, subsequently identified as different forms of oxalate by scanning electron microscopy and energy dispersive X-ray micro-nutrient analysis. Twenty two isolates were further tested for crystal formation in TCP and HWA plates. Within 120 days of inoculation, abundant crystals were formed in TCP and HWA plates by all four isolates of Tor-97-4. Ha- 96-3 and two isolates of R croceum produced intermediate amounts of crystals on both TCP and HWA plates. Ha-96-1 and T fibrillosa produced low amounts of crystals while no crystal, formation was observed by any of the other isolates. Formation of calcium oxalate crystals in the TCP medium clearly indicates the ability of these fungi to release P in soluble form, ‘butHWA contains a large pool of calcium in the form of calcium carbonate, which makes it difficult to interpret the origin of calcium in these crystals. HWA contains P bound in compounds like apatite with extremely low solubilities (Steenari and Lindqvist, 1997), but in the present study, mycelia of Tor-97-4 from HWA plates showed a significantly higher concentration of P compared to P. croceum or Ha-96-3. This provided evidence that Tor-97-4 isolates were able to solubilise P from otherwise insoluble complexes by calcium oxalate formation. Many mycorrhizal and non-my corrhizal soil fungi have been reported to solubilise inorganic P by formation of calcium. oxalate (Lapeyrie et al,, 1991; Sayer and Gadd, 1997) and this mechanism may be more important in high pH environments with high kwels of calcium. Ectomycorrh~al fungi may behave differently in pure culture studies than in symbiosis with plants, therefore fkrther investigations were performed using intact symbiotic systems.

Nutrient uptake from hardened wood ash – by mycorrhizal and non-mycorrhizal spruce seedlings In order to examine the abilities of Tor-97-4, Ha-96-3 and P. crocewn mycelia (in intact symbiotic systems) to mobilise nutrients in HWA, a specially designed pot-system was used (Appendix, Fig. 1). In this experiment, ash application had a strong positive effect on plant biomass (Appendix, Fig. 2) indicating the importance of wood ash as a nutrient source. Mycomhizal seedlings inoculated with Tor-97-4 grew better than those inoculated with P. croceurn in the presence of ash in the high nitrogen treatment. Similarly, in the competition study (V) much higher plant biomass in Tor-97-4 pre-colonised seedlings grown at the high nitrogen level with ash indicate that this fungus is capable of mobilizing and capturing nutrients not only from the wood ash (IV) but has also taken up N horn the substrate to a larger extent than other seedlings. There was no significant difference in plant biomass for Tor-97-4 pre-colonised seedlings grown in non-ash amended substrates (LN –A, HN -A) suggesting that the N supply was adequate, and the growth was limited by other nutrients which were supplemented by the ash. In the ash treatment, N addition had a significant positive effect on growth, indicating that N became a growth limiting factor due to ash induced growth enhancement. Improved plant growth in response to ash fertilisation has been reported both in field and laboratory studies (Unger and Femandez, 1990; Ferm et al., 1992; Jacobson and Ring, 1995; Voundi Nkana et al., 1998).

30 In the nutrient uptake experiment (Appendix) analysis of soil solution from the mycelial compartments of the high nitrogen pots showed significantly higher P04 concentration in the Tor-97-4 treatments than in other mycorrhizal or non-my corrhizal treatments. This suggests that Tor-97-4 stimulated the P release from the ash although there was no indication that the fungus transported more P to the plant compared to the other tested fungi or non-my corrhizal roots. The release of P fkom the ash may have been induced by exudation of oxalate by roots or fungi. Griffiths et al. (1994) reported significantly higher concentrations of PO1 and oxalate in soil solution of soils colonised by mat forming ectomycomhizal fungi compared to non-mat soils. Oxalate concentration was estimated in the soil solution in the present experiment but no distinct trends were found and it can be expected that the majority of the produced oxalate would precipitate into calcium oxalate in the presence of ash.

31 Conclusions

The results so far obtained suggest that integrated forest residue harvesting and compensatory ash addition may have an effect on mycorrhizal fimgi, partly by affecting the densities of mycorrhizal roots, and partly by inducing shifts in community structure. Removal of residues had a significant effect on mycorrhizal density in one experiment (II) while ash application had a small effect on community structure in another study (III). However it should be stressed that there are so far no combined field experiments integrating the effects of these two treatments and it is thus difficult to draw firm conclusions regarding the long-term impact of such an integrated management practice. Possible effects on the organic matter capital of forests will depend on the intensity and type of res idue harvesting. The goal to increase national energy production from forest residues by a further 25-35 Twh should be seen in relation to the possible effects on humus thickness, drought stress and community structure.

There is mounting evidence to suggest that ectomycorrhizal fungi play an important role in mobilisation of primary elements from minerals as well as their uptake from soil solution. These organisms may play a role in mobilisation of nutrients from ash granules as well as contributing to efficient utilisation of available base cations in situations of intensive biomass harvesting.

In spite of the fact that the molecular methods developed over the past 10 years now enable us to identify mycorrhizal species on roots with high resolution it is still not possible to culture many common ectomycorrhizal species and investigate their function in controlled laboratory experiments. The species we examined in this thesis responded to ash in contrasting ways ranging from complete cessation of growth to active colonisation and mobilisation of nutrients. Improved understanding of the sustainability of biofuel harvesting fi-om :forests and its possible effects on ectomycorrhizal biodiversity thus require a much more complete understanding of the fictional role and sensitivity of individual species.

32 Acknowledgements

I would like to extend my deepest appreciation to all the people, both at the Department of Microbial Ecology and outside, who made my stay in Lund pleasant and enjoyable. I particularly like to thank

Roger Finlay (my main supervisor), for providing me the opportunity to do my doctoral work in Lund, for your support, guidance and encouragements over the years, Susanne Erland (my co-supervisor), for introducing me to the exciting world of PCR for your guidance and help in several different ways, HiWan Wallander (my co-supervisor), my main source of inspiration during the functional studies of ectomycomhiza – for sharing the excitement of new results and stimulating discussions, Bengt !Mderstrdm and Anders TunIid (heads of the department), for your excellent adminktration and help, Erland B35th, for your interesting talks and comments, Ann-Margret, for teaching me how to synthesise mycorrhizas and providing me the secret recipes - for your care and concern and discussions on life, Annette Persson, for your excellent skills in secretarial work, Tina (my former colleague), for your help during my early days in Sweden and teaching me usefi.d phrases in Swedish, Tommy Olsson, for efilcient help in elemental analyses, Ann-Mari Fransson, for help in the data analysis, Date, my office- and lab-mate, for sharing the laughs and sorrows, David, for helping me in the field sampling, Dag, for being a good backup to rescue my computer, Johan, for DNA sequencing of the ‘ash fungi’, Ingrid, for always being very kind to me, Peter, for your friendship and discussions, Pal Axel, Katarina, Karin, Christina and Maja, for creating a fliendly environment in the department, Umm, my very sympathetic and humane friend, for your deepest concern on the human rights in the Third Worl& Luis and Natasha, for your tilendshlp and care, Rukhsana Bajwa and Jahan Zeb Naz, for your help in the tough times and pulling me out of ‘the crisis’ and never letting me down, to my family for your prayers, love and support.

This project was financed mainly by the Swedish National Energy Administration (STEM); the Swedish Council for Forestry and Agricultural Research (SJFR) and the Swedish Institute (S1) also contributed.

ShaMd Mahmood

33 References

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