Research Collection
Doctoral Thesis
Effects of nitrogen-addition and irrigation on the structure and function of ectomycorrhizal communities
Author(s): Hutter, Sylvia Christa
Publication Date: 2014
Permanent Link: https://doi.org/10.3929/ethz-a-010140121
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DISS. ETH Nr. 21469
EFFECTS OF NITROGEN-ADDITION AND IRRIGATION ON THE STRUCTURE AND FUNCTION OF ECTOMYCORRHIZAL COMMUNITIES
Abhandlung zur Erlangung des Titels
DOKTORIN DER WISSENSCHAFTEN der ETH ZÜRICH
(Dr. sc. ETH Zürich)
vorgelegt von
SYLVIA CHRISTA HUTTER
Mag. rer. nat., Karl-Franzens-Universität
geboren am 03.04.1981
österreichische Staatsbürgerin
angenommen auf Antrag von
Prof. Markus Aebi, Referent Dr. Martina Peter, Korreferentin Dr. Jean Garbaye, Korreferent Prof. Rosmarie Honegger, Korreferentin
2014
Table of contents
Summary 2
Zusammenfassung 7
General Introduction 13
Chapter 1 30
Enzyme activities show a stronger dependency on ectomycorrhizal fungal morphotypes than on long-term nitrogen-addition and season in a spruce forest
Chapter 2 71
The ectomycorrhizal fungal community of a drought-stressed Scots pine forest is functionally resilient to irrigation
General Discussion and Conclusion 112
Danksagung 122
1
Summary
Ectomycorrhizal (ECM) fungi live in symbiosis with most temperate forest trees. In this partnership, the fungus increases the water and nutrient uptake of the tree and in return receives sugars from the plant. Due to environmental factors such as nitrogen deposition, drought periods or season fungal communities can change their structure, including the community composition, the species richness, and the abundance of individual ECM species. Little is known, however, about both the functional response of these fungi to environmental impacts, and about the distribution of the function of
ECM morphotypes within their communities. Relevant functional traits include for instance the activities of extracellular enzymes involved in the nutrient acquisition from organic matter.
In this doctoral thesis the following questions were investigated:
Do factors such as long-term nitrogen addition in a spruce forest, irrigation in a
dry pine forest and seasons such as spring and autumn change the structure
of ECM communities?
Do the functional abilities of the ECM morphotypes and the communities differ
when tested through enzyme activities?
If the functional abilities differ, can we relate any pattern of the functional profile
or functional plasticity of the ECM morphotypes to their abundance reaction
due to the factors?
The environmental factors fertilization, drought and spring may facilitate a reduction in the carbon flow from the tree to the fungus. Due to fertilization sufficient inorganic nutrients, particularly nitrogen, are available, which may cause the trees to limit the carbon allocation to their partnering fungi. Drought leads to a decrease in photosynthesis, resulting in a scarcity of sugars produced by trees, which 2 subsequently might reduce the carbon flow to the fungi. Similarly, because sugar reserves are needed for building new biomass and the available sunlight is insufficient for maximum sugar production by photosynthesis in spring, trees may pass on less sugar to their partnering fungi during this season. From these arguments, three hypotheses can be put forward in general. First, we expected that fungi that have a high ability to degrade organic carbon, and can therefore supply themselves with carbon, are favoured in environments where carbon supply from trees is limited. This implies that a higher abundance of such species should be observed in fertilized plots, during drought periods and in spring. Second, for ECM fungi which show no abundance changes in response to these factors, a high ability for adaptation reflected by plastic enzyme activities might be present. Third, carbon limitation might favour ECM fungi that are less carbon demanding because they produce comparatively little external mycelium such as the so-called contact or short- distance exploration types.
Specifically for the response of ECM fungi to fertilization, we can put forward two further hypotheses. First, fungi that possess a high ability to degrade organic nitrogen can be expected to show reduced abundances in fertilized plots. Their special ability would not be advantageous in an environment with high availability of inorganic nitrogen. Second, fungi that show increased abundances in the nitrogen-addition plots are expected to be competent in degrading organic phosphate, since this element is likely to be a limiting growth factor in such an environment.
Specifically for the response of ECM fungi to drought, an additional hypothesis can be stated. Enzyme activities for phosphate and nitrogen mobilization can be expected to be higher in ECM fungi favouring dry conditions compared to other fungi, since a higher nutrient status enhances drought tolerance.
3
We tested these hypotheses in two experiments. Chapter one deals with a long-term high nitrogen deposition experiment, which was simulated in a Swiss spruce forest by fertilizing 150 kg nitrogen ha-1a-1. In chapter two an irrigation experiment is described, which was carried out in a pine dominated forest in one of the driest valleys in
Switzerland.
In both experiments, the ECM communities were investigated by visual classification, by ITS sequencing, and by counting of ECM root tips. The functional response was determined by measuring activities of enzymes involved in organic matter degradation. The measurements were performed four times in two seasons (spring
2008 and 2009, autumn 2007 and 2008).
The ECM communities changed drastically in response to nitrogen addition and moderately in response to irrigation and season. All three factors (nitrogen addition, irrigation and season) affected the relative abundances of ECM morphotypes.
Moreover, the number of ECM root tips and the species diversity declined after nitrogen addition. During spring, the species richness increased compared to autumn in the irrigation experiment, whereas irrigation did not lead to a change in diversity.
In both studies, enzyme activities per mm2 of the ECM communities differed to a little degree between treatment and control plots. Small but significant increases were observed for sugar degrading enzymes due to nitrogen addition and in the dry controls of the irrigation study. The phosphatase increased its activity due to spring compared to autumn in the nitrogen-addition study and increased in the dry controls of the irrigation experiment. Moreover, leucine aminopeptidase, responsible for protein degradation, increased in spring compared to autumn in both studies. Total enzyme activities of the ECM communities cumulated over all ECM root tips were drastically reduced after nitrogen addition because the number of ECM root tips was
4 reduced by more than half compared to the control plots. No significant change was observed in the total enzyme activities in the irrigation experiment.
All ECM morphotypes were able to produce all of the investigated enzymes, but the measured values differed for each morphotype. However, no simple relation was found between the enzyme activities and the abundance reactions of the ECM morphotypes due to the investigated factors. Within individual ECM morphotypes, several significant differences of enzyme activities were observed due to nitrogen addition, irrigation and season. In the nitrogen-addition experiment, enzyme activities mostly increased due to nitrogen addition and spring. In the irrigation study, enzyme activities were higher in the dry controls compared to irrigated plots and again in spring compared to autumn.
Our results over both studies indicate that the functional traits of the ECM communities were maintained, although a shift in the ECM fungal composition was observed to a high degree in the nitrogen-addition study and to a moderate degree in the irrigation study and due to season. This suggests redundant functional abilities among ECM morphotypes and therefore can be interpreted as a resilient reaction of these ecosystems. However, since the number of ECM root tips was drastically reduced due to nitrogen-addition, the total enzyme activities were diminished, which might influence the nutrient cycles in this ecosystem. This was already reflected as reduced concentrations of several elements such as phosphate in the needles of the nitrogen-addition trees. In the irrigation study no change in total enzyme activities was observed. This functional and structural intact ECM community in the drought- stressed ecosystem might have facilitated the previously observed fast recovery of drought stressed Scots pine trees.
Several ECM morphotypes with abundance changes and stable abundances exhibited a high degree of adaptation in their functional traits. The results do not 5 agree with the hypothesis that ECM morphotypes showing stable abundance exhibit a high degree of plasticity for adapting to changing conditions. In contrast, ECM morphotypes with changes in abundance are also able to adapt to varying conditions.
In the present studies, most of the ECM morphotypes did not show the expected patterns in their enzyme activities related to their changed abundances, which could be explained by a reduced carbon supply as in our hypotheses. Therefore, other factors seem to be more important for shaping the ECM communities. These factors may be related to the efficiency of the ECM fungus to deliver nutrients or water to its partnering plant. For instance, future studies could measure the amount of nitrogen the fungus delivers to its host through isotopic tracing, exploiting the natural occurring
15N in the fungi and plants. Nevertheless, it would be interesting to monitor the structure and function of the ECM communities with the same approach as used in this thesis at the former nitrogen-addition experiment after several years, and after the termination of the irrigation experiment.
6
Zusammenfassung
Ektomykorrhiza (EKM) wird eine in gemässigten Regionen weit verbreitete Symbiose zwischen Bäumen und Bodenpilzen genannt. In dieser Symbiose versorgt der Baum den Pilz mit Zucker, den er aus der Photosynthese gewinnt. Im Gegenzug wird seine
Nährstoff- und Wasseraufnahme durch den Pilz erhöht. Viele verschiedene Pilzarten können einen Wald besiedeln und so entsteht eine diverse Pilzgemeinschaft. Durch
Faktoren wie erhöhter Stickstoffeintrag, Trockenheit oder Jahreszeit, können sich diese Pilzgemeinschaften verändern. Das äussert sich in Veränderungen der
Häufigkeit einzelner Pilzarten und der Artanzahl. Wie sich aber diese Faktoren auf die funktionellen Aspekte der Pilzgemeinschaften auswirken, wurde bislang wenig untersucht. Auch wie die Funktionen der verschiedenen Pilzarten innerhalb der
Gemeinschaften verteilt sind, steht erst am Beginn der Forschung. Bei messbaren funktionellen Eigenschaften der Pilzarten handelt es sich zum Beispiel um die
Aktivitäten von Enzymen, die im Abbau von organischem Material involviert sind.
In dieser Doktorarbeit wurden folgende Fragen untersucht:
Bewirken Faktoren wie Langzeitstickstoffeintrag in einem Fichtenwald,
Bewässerung in einem trockenen Föhrenwald und die Jahreszeiten Frühling
und Herbst Veränderungen der EKM Gemeinschaften?
Unterscheiden sich die Funktionen der Pilzarten und der Gemeinschaften, die
durch Enzymaktivitäten erhoben werden?
Zeigen diejenigen Pilzarten, die durch diese Faktoren eine Abundanzänderung
aufweisen, ein einheitliches Muster in ihren Enzymaktivitäten?
Wenn sich die funktionalen Fähigkeiten einzelner Pilzarten durch die Faktoren
ändern können, kann man diese Plastizität der Enzymaktivitäten mit einer
7
stabilen Abundanz der EKM Morphotypen infolge dieser Faktoren in
Beziehung bringen?
Durch Stickstoffdüngung, Trockenheit und Frühling ist eine Reduktion des
Kohlenstoffflusses vom Baum zum Pilz zu erwarten. Es wird vermutet, dass durch die Stickstoffdüngung der Baum genügend Stickstoff zur Verfügung hat und daher seine Symbionten, die insbesondere für die erhöhte Nährstoffzufuhr bei Stickstoff limitierenden Verhältnissen sorgen, mit weniger Kohlenstoff versorgt. Trockenheit kann zu Minderung der Photosynthese führen, was eine Reduktion der
Zuckerproduktion zur Folge hat. Wenn dem Baum wenig Zucker zur Verfügung steht, könnte die Zuckerabgabe zum Pilz vermindert werden. Der Kohlenstofffluss zu den
Pilzen könnte auch im Frühling vermindert sein, da die Zuckerreserven der Bäume für die Bildung von neuer Biomasse benötigt werden und noch nicht genügend
Sonnenlicht für ein Maximum an Photosynthese vorhanden ist. Demzufolge wurden drei generelle Hypothesen formuliert. Erstens erwarten wir, dass diejenigen Pilzarten, die eine hohe Fähigkeit zur organischen Kohlenstoffdegradation besitzen und sich daher selbst mit Kohlenstoff versorgen können, in jener Umgebung favorisiert werden wo die Kohlenstoffgabe der Bäume reduziert ist. Dies würde bedeuten, dass eine höhere Häufigkeit dieser Pilzarten in den gedüngten Flächen, in den trockenen
Flächen und im Frühling beobachtet werden müsste. Zweitens, bei den Pilzarten, die keine Veränderung der Häufigkeit durch die zuvor erwähnten Faktoren zeigen, könnte sich eine hohe Anpassungsfähigkeit an die veränderten Bedingungen durch flexible Enzymaktivitäten äussern. Drittens, die Kohlenstoffreduktion durch den Baum könnte EKM Pilze favorisieren, die wenig Kohlenstoff benötigen, wie vielleicht jene die wenig externes Mycel produzieren, z.B. Kontakt oder Kurze-Distanz
Explorationstypen.
8
Speziell für die Reaktion der EKM Pilze auf Stickstoffdüngung formulieren wir zwei weitere Hypothesen. Erstens erwarten wir, dass Pilzarten, deren Häufigkeit durch
Stickstoffzugabe zurückgeht, eine hohe Fähigkeit zum Abbau von organischem
Stickstoff aufweisen. Diese Fähigkeit wäre nicht vorteilhaft in einer Umgebung mit einer hohen Verfügbarkeit von Stickstoff. Zweitens, die Pilzarten, deren Ausbreitung hingegen durch Stickstoffgabe zunimmt, sollten eine hohe Fähigkeit aufweisen, organisches Phosphat abzubauen, da Phosphat in einer hohen anorganischen
Stickstoffumgebung limitierend werden könnte.
Speziell für die Reaktion der EKM Pilze auf Trockenheit kann eine weitere Hypothese formuliert werden. Die Pilzarten, die durch Trockenheit favorisiert werden, könnten eine erhöhte Fähigkeit zur Nährstoffmobilisierung (Stickstoff und Phosphat) aus organischem Material im Vergleich zu anderen aufweisen, da ein erhöhter
Nährstoffstatus für eine höhere Trockentoleranz sorgt.
Die beschriebenen Erwartungen wurden in zwei Experimenten getestet. Kapitel 1 handelt von einem Stickstoffeintrag-Langzeitexperiment, welches in einem subalpinen Fichtenwald im Kanton Freiburg, CH durchgeführt wurde. Kapitel 2 beschreibt die Ergebnisse aus einem Bewässerungsexperiment in einem von Föhren dominierten Wald im Walliser Rhonetal, einem der trockensten Täler der Schweiz.
In beiden Experimenten wurden die EKM Gemeinschaften durch visuelle
Klassifizierung in so genannte EKM Morphotypen, ITS Sequenzierung und zählen der EKM Wurzelspitzen untersucht. Die funktionelle Reaktion wurde durch Messung von Enzymaktivitäten an den EKM Wurzelspitzen erhoben. Die Messungen wurden viermal während zwei Jahreszeiten durchgeführt (Frühling 2008, 2009 und Herbst
2007, 2008).
Die EKM Gemeinschaften veränderten sich drastisch in Folge des
Langzeitstickstoffeintrages und moderat durch Bewässerung und Jahreszeit. Der 9
Langzeitstickstoffeintrag wirkte sich negativ auf die Diversität und Anzahl der EKM
Wurzelspitzen aus. Frühling führte im Bewässerungsexperiment zu einer Erhöhung des Artenreichtums, während Bewässerung keine Veränderung der Diversität bewirkte. Alle drei genannten Faktoren führten jedoch zu Veränderungen von
Häufigkeiten einzelner EKM Morphotypen.
In beiden Studien wurden geringe Veränderungen der Enzymaktivitäten pro mm2
Wurzelspitzenfläche der Pilzgemeinschaften beobachtet. Kleine aber signifikante
Erhöhungen von Zucker abbauenden Enzymen wurden durch Stickstoffdüngung und in den trockenen Kontrollen des Bewässerungsexperimentes gemessen. Das
Phosphatase Enzym erhöhte seine Aktivität während des Frühlings verglichen mit
Herbst im Stickstoffeintrag-Langzeitexperiment und in den trockenen Kontrollen des
Bewässerungsexperiments. Ausserdem erhöhte sich die Leucin
Aminopeptidaseaktivität im Frühling verglichen mit Herbst in beiden Studien. Wenn man die Enzymaktivitäten der Pilzgemeinschaften mit der Anzahl an vorhandenen
EKM Wurzelspitzen hochrechnet, ergibt das für die mit Stickstoff gedüngte EKM
Gemeinschaft eine erhebliche Verminderung der Enzymaktivitäten, da die Anzahl der
Wurzelspitzen durch Stickstoffzugabe auf weniger als die Hälfte reduziert wurde.
Beim Bewässerungsexperiment änderte sich diesbezüglich nichts.
Alle EKM Morphotypen waren in der Lage die Enzyme zu produzieren und die
Enzymaktivitäten zwischen den Morphotypen zeigten erhebliche Unterschiede. Es konnte jedoch kein Zusammenhang der Enzymaktivitäten mit den veränderten
Abundanzen der Morphotypen, die durch die untersuchten Faktoren zustande kamen, festgestellt werden. Innerhalb einzelner EKM Morphotypen konnten signifikante Unterschiede durch Stickstoffdüngung, Bewässerung und Jahreszeit beobachtet werden. Beim Stickstoffexperiment wurden meist erhöhte
Enzymaktivitäten durch Stickstoffgabe und im Frühling festgestellt. Beim 10
Bewässerungsexperiment erhöhten sich einige Enzymwerte in den trockenen
Kontrollen und ebenfalls im Frühling verglichen mit Herbst.
Unsere Resultate über beide Studien zeigen, dass die funktionellen Merkmale der
EKM Gemeinschaften erhalten bleiben, obwohl eine Veränderung in der
Zusammensetzung im grossen Ausmass durch Stickstoffzugabe und zu einem geringeren Ausmass durch Bewässerung sowie durch die Jahreszeiten zu beobachten war. Dies deutet darauf hin, dass die funktionalen Fähigkeiten der
Pilzarten redundant sind. Eine mögliche Interpretation ist daher eine resiliente
Reaktion dieser Ökosysteme infolge der untersuchten Faktoren. Die Anzahl EKM
Wurzelspitzen wurde in den Stickstoffflächen drastisch reduziert und in Folge auch die totalen Enzymaktivitäten, was zu einer Beeinflussung der Stoffkreisläufe in diesem Ökosystem führen könnte. Eine Reduktion von Elementen wie z.B. Phosphat in den Nadeln der Bäume in den Stickstoffflächen deutet bereits darauf hin. Beim
Bewässerungsexperiment dagegen, wurde keine Veränderung der totalen
Enzymaktivitäten beobachtet. Die schnelle Erholung der trocken gestressten Kiefern in diesem Areal kann durch die noch funktionell und strukturell intakte EKM
Gemeinschaft erklärt werden.
Einige EKM Morphotypen die ihre Häufigkeiten geändert haben aber auch einige die eine stabile Abundanz äusserten, zeigten ein hohes Mass an Anpassung in ihren
Funktionen. Diese Resultate unterstützen nicht die Hypothese, in der nur die
Pilzarten, die eine stabile Abundanz aufweisen, auch eine hohe Anpassungsfähigkeit gegenüber sich veränderten Umweltbedingungen besitzen würden. Also haben auch
Pilzarten, die eine veränderte Abundanz gegenüber den untersuchten Faktoren aufweisen, die Fähigkeit ihre Enzymaktivitäten an sich ändernde
Umweltbedingungen anzupassen.
11
Bezüglich der möglichen Kohlenstoffreduzierung der Bäume zu ihren Pilzen durch die untersuchten Faktoren können keine generellen Schlussfolgerungen gezogen werden. Die meisten EKM Morphotypen zeigten keine systematischen Muster in ihren Enzymaktivitäten im Zusammenhang mit den veränderten Abundanzen, was die Kohlenstoffreduzierung nach unseren Hypothesen erklären könnte. Daher könnten andere Faktoren, die die EKM Gemeinschaften formen, wichtiger sein. Die
Effizienz der Nährstoff- und/oder Wasserlieferung vom Pilz zum Baum könnte so ein
Faktor sein. Zum Beispiel könnten zukünftige Studien die Stickstoffmenge, die der
Pilz zum Baum liefert, mit natürlich auftretenden 15N in den Pilzarten und deren
Partnern messen. Trotzdem wäre es auch spannend mit den gleichen Methoden, die in dieser Dissertation zum Einsatz kamen die EKM Gemeinschaften des ehemaligen
Stickstoffexperiments nach einigen Jahren und nach Ende des
Bewässerungsexperiments wieder zu untersuchen.
12
General introduction
Ectomycorrhizal fungi
Most tree species in temperate regions live in symbiosis with ectomycorrhizal (ECM) fungi. The fungal hyphae and the fine root tips of the trees build the so-called ectomycorrhizal organ. From a fungal spore or from an already established fungus near the newly growing root tip to be colonized, the hyphae form a mantle around the root tip and grow between the plant cells. In this area, called the Hartig net, a bidirectional movement of nutrients and water takes place. Sugars produced by the tree from photosynthesis flow to the fungus, and water and nutrients such as nitrogen and phosphorus move to the plant. The fungi mostly take up water and nutrients by the external fungal mycelium, which exploits the soil surrounding the colonized root tips. Therefore, the fungi are often seen as extensions of the root system of the tree
(Smith and Read 2008). In addition to this conventional view of the symbiosis, several pieces of evidence suggest that some ECM fungi also gain carbon from soil organic matter and thus are able to act as saprotrophs (Koide et al. 2008, Courty et al. 2010).
ECM community assessment
Large numbers of ECM species may associate with the root system of the same tree, successions having been described from young to old growth forests, as studied and summarized by Dahlberg (2001), Twieg (2007) and Egli (2011). This immense underground fungal diversity can only be studied with molecular tools. Fruiting body surveys of basidio- and ascomycetes (macromycetes), whose usually seasonal formation and appearance depend on diverse, as yet partly unknown factors do not adequately mirror the species richness and diversity underground. Several ECM
13 fungi have subterranean fruiting bodies or do not form any on a regular basis
(Dahlberg 2001, Horton et al. 2001, Peter et al. 2001a).
Through the development of DNA-based methods, it is possible to identify ECM fungi at their species level (Gardes et al. 1993). For identification, mostly the internal transcribed spacer (=ITS) region of the fungal DNA is amplified through PCR and sequenced. The obtained DNA sequences can be compared with known sequences of fungi in databases, making it possible to gain a species name (Horton et al. 2001).
Another approach for studying the ECM community structure is to visually characterize the fungi via their appearance into so-called morphotypes according to
Agerer (1987-2012). Some ECM morphotypes display such a unique look that it is easy to determine their species names. For instance, Russula integra exhibits a brown greenish mantle and yellowish pustules on its surface. However, for most of the ECM fungi, it is difficult to achieve a species name by visual classification (Peter et al. 2001b), but a grouping into a genus is mostly achievable. In the current thesis a combination of morphotyping and ITS sequencing was used for determining species names.
Reaction of ECM communities to disturbances
The reaction of ECM communities due to different disturbances has been studied extensively, and it was found that several factors such as clear cutting (Byrd et al.
2000), heavy metal pollution (Hui et al. 2011) and fire (Kipfer et al. 2011) can change the structure of ECM communities. Another important factor influencing ECM community composition is atmospheric nitrogen deposition (Peter et al. 2001a).
Nitrogen pollutants, as emitted by combustion processes, intensive animal husbandry or synthetic fertilizers (Galloway et al. 2008, Davison 2009) were suspected to be a major factor in this development (reviews: Egli 2011, Lilleskov et al. 2011). In
14 Switzerland, anthropogenic nitrogen deposition has increased over the last century and currently ranges from under 5 kg nitrogen ha-1 yr-1 in mountain regions to levels over 40 kg nitrogen ha-1 yr-1 in densely populated areas (Figure 1). Therefore, field experiments were set up to test the impact of nitrogen pollutants on the above- and belowground diversity of ECM partners of forest trees (Peter et al. 2001a, Avis et al.
2003). Chapter 1 focuses on such a long-term experimental site in a Swiss sub alpine forest ecosystem with about 17.5 kg nitrogen ha-1 yr-1. On this experimental site, the aboveground ECM diversity was studied with accurate fruiting body surveys and the belowground diversity with comparative ITS analyses prior to and after the addition of substantial amounts (150 kg ha-1 yr-1) of nitrogen fertilizer (Peter et al.
2001a, Gillet et al. 2010). Gillet et al. (2010) explored the shift in community structure of ECM and saprobic fungi. Long-term nitrogen addition can lead to a decrease in species number, a decrease in ECM root tips and a change in the dominant species
(Frey et al. 2004, Cox et al. 2010, Kjoller et al. 2012). It is assumed that trees reduce belowground carbon allocation in response to higher nitrogen concentrations in the soil (Högberg et al. 2007, Demoling et al. 2008). Moreover, the demand for sugars used for nitrogen assimilation by the fungi might increase, while this would reduce the amount of carbon available for fungal growth (Wallander et al. 1998). The change in ECM community structure due to long-term nitrogen addition might be explained by the functional ability of some ECM fungi to directly use soil organic matter as an additional carbon source (Peter et al. 2001a, Gillet et al. 2010).
15
Figure 1. Nitrogen deposition map of Switzerland; (Source: www.bafu.admin.ch)
A number of studies found that the community structure of ECM fungi can also change in response to drought (Shi et al. 2002, Swaty et al. 2004, Buée et al. 2005,
Richard et al. 2011). All of these studies reported significant abundance changes of
ECM fungi; with some species increasing in abundance, and some decreasing due to drought. It is currently unclear why ECM fungi change their abundance, but some hypotheses have been put forward. For instance, drought periods may be advantageous for ECM fungi with the ability to directly derive sugars from soil organic matter, because photosynthesis rates of trees can reduce during drought periods.
Thus, sugar production rates decline (Breda et al. 2006, McDowell et al. 2011,
McDowell et al. 2013) and trees may pass on less sugar to their partnering ECM fungi.
Drought was suggested to be the main driver for high mortality rates of Scot pines in the inner alpine valleys in central Europe (Bigler et al. 2006, Dobbertin et al. 2005,
16 Gonthier et al. 2010). To study drought effects on these pine ecosystems, a long term irrigation experiment was established in the pine-dominated Pfynwald in an inner alpine Swiss valley, which is characteristic by low precipitation of about 600 mm per year (Figure 2) and high temperature amplitudes (Figure 3). Dobbertin et al.
(2010) and Eilmann et al. (2010) reported an instant growth response of the drought released trees, whereas belowground no significant change in fine root biomass was observed due to irrigation (Brunner et al. 2009).
Figure 2. Precipitation map of Switzerland; (Source: www.meteoschweiz.ch)
17
Figure 3. Climatic chart of Sion (Valais, Switzerland) from 1981-2010; the thick red curve displays mean monthly temperatures; the red lines above and below the thick curve show mean maximum and minimum temperatures; blue bars indicate mean monthly precipitation; (Source: www.meteoschweiz.ch)
In response to the varying conditions in different seasons, ECM communities can alter their structure throughout the year (Buée et al. 2005, Courty et al. 2008). The carbon supply to ECM fungi by trees may be reduced in spring because sugar reserves are needed for building new biomass and sunlight is still insufficient for maximum photosynthesis and consequently high sugar production rates (Pritsch and
Garbaye 2011). Again, this could favour species able to supply themselves with sugars by breaking down soil organic material.
Enzyme activities as functional traits of ECM fungi
In contrast to the approaches developed to study ECM communities at different taxonomic levels in the field, it is still quite a challenge to study their functional traits.
Functional traits may include, among others, fungal carbon demand from the tree, the efficiency of the fungi in transporting nutrients and water to the tree, or their ability to decompose organic matter (Koide et al. 2007, Courty et al. 2010). Laboratory-based studies have demonstrated that many ECM fungi are able to degrade complex
18 macromolecules found in organic matter (reviewed by Talbot et al. 2010). To achieve the decomposition of complex macromolecules, the fungi release enzymes into their surrounding environment. Recently, a method was developed and optimized by
Pritsch et al. (2004, 2011) to measure enzyme activities involved in the degradation of typical soil components on the surface of ECM root tips from the field. The advantage of this method is the rapid measurement of hundreds of ECM root tips for different enzymes (Koide et al. 2007). A downside of the method is that the enzyme activities are measured at the mantle region on the ECM root tip of the fungus, whereas enzyme activities of the mycelium exploiting the soil cannot be measured.
This might be a problem when studying ECM fungi that exhibit much external mycelium with long vessel-like structures, but not when studying ECM fungi with little external mycelium (Koide et al. 2007).
The enzyme activities of ECM fungi are dependent on the fungal species (Courty et al. 2005), environmental factors such as soil properties (Courty et al. 2005) or season
(Buée et al. 2005), and human disturbances such as fertilization (Rineau and
Garbaye 2009, Jones et al. 2012) or a declining tree density due to forest management (Mosca et al. 2007). For instance, Courty et al. (2005) found that within a single ECM fungus, the enzyme activities can change due to location and soil profile. Rineau and Garbaye (2009) suggested that some ECM species act as generalists exhibiting low activities of all enzymes, whereas some act as specialists, revealing high activities of a few selected enzymes.
On the level of ECM communities, Rineau and Garbaye (2009) observed a significant impact of liming on several enzyme activities, suggesting a reaction of the ECM community towards increased mobilization of nutrients from soil organic matter after liming. In contrast, Jones et al. (2010) observed a minor reaction of the enzyme activities of the ECM community due to wildfire and clear cutting, suggesting a
19 functional resilient behaviour to these impacts. Summarizing prior studies (Courty et al. 2005, Courty et al. 2006, Buée et al. 2007, Rineau and Garbaye 2009) Pritsch et al. (2011) described the enzyme activities of the ECM communities as redundant in essential functions such as phosphatase activities and selective in other functions such as laccase activity. Moreover, based on the work of Courty et al. (2005) and
Buée et al. (2007), they suggested a certain ability of individual ECM fungi to adapt to a changing environment.
The methodical approach to study functional traits of ECM fungi by Pritsch et al.
(2004, 2011) was adopted within this thesis, and the following eight enzymes involved in the degradation of soil organic matter, were chosen for measurement: acid phosphatase that assists the liberation of free phosphate from organic molecules; leucine-aminopeptidases that catalyzes the digestion of proteins through the break down of peptide bonds; laccase whose activity leads to the breakdown of phenolic macromolecules such as lignin; the five enzymes N-acetyl-glucosaminidase, cellobiohydrolase, beta-glucosidase, xylosidase, and glucuronidase which are responsible for the hydrolysis of glycosidic bonds in carbohydrates. N-acteyl- glucosaminidases are involved in the degradation of chitin, which is a component of the cell walls of fungi and of exoskeletal parts of some animals. The remaining enzymes are involved in the degradation of cellulose and hemicelluloses (Pritsch et al. 2004, Courty et al. 2010, Pritsch and Garbaye 2011, Pritsch et al. 2011). The resulting enzyme activity profiles can be seen as functional traits of single ECM fungi or of whole ECM communities (Cullings et al. 2009, Jones et al. 2010).
Exploration strategies as functional traits of ECM fungi
Agerer (2001) has developed a classification system with the exploration behaviour based on the amount, structure and appearance of the external mycelium of ECM
20 fungi (Figure 4). Whereas contact exploration types with very little external mycelium emanating from the ECM root tip, might explore the soil only in their direct environment, long-distance exploration types with vessel-like structures might be able to explore the soil in quite some distance from the ECM root tip. It has been suggested that fungi with much external mycelium such as the long-distance exploration types have a high demand of carbon from the tree to form these structures (Lilleskov et al. 2011). Therefore, if carbon allocation from trees to their symbionts is reduced, fungi with little external mycelium such as contact and short- distance exploration types might be favoured. Lilleskov et al. (2011) further proposed that so-called medium fringe exploration types may be sensitive to nitrogen addition, since several species found to be nitrogen-sensitive exhibited this kind of exploration type. It has also been suggested that long-distance exploration types maybe specialized in water uptake and therefore might be more abundant in dry regions
(Garbaye 2000, Lehto et al. 2011).
Agerer et al. (2000, 2001) further related the exploration types to their potential function in the ecosystem. For instance, they showed that the long-distance exploration type, as within the Boletus, lack laccase activity, whereas Lactarius and
Russula mostly belonging to the contact exploration type exhibited high laccase activity, which is involved in the degradation of lignins. This ability may support nutrient acquisition for these fungi, whereas the lack of laccase for the long-distance exploration type might be compensated through the larger surface area and greater range of spread (Agerer et al. 2000, Agerer 2001). Additionally, Hobbie and Agerer
(2010) related δ15N values in fruiting bodies with their exploration strategies. They found higher levels of δ15N in fungi which exhibited high biomass exploration types such as medium-distance mat and fringe and long-distance exploration types compared to fungi with low biomass such as contact, short and medium-distance
21 smooth exploration types. They suggested two different strategies for the nitrogen mobilisation of the high biomass fungi and the low biomass fungi, whereby the latter focus on labile nitrogen compounds and the former focus on nitrogen caught in molecules like proteins and chitin. Hobbie et al. (2014) further related δ15N fungal patterns with different soil depths and found that hydrophobic ECM fungi (belonging to long-distance and medium-distance exploration types) assimilate their nitrogen from deeper horizons. In contrast, two hydrophilic ECM fungi (belonging to contact, short and medium-distance smooth exploration types) mobilised their nitrogen from shallower and two ECM fungi from deeper horizons. Finally, Tedersoo et al. (2012) related the exploration types to potential enzyme activities of ECM fungi and found that exploration types with much mycelium have higher activities compared to exploration types with little mycelium except for laccase.
22 a)
b) c)
d) e)
Figure 4. Different ECM fungi on a fine root a) and different exploration types after Agerer (2001): b) Craterellus lutescens: contact exploration, c) Cenococcum geophilum: short- distance exploration, d) Cortinarius sp.: medium-distance fringe exploration, e) Boletus edulis: long-distance exploration; 23 Thesis outline
The aim of this thesis was to improve the understanding of the structure and function of ECM communities subjected to disturbances such as long-term nitrogen addition and irrigation. In addition, the seasonal impacts of spring and autumn were studied.
Therefore, the following questions were addressed:
Do factors such as long-term nitrogen addition in a spruce forest, irrigation in a
dry pine forest and seasons such as spring and autumn change the structure
of ECM communities?
Do the functional abilities of the ECM morphotypes or communities differ when
tested through enzyme activities?
If the functional abilities differ, can we relate any pattern of the functional profile
or functional plasticity of the ECM morphotypes to their abundance reaction
due to the factors?
Two independent studies were performed and are presented in two chapters:
Chapter 1 examines the taxonomic and functional reaction of an ECM community to long-term nitrogen addition in a sub alpine Norway spruce forest. Species identification was performed through visual classification of ECM fungi and ITS sequencing. Moreover, for quantification of community structure, ECM root tips were counted. Functional traits were investigated by measuring enzyme activities relevant in the degradation of soil organic matter. Chapter 2 describes the effect of irrigation on the taxonomic and functional structure of an ECM community in a drought exposed Scots pine dominated forest in the Central Alps of Switzerland. The methodical procedure was the same as for Chapter 1.
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29
Chapter 1
Enzyme activities show a stronger dependency on ectomycorrhizal fungal morphotypes than on long-term nitrogen-addition and season in a spruce forest
Sylvia Hutter, Simon Egli, Renaud Maire, Rosmarie Honegger, Jean Garbaye and
Martina Peter
Summary
Anthropogenic nitrogen deposition in terrestrial ecosystems is a major ecological issue. It is well documented that ectomycorrhizal (ECM) communities can change due to enhanced nitrogen availability, whereas little is known about their functional response.
Enhanced nitrogen deposition was simulated in a spruce forest by adding 150 kg nitrogen ha-1 yr-1 for 12 years starting in 1997. ECM root tip samples were taken four times over two years in two different seasons (autumn 2007 and 2008, spring 2008 and 2009) from nitrogen treated and control plots. For ECM community analysis,
ECM root tips were quantified, classified into morphotypes and identified via ITS sequencing. As functional traits, secreted enzyme activities were determined from
ECM morphotypes.
The ECM communities differed between control and nitrogen-addition plots. The community of the nitrogen-treated plots showed a decrease in species richness, a reduced number of ECM root tips and different dominant ECM morphotypes. The enzyme activities per mm2 of the ECM communities were similar although different
30 species contributed to these functions. However, since the number of root tips was strongly reduced due to nitrogen addition, a decrease of the total enzyme activities on the roots within the nitrogen-addition plots was evident. On the level of individual
ECM morphotypes, enzymatic profiles were mainly determined by species rather than by nitrogen addition, season or nitrogen sensitivity of a particular species.
Our results indicate that functions were taken over by functional redundant but better adapted ECM morphotypes at higher nitrogen levels. However, the decrease in species richness renders the ecosystem more vulnerable to additional stresses since the reduced species pool might not allow further adaptation. In addition, the decrease in total enzyme activities of the nitrogen-addition community may reduce the decomposition of the organic matter and the acquisition of organically bound carbon, nitrogen and phosphate in the soil, which results in an imbalanced nutrient status of the trees in this Norway spruce forest ecosystem.
Keywords: biotrophic-saprotrophic continuum, ectomycorrhizal morphotypes, exploration types, enzyme activities, nitrogen addition
31 Introduction
Fungi are key players in forest ecosystems, as nutrient cycler via decomposition of litter and wood, and as mycorrhizal symbionts of host trees. In temperate climates most forest trees associate with ECM fungi, the result being characteristic symbiotic phenotypes: the youngest lateral roots change their growth pattern while getting ensheathed by a fungal partner. The tight fungus-root cell interface at the so-called
Hartig net is the site of exchange of photosynthates from the plant and nutrients, mainly nitrogen compounds and phosphates, which have been mobilized in the soil by the fungal partner (Smith and Read 2008).
In the late 1980ies, a reduction of ECM fruiting bodies was observed in European forests and the long-term increase of nitrogen deposition was assumed to be the main reason (Arnolds et al. 1991). The combustion of fossil fuels and intensive agricultural activities are the main sources for nitrogen release into the atmosphere and consequently for accumulation on the ground. It is estimated that by the year
2050 up to 50 kg nitrogen ha-1 yr-1 will be observed in several regions of the world and even higher levels in forest ecosystems (reviewed by Galloway et al. 2008). In areas with intensive livestock farming in Switzerland, levels already reached 60 kg nitrogen ha-1 yr-1, whereas in mountain areas rates were comparatively low at 5 kg nitrogen ha-1 yr-1 (Bassin et al. 2007).
Various field experiments were set up to examine the impact of increased nitrogen input on ECM communities above- and belowground (Peter et al. 2001, Lilleskov et al. 2002, Avis et al. 2003, Avis et al. 2005, Clemmensen et al. 2006, Taniguchi et al.
2007, Borja et al. 2009, Wright et al. 2009). Short-term observations aboveground revealed a fast reduction of species diversity and a change in sporocarp dominance.
Belowground, the short-term ECM community change was less pronounced (Karen
32 and Nylund 1997, Peter et al. 2001), but larger changes were observed as more time progressed (Frey et al. 2004, Cox et al. 2010). In addition to a changed community composition, the number of ECM root tips changed along a nitrogen deposition gradient, with lowest numbers found in the area with highest nitrogen deposition
(Kjoller et al. 2012). It is assumed that higher nitrogen concentrations in soils lead to a reduced belowground carbon allocation by hosts (Högberg et al. 2007, Demoling et al. 2008) and a higher demand of sugars for nitrogen assimilation in the fungus itself, which reduces the amount of carbon available for fungal growth (Wallanda et al.
1998). ECM fungi which increase in abundance after nitrogen addition (=nitrophilic fungi) might therefore show a high functional ability to decompose soil organic matter as an additional carbon source (Peter et al. 2001, Gillet et al. 2010). Alternatively, nitrophilic fungi might be better adapted to lower carbon supply from the tree because they produce little external mycelium such as the contact or short-distance exploration types (Hobbie et al. 2010, Lilleskov et al. 2011). In contrast, fungi featuring much external mycelium, in particular the medium-distance fringe exploration type (Agerer 2001), showed reduced abundances after nitrogen addition
(=nitrophobic fungi) (Lilleskov et al. 2011). A further trait for nitrophobic fungi was supposed to be a specialization in acquiring nitrogen from organic matter (Lilleskov et al. 2011).
ECM fungi are able to mobilize organic nitrogen, phosphate and carbon from complex macromolecules like proteins and sugars through secreted enzymes (Koide et al. 2008, Talbot et al. 2010). A method to assess such enzyme activities on the surface of single ECM root tips from the field was developed by Pritsch et al. (2004) and improved by Courty et al. (2005) and Pritsch et al. (2011). Using this approach, the activity of enzymes involved in the degradation of cellulose, hemicellulose, lignin, chitin, proteins and organic phosphates are measured. The resulting enzyme
33 activities of single ECM fungi or whole communities can be seen as functional traits
(Cullings et al. 2009, Jones et al. 2010). Several studies showed that these traits are dependent on the fungal species and on environmental factors (Courty et al. 2005,
Buée et al. 2007, Mosca et al. 2007, Rineau and Garbaye 2009, Jones et al. 2010).
However, the effects of enhanced nitrogen deposition on such traits are little understood (Lilleskov et al. 2011). Although several studies showed changes in enzyme activities measured in the soil of nitrogen input experiments (Carreiro et al.
2000, Saiya-Cork et al. 2002, Frey et al. 2004, Waldrop et al. 2006, Keeler et al.
2009, Lucas et al. 2008), little is known in this respect at the level of single ECM fungi and their communities. So far, only Jones et al. (2012) reported increases in enzyme activities of ECM fungi in response to nitrogen addition.
In 1997 a field experiment with continuously high nitrogen input was set up at the
Parabock site in a sub alpine spruce forest in Switzerland. The short-term response of the above- and belowground ECM community was reported by Peter et al. (2001).
Aboveground, sporocarp surveys showed a sharp decline of species richness and the number of fruiting bodies produced after one year of fertilization. Belowground, the ECM composition changed slightly after two years. Some species of
Thelephoraceae and Atheliaceae increased in their abundance, whereas others such as members of the Russulaceae decreased, but no reduction in diversity was observed. Gillet et al. (2010) noted a persistent reduction of aboveground ECM richness and sporocarp density over 10 years of nitrogen treatment.
In the present study, the belowground ECM diversity and community structure were re-examined at the Parabock site after 10, 11 and 12 years, respectively, and compared with the well documented situation at the beginning of the experiment. We additionally focused on the following questions: 1) Do the activities of enzymes, as secreted by ECM root tips for the degradation of organic matter, change in response
34 to nitrogen fertilization and season? 2) Are relations found between the mycorrhizal exploration types, i.e. the amount, organization and appearance of the external mycelium of ECM root tips (Agerer 2001), and an either nitrophilic, nitrogen-tolerant or nitrophobic nature of the respective ECM species? Such presumed relations were summarized by Lilleskov et al. (2011).
We hypothesized that in the nitrogen-addition plots the ECM morphotype diversity and the amount of ECM root tips decrease and a change in dominant ECM morphotypes occurs. Consequently the functional traits measured as enzyme activities on root tips are expected to differ between control and nitrogen-addition communities. We further hypothesized that the ECM community in fertilized plots tends to be composed of ECM morphotypes which show one or several of the following traits: (i) they exhibit little external mycelium, (ii) are specialized in carbon and phosphate acquiring enzymes, and (iii) are less competent in mobilizing nitrogen from soil organic matter. We expected that ECM morphotypes which are negatively impacted by increased mineral nitrogen levels, so-called nitrophobic ECM morphotypes, tend to be specialized in degrading nitrogen-rich organic sources such as chitin and proteins but are weak in carbon releasing enzymes and/or exhibit exploration types with much external mycelium.
35 Material and methods
Study site
The experimental site Parabock is located in Switzerland (canton of Freiburg,
Moosboden, community of Cerniat near Bulle) and contains exclusively Norway spruce (Picea abies), planted as 5 year old saplings in 1960. The stand is located in a Flysch zone 1350 m above sea level and the soil is dystric gleysol (FAO-UNESCO
1997). The mean annual precipitation is about 2000 mm. The average atmospheric nitrogen input was estimated to be 20 kg nitrogen ha-1 yr-1(BAFU 2007).
Experimental design
To assess the effects of nitrogen addition, we used a clumped segregation design with four replicates. The size of each replicate was 8 m × 8 m, divided in subplots of
1 m2. The four replicates were situated next to each other resulting in two 16 m × 16 m quadrats, one used as control and one for the nitrogen treatment. A buffering zone of 10 m separated the two quadrats. We chose a segregation design to avoid the risk of the control replicates being affected by nitrogen leaching. In order to simulate a constant high nitrogen deposition (150 kg nitrogen ha-1 yr-1), a slow-release fertilizer in solid, globular form was used that steadily released the nitrogen (NH4NO3) during approximately six months. The fertilizer was manually distributed twice a year (May and October) from 1997 until the end of the experiment in 2009.
Element analysis of soil and needles
Soil samples for chemical analyses were taken in fall 2007. Eight evenly distributed, individual samples (0-10 cm) were taken from each of the four replicate plots per treatment and pooled together to one control and one fertilized samples. From each
36 of the sieved and afterwards oven dried samples, 500 g were analyzed by INRA
Nancy. For needle analysis, branches from four control trees and four fertilized trees were sampled randomly in fall 2008. Current year needles were removed from the branches, oven dried for 24 hours at 80°C and ground to a powder. Needle powder was analyzed by INRA Nancy.
Sampling and processing of soil cores and ECM root tips
To assess the ECM community structure, 48 soil cores (6 per replicate plot) were systematically sampled four times in two seasons: October 2007, May 2008, October
2008 and May 2009, giving a total of 192 soil cores. A corer was used to extract each soil core with the length of 10 cm and the diameter of 4.4 cm (60.8 cm3). Soil cores were transported in plastic bags in a cool box and stored at 4°C. The soil core processing started the following day and was finished latest after three weeks.
For classification into different ECM morphotypes, the soil cores were broken, the
ECM roots collected and washed under tap water. Each ECM root tip was classified under a dissection microscope in a Petri dish filled with tap water. Our classification scheme, using Agerer’s (1987-2012) method, included two main features: (1) colour, texture and shape of the ECM root tips and (2) presence and appearance of external hyphae and / or rhizomorphs. Moreover, the exploration type according to Agerer
(2001) of each ECM morphotype was classified based on the structure, length and amount of external mycelium. Although ECM roots were carefully washed, some of the mycelium might have been washed away. Therefore, our exploration types could differ from those suggested by Agerer (2001). In total, 79101 ECM root tips were visually classified into ECM morphotypes. Since it is difficult to identify ECM species by morphotyping, the internal transcribed spacer (ITS) region of one tip per ECM morphotype of a soil core was sequenced.
37 For sequencing, DNA was extracted from a total of 932 ECM root tips after enzyme activities were measured, using the DNeasy 96 Plant Kit from Qiagen. DNA amplifications through PCR were conducted with the universal fungal primer ITS1F and ITS4 with the program suggested by Gardes and Bruns (1993). The primer
ITS1F and the Big Dye Kit (Life technologies, Carlsbad CA) for cycle sequencing were used for the sequencing reaction and an ABI3700 automated sequencer (Life technologies, Carlsbad CA) was used for performing the electrophoresis. A total of
339 samples were successfully sequenced and compared to the databases Unite and/or NCBI. We used a cut off value of 97 % for species identification and 95 %-97
% to assign a genus. To separate two sequences with shared less then 97 % identity, a number was added to the name, e.g. Russula sp.1. In some cases, sequences were compared and named after sporocarp references collected from the same site (c.f. Gillet et al. 2010).
Since ECM morphotypes consisted of several sequence determinations we assigned a species name if more than half of the sequences belonged to one species. If this was not achieved, the ECM morphotype was called after the most dominant genus, as for Cortinarius spp. and Pseudotomentella spp. One ECM morphotype was called
Russula-like, since it could be assigned to neither a species nor a genus but Russula species were dominant (Table S2).
To validate the correctness of morphotyping within a soil core, 3-5 tips of the same
ECM morphotype in a soil core were sequenced. ITS-sequencing proved that morphotype assignment within a soil core was reliable to assess different ECM fungi
(data not shown), and therefore all diversity indices were calculated on the basis of
ECM morphotypes.
We termed species as “nitrophilic” if their abundance increased after nitrogen- addition compared to the control plots as well as compared to their abundance prior
38 to the nitrogen treatment (Peter et al. 2001). Species which showed reduced abundance were called “nitrophobic” and species whose abundance was not changed due to the treatment were called “nitrogen tolerant”.
Enzymatic assay on individual ECM root tips
According to the visually estimated abundance of ECM morphotypes in a soil sample, between 3 and 21 root tips per ECM morphotype were excised and individually placed in a 96-well plate. Enzymatic activities were determined as described by
Pritsch et al. (2011). The following substrates were used for the enzymes: MU–β-d- glucopyranoside (MU–Gls) for β-glucosidase, MU–N-acetyl-β-d-glucosaminide (Mu–
Nag) for N-actetyl-β-d-glucosaminidase, MU–cellobioside (MU–Cel) for cellobiohydrolase, MU–β-d-glucoronide hydrate (MU–Glr) for glucuronidase, xylopyranoside (MU–Xyl) for xylosidase, 7-amino-4-methylcoumarin (AMC-Leu) for leucine aminopeptidase and 2-2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) for laccase activities. All substrates were purchased at Sigma-Aldrich Chemie GmbH
Buchs, Switzerland. Fluorescence measurements were carried out at 368 nm excitation and 465 nm emission with a microplate reader Tecan Infinite M200 (Tecan
GmbH, Germany). Photometric measurements for ABTS discoloration were performed at 425 nm with the same microplate reader. The projection surface area of each ECM root tip was determined using the automated image analysis software
WinRhizo Version 4.1c (Regent Instruments, Canada). The enzyme activities were expressed as MU and AMC release or ABTS oxidation (discoloration) in pmol per projection area (mm2) and time (min) per excised root tip or as pmol per minute multiplied by the number of ECM root tips. Glucuronidase activities were omitted from all analyses since the substrate MU–β-d-glucoronide hydrate probably degraded in the course of our study which resulted in meaningless measurements.
39 Calculations and statistical analysis
Diversity indices (species richness, Simpson index of diversity, Shannon index of diversity and rarefied ECM morphotypes) were calculated per replicate plot of a sampling. Since the control plots and nitrogen-addition plots revealed an unequal number of ECM root tips, we calculated the rarefied number of species using the analytical equation proposed by Sanders et al. (1968). In this method, due to the set- up of the equation, the rarefied species number can only be calculated for a lower number of individuals than actually observed in the plot. Thus, the rarefied species number was only calculated for the control plots, using the lower number of individuals observed in the corresponding nitrogen-addition plot. In addition, we used a stochastic method as follows. First, we assigned a probability of occurrence to each
ECM morphotype by dividing the number of root tips associated with this ECM morphotype by the total number of root tips as observed in the control plot. Second, using the probabilities, an ECM morphotype was randomly selected m times, where m was the number of observed root tips in the associated nitrogen-addition plot.
Thus, we obtained a random rarefied species distribution. Third, the process was repeated 10,000 times, and the average species numbers and their standard deviations were calculated.
We used two-way analysis of variance (ANOVA) to test the factors nitrogen addition and season on diversity indices, the total number of ECM root tips and the enzyme activities on mm2 and total scale of the ECM communities within the six soil cores of a replicate plot per sampling (n=8, 4 replicate plots, 2 samplings per season). To be able to test the impact of the nitrogen addition and season on relative abundances
(=percentage of ECM root tips of an ECM morphotype over all counted ECM root tips in a replicate plot) and enzyme activities of single ECM morphotypes, we averaged these values within a replicate plot over both samplings of a season, because not all
40 species were found in each sampling. The lumping over both samplings within a season provided more stable numbers of n (2-4) for the two-way ANOVA. To test differences in the activities of single enzymes among ECM morphotypes independently of the treatments (averaged per replicate plot of a season, n=13-16) one-way ANOVA was used. The enzyme activities were Logn transformed prior to the statistical tests. The program SPSS 19 was used for all statistical analyses.
To assess the influence of season, treatment and ECM morphotypes on the enzyme activity profiles, principal component analysis (PCA) in Canoco for Windows 4.5 was performed. The mean activities of each of the nine most abundant ECM morphotypes were calculated per treatment for both seasons (spring and autumn) resulting in a data matrix of seven columns (7 enzymes) and 36 rows (9 morphotypes × 2 treatments × 2 seasons). To evaluate relationships among enzyme activity profiles, enzymes were treated as variables (species) and ECM morphotypes as objects
(samples). Samples were centred and standardized, species centred by species and data Logn transformed.
41 Results
Impact of nitrogen-addition and season on element contents and ECM community structure
Our results show severe impacts on the nutritional status of the host trees. Element contents in Norway spruce needles revealed that phosphate levels were significantly lower in needles from nitrogen-addition plots compared to needles from control plots
(Table S2). Also micro nutrients such as Ca, K, Mg and Mn measured in the needles indicated a tendency to be lower in nitrogen-treated plots compared to the control plots (Table S2). Results from soil analysis indicated a decrease of Ca and of the ph due to nitrogen addition (Table S2).
45 different fungal species were found in the control plots, whereas in the nitrogen- addition plots 38 were identified (Table S1). Nitrogen addition reduced the number of
ECM root tips to less than half of what was observed in the control plots (Table 1), and changed the dominant ECM morphotypes (Figure S1). Two-way ANOVA on treatment and season revealed significant treatment effects for many ECM morphotypes, whereas significant seasonal effects were only observed for
Pseudotomentella spp. (Table 1). The most abundant ECM morphotypes found in the control plots were Tomentella stuposa with an abundance of 26 %, Amphinema byssoides (23 %) and Russula integra (12 %). All three species were drastically less abundant after nitrogen addition (2 %, 5 %, 4 %, respectively) and they are therefore called nitrophobic species. The dominant morphotypes in fertilized plots with increased abundances after nitrogen addition (=nitrophilic) were Hygrophorus pustulatus (22 %), Pseudotomentella spp. (21 %) and Tylospora asterophora (17 %).
Clavulina cristata, Cortinarius spp. and Russula-like revealed similar abundances in both treatments (= nitrogen tolerant) (Figure S1). It has to be noted that in the ECM
42 morphotypes Cortinarius spp. and Russula-like the set of species lumped together differs in control and nitrogen-addition plots. For instance the ECM morphotype
Cortinarius spp. consists of nine Cortinarius species, whereby six occur in the control plots and three in the nitrogen-addition plots (Table S1).
The nitrophilic ECM morphotypes exhibited contact exploration types (Tylospora asterophora and Hygrophorus pustulatus) and a medium-distance smooth exploration type (Pseudotomentella spp.). Nitrophobic ECM morphotypes formed two contact exploration types (Tomentella mucidula and Russula integra) and a medium- distance fringe exploration type (Amphinema byssoides). Tolerant ECM morphotypes showed two contact exploration types (Clavulina cristata and Russula–like) and one medium-distance fringe exploration type (Cortinarius spp.).
43 Table 1. Relative abundances of dominant ECM morphotypes in control and nitrogen-addition plots for both seasons (n=2-4, mean ± SE in %), the number of ECM root tips, the rarefied number of ECM morphotypes and diversity indices (n=8, mean ± SE). The amount and structure of the external mycelium of the ECM morphotypes are specified as exploration types. Results of the two-way ANOVAs are given as p-values.
Nitrogen-addition Nitrogen-addition Control Control P value Sensitivity to ECM morphotype nitrogen-addition Expl. type Spring Autumn Spring Autumn Treatment Season Amphinema byssoides Nitrophobic Med. fringe 6.3 (5.1) 5.7 (3.1) 22.6 (2.0) 23.4 (2.5) <0.001 0.965
Russula integra Nitrophobic Contact 6.4 (3.1) 3.8 (1.9) 12.3 (2.2) 11.7 (1.6) 0.015 0.519
Tomentella stuposa Nitrophobic Contact 4.1 (1.4) 4.5 (2.7) 23.0 (3.4) 30.0 (2.5) <0.001 0.252
Hygrophorus pustulatus Nitrophilic Contact 18.4 (5.4) 22.3 (7.7) 1.3 (0.3) 2.9 (2.5) 0.003 0.577
Pseudotomentella spp. Nitrophilic Med.smooth 29.6 (6.0) 15.4 (2.8) 5.8 (1.6) 2.8 (0.9) <0.001 0.028
Tylospora asterophora Nitrophilic Contact 10.1 (2.8) 21.7 (9.4) 6.8 (1.9) 5.3 (2.4) 0.082 0.346
Clavulina cristata Tolerant Contact 3.2 (1.6) 14.8 (8.8) 11.8 (3.9) 10.9 (4.8) 0.850 0.475
Cortinarius spp. Tolerant Med. fringe 4.3 (1.8) 6.1 (3.4) 3.5 (1.7) 5.5 (2.0) 0.778 0.428
Russula-like Tolerant Contact 13.4 (6.6) 10.0 (4.9) 8.6 (1.7) 3.7 (1.6) 0.262 0.393
Number of ECM root tips 1306.9 (204.7) 1812.5 (304.0) 3191.0 (395.6) 3570.8 (329.3) <0.001 0.172
Number of ECM morphotypes 7.6 (0.5) 7.5 (0.5) 9.4 (0.4) 9.0 (0.3) <0.001 0.547
Rarefied number of ECM morphotypes analytic n.a. n.a. 9.3 (0.4) 9.0 (0.3) n.a. n.a.*
Rarefied number of ECM morphotypes stochastic 7.6 (0.5) 7.5 (0.5) 9.2 (0.4) 8.9 (0.3) 0.001 0.600
Simpson index of diversity 0.7 (0.0) 0.7 (0.0) 0.8 (0.0) 0.8 (0.0) 0.005 0.170
Shannon index of diversity 1.6 (0.1) 1.5 (0.1) 1.9 (0.0) 1.7 (0.0) 0.001 0.200 *n.a. =not applicable
44 Effects of nitrogen-addition and season on enzyme activities
Although the ECM morphotype dominance changed due to nitrogen addition, enzyme activities per mm2 root-tip surface of the communities were not markedly altered
(Figure 1). Small but significant differences were observed for the cellulose degrading enzymes. Glucosidase and cellobiohydrolase showed a significant increase in the nitrogen-addition plots (up to a factor of 1.6). Moreover, the phosphatase and leucine aminopeptidase activities exhibited a significant increase in spring compared to autumn. A significant decrease in total enzyme activity of the
ECM community (=all ECM morphotypes together including their number of ECM root tips, expressed as pmol/min* ECM root tips) was evident after nitrogen addition due to the reduced number of fine roots (Figure 2).
4.5 * Nitrogen-addition Spring 4 Nitrogen-addition Autumn
2 Control Spring 3.5 ** Control Autumn 3
2.5 *** 2 ***
1.5
1 enzyme activity pmol/min/mm activity enzyme 0.5
0 Pho Leu Nag Cel Gls Xyl Lac
Figure 1. Enzyme activities per mm2 root tip surface (n=8, ±SE) averaged over all ECM morphotypes in nitrogen-addition and control plots of both seasons. The values are given Logn transformed for a better overview. Enzyme abbreviations: Pho: acid phosphomonoesterase; Leu: leucine aminopeptidase; Nag: N- acetylglucosaminidase; Cel: cellobiohydrolase; Gls: glucosidase; Xyl: xylosidase; Lac: laccase; A two-factor ANOVA revealed significant treatment (Cel and Gls) and season (Pho and Leu) effects reported as asterisks:***: P < 0.001, **:P < 0.01, *:P < 0.05.
45 Pho Spring Leu
addition Nag Nitrogen- Cel Gls Autumn Xy l addition Nitrogen- Lac
Spring
Autumn Control Control
0 50000 100000 150000 200000 250000 300000 350000 total enzyme activity (pmol/min*ECM tips)
Figure 2. Total enzyme activities of all ECM morphotypes multiplied by their number of ECM root tips (n=8 ± SE). The two-way ANOVA revealed a significant nitrogen addition effect with p= <0.001. Enzyme abbreviations: Pho: acid phosphomonoesterase; Leu: leucine aminopeptidase; Nag: N- acetylglucosaminidase; Cel: cellobiohydrolase; Gls: glucosidase; Xyl: xylosidase; Lac: laccase.
PCA revealed a clustering of the enzyme activities according to ECM morphotypes rather than according to treatment or season (Figure 3a). Tomentella stuposa and
Pseudotomentella spp. were clearly separated from other morphotypes on the first axis. This was mainly due to their low laccase activity, which correlated strongly with the first PCA axis. Cortinarius spp. was separated from the other ECM morphotypes mainly on the second axis, which was partly due to their high leucine aminopeptidase activity. All other ECM morphotypes (Tylospora asterophora, Russula integra,
Russula-like, Hygrophorus pustulatus, Clavulina cristata and Amphinema byssoides) revealed relatively constant profiles over the different treatments, showed similar profiles among each other and were therefore intermingled on the PCA graph of the first two axes. Figure 3b shows the same analysis, but ECM morphotypes were labelled as exploration types and according to their sensitivity to nitrogen addition. No grouping of enzyme activity profiles was observed based on the sensitivities of ECM morphotypes to nitrogen addition or based on the exploration types.
46
Tylospora asterophora 1.5
Cortinarius spp. Leu Tomentella stuposa
Pseudotomentella spp.
Russula integra
Lac Russula-like Pho Amphinema byssoides Clavulina cristata Cel Hygrophorus pustulatus Nag Xyl Gls -1.0 1.5
-1.0 2.0
Leu
Lac Pho
Cel Nag -1.0 Xyl Gls
-1.0 2.0
Figure 3. a) Principle component analysis (axis one: 69 %, axis two 16.6 % of variation) indicating the effect of ECM morphotypes, nitrogen-addition and season on seven enzyme activities. Each data point corresponds to the mean enzyme activities of the dominant ECM morphotypes in control and nitrogen-addition plots during spring and autumn. Light blue symbols: control spring; blue: control autumn; light red: nitrogen addition spring, red: nitrogen addition autumn. b) The same PCA mentioned above demonstrating the ECM morphotypes marked as nitrophobic, nitrophilic or tolerant to nitrogen addition and indicating their exploration types. Blue: nitrogen tolerant; orange: nitrophilic; green: nitrophobic; circles: contact exploration types; squares: medium fringe exploration types; diamonds: medium smooth exploration types;
47 The relatively constant enzyme activity profiles of the different ECM morphotypes throughout the treatment combinations can also be seen in Figure 4. The Cortinarius spp. show the most evident exception to this with significantly higher activities in nitrogen-addition plots. More plastic enzyme activities were also exhibited Tomentella stuposa (=nitrophobic) and Pseudotomentella spp. (=nitrophilic) with an activity increase of some enzymes (Figure 4, Table 2). The remaining ECM morphotypes
(Tylospora asterophora, Amphinema byssoides, Clavulina cristata, Hygrophorus pustulatus, Russula integra and Russula-like) revealed no or few enzymes with significant differences in activities. An interaction between treatment and season was observed mainly in laccase activities for Hygrophorus pustulatus and Amphinema byssoides (Table 2).
Clear differences were evident for enzyme activities among ECM morphotypes independently of the treatments (Figure S2). The highest variability of activities among ECM morphotypes was observed for laccase and leucine aminopeptidase.
For instance, the laccase activity of Tomentella stuposa with 3.5 pmol/mm2/min differed significantly by a factor of 45 compared to Russula integra with 155.3 pmol/mm2/min.
48 Nitrophobic Nitrophilic Tolerant
Spring Spring Spring
Autumn Autumn Autumn
Pho Pho Spring Leu Pho Leu Spring Leu Tomentella stuposa Nag Spring Nag Nag Cel Cel Clavulina cristata Gls Cel Gls Gls Xyl Hygrophorus pustulatus CCNN Autumn Xyl Xyl Lac CCNN Autumn Lac
CCNN Autumn Lac
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 2 0 50 100 150 200 250 300 350 2 enzyme activity pmol/min/mm enzyme activity pmol/min/mm 2 enzyme activity pmol/min/mm
Spring Spring Spring
Autumn Autumn Autumn Pho Pho Leu Leu Nag Nag Pho Spring Leu Spring Cel Cel Russula-like Spring Gls Gls Nag Cel Xyl Pseudotomentella spp. Xyl Amphinema byssoides Amphinema Lac Lac Gls
CCNN Xyl CCNN Autumn Autumn CCNN Autumn Lac
0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 2 enzyme activity pmol/min/mm 2 2 enzyme activity pmol/min/mm enzyme activity pmol/min/mm
371 480 Spring Spring Spring 375 Autumn Autumn Autumn Pho Spring Leu Pho Pho Nag Leu Russula integra Russula Spring Spring Leu Cel Nag
Cortinarius spp. Nag Gls Cel Cel Xyl Tylospora asterophora Gls Gls
CCNN Autumn Lac Xyl Xyl CCNN Autumn Lac CCNN Autumn Lac 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 2 0 50 100 150 200 250 300 350 enzyme activity pmol/min/mm 2 enzyme activity pmol/min/mm 2 enzyme activity pmol/min/mm
Figure 4. Enzyme activities per mm2 root tip surface of dominant nitrophobic, nitrophilic and nitrogen tolerant ECM morphotypes (n=2-4). Enzyme abbreviations: Pho: acid phosphomonoesterase; Leu: leucine aminopeptidase; Nag: N-acetylglucosaminidase; Cel: cellobiohydrolase; Gls: glucosidase; Xyl: xylosidase; Lac: laccase;
49 Table 2. Effects of nitrogen addition, season and their interaction on individual and overall enzyme activities as measured in pmol/min/mm2 of ECM morphotypes (n=2- 4). Two-way ANOVAs revealed significant differences and are reported as follows: +++, - - - or ***: P < 0.001; ++, - - or **: P < 0.01; +, - or *:P < 0.05; +: higher activity in treatment (above) or spring (below); -: higher activity in control (above) or autumn (below); *significant interaction; Enzyme abbreviations: Pho: acid phosphomonoesterase; Gls: glucosidase; Xyl: xylosidase; Nag: N- acetylglucosaminidase; Leu: leucine aminopeptidase; Lac: laccase; Cel: cellobiohydrolase; The amount and structure of the external mycelium of the ECM morphotypes is specified as exploration types in the brackets.
Sensitivity to Overall nitrogen- Exploration enzyme addition type Pho Gls Xyl Nag Leu Lac Cel activity Factor treatment Amphinema byssoides Nitrophobic Med. fringe - + Russula integra Nitrophobic Contact - + Tomentella stuposa Nitrophobic Contact ++ + + ++ Hygrophorus pustulatus Nitrophilic Contact ++ + Pseudotomentella spp. Nitrophilic Med. smooth ++ + - - + Tylospora asterophora Nitrophilic Contact + ++ Clavulina cristata Tolerant Contact Cortinarius spp. Tolerant Med. fringe ++ ++ ++ ++ ++ ++ +++ Russula-like Tolerant Contact Factor season Amphinema byssoides Nitrophob Med. fringe - - - Russula integra Nitrophob Contact + + Tomentella stuposa Nitrophob Contact + Hygrophorus pustulatus Nitrophil Contact - - - - Pseudotomentella spp. Nitrophil Med. smooth + Tylospora asterophora Nitrophil Contact ++ Clavulina cristata Tolerant Contact Cortinarius spp. Tolerant Med. fringe Russula-like Tolerant Contact + Treatment x season interaction Amphinema byssoides Nitrophob Med. fringe ** Russula integra Nitrophob Contact Tomentella stuposa Nitrophob Contact * Hygrophorus pustulatus Nitrophil Contact ** Pseudotomentella spp. Nitrophil Med. smooth Tylospora asterophora Nitrophil Contact ** Clavulina cristata Tolerant Contact Cortinarius spp. Tolerant Med. fringe Russula-like Tolerant Contact
50 Discussion
Peculiarities of the Parabock experimental site with spruce
The Swiss plateau is a densely populated area with intense private traffic and animal husbandry. The Parabock experimental site is located within a coherent pre-alpine forest area adjacent to the plateau, in a region (Gruyère district) which is well known for intense animal husbandry, and is situated near an intensely used highway. Thus the Parabock experimental site receives approx. 17.5 kg nitrogen ha-1 yr-1 as atmospheric nitrogen pollutants, an estimated 10-fold rate as in pre-industrial times.
Consequently, the control plots are not oligotrophic sites, and, as pointed out by
Lilleskov et al. (2011), extremely nitrophobic ECM fungi such as Piloderma or
Tricholoma spp. are missing above- and belowground in the study of Peter et al.
(2001), whereas in the present investigation one Piloderma species was found in both control and nitrogen-addition plots. As summarized by Braun et al. (2010), nitrogen pollution is substantial throughout Switzerland, only high alpine areas receiving less that 10 kg ha-1 yr-1 of atmospheric nitrogen pollutants. Thus, the
Parabock site is well suited for a long-term experiment on the impact of even higher levels of nitrogen fertilization and reflects the above- and belowground ECM diversity in a characteristic Swiss forest ecosystem.
Comparing the response of nitrogen addition on ECM communities above- and belowground
Consistent with our hypothesis and other studies (Fransson et al. 2000, Frey et al.
2004, Treseder et al. 2004, Cox et al. 2010, Kjoller et al. 2012), long-term nitrogen addition reduced the belowground ECM richness, decreased the number of ECM root tips to less than half and changed the ECM morphotype dominance. The
51 aboveground ECM community of this experimental site also exhibited a constant reduction of ECM species richness and sporocarp density over the first 10 years of nitrogen addition. However, one fungus, Hygrophorus pustulatus, clearly seemed to profit from nitrogen addition (Gillet et al. 2010). Belowground, the fungus also responded slightly positive at the beginning of this experiment (Peter et al. 2001) and belonged to the most abundant fungi in the nitrogen-treated plots in the present study. Another fungus, Thelephora palmata showed an increase of produced sporocarps in the nitrogen-treated plots in the last three years of the study, 2005 until
2007 (Gillet et al. 2010). In the present study this species was included in the ECM morphotype of Pseudotomentella spp. (Table S2). The sequence data in this ECM morphotype showed that Thelephora palmata was also more prominent in the nitrogen-addition plots compared to the control plots, which support the observations aboveground. Cox at al. (2010) also showed a positive response of Thelephora spp. to increased nitrogen levels. Russula integra decreased belowground at the beginning of this experiment after nitrogen addition (Peter et al. 2001). In our study and in the long-term aboveground observations (Gillet et al. 2010), this fungus was rare in the nitrogen-addition plots, whereas in the control plots it was much more abundant. The literature so far reports a mixed response of Russula species to nitrogen addition, ranging from positive over absent to negative (Lilleskov et al.
2011). Clavulina cristata exhibited similar abundances belowground in control and nitrogen-addition plots in the present study. Aboveground, this fungus exhibited a drastic reduction after nitrogen addition (Gillet et al. 2010). Similarly, Cortinarius spp. were negatively affected aboveground, whereas belowground they seem to be tolerant. However, since we lumped all Cortinarius spp. into one ECM morphotype, some species most likely are sensitive to nitrogen addition. For instance, Cortinarius decipiens and Cortinarius flexipes appeared only in the control plots belowground
52 and aboveground, which points to nitrophobic behaviour. In contrast, Cortinarius anomalus was detected in the nitrogen-addition plots, again belowground and aboveground. In the literature, Cortinarius spp. are mostly classified as sensitive to nitrogen addition (Lilleskov et al. 2011). In contrast, Cox et al. (2010) found no relationship of Cortinarius spp. with nitrogen availability but reported low abundances of this genus across all studied plots of Pinus sylvestris forests with elevated levels of nitrogen. Our results support the fact that different species within a genus can respond differently to nitrogen addition as has already been suggested by Cox et al.
(2010).
Impact of nitrogen-addition on enzyme activities of ECM communities
Although the enzyme activity profiles differed for different ECM morphotypes, and control and nitrogen-addition plots exhibited different dominant ECM morphotypes, the enzyme activities per mm2 of the communities were not significantly changed by nitrogen addition. This indicates that the ecological functions were taken over by functionally redundant but better adapted species after long-term high nitrogen input.
Species-specific enzyme profiles with overlaps in functions and the adaptation of the ecosystem to environmental conditions by changed ECM communities was described in several previous studies (Rineau and Garbaye 2009, Courty et al. 2010, Diedhiou et al. 2010, Jones et al. 2010, Jones et al. 2012), and might therefore be a general characteristic of ECM communities.
The only enzymes that showed a significant increase in activities after nitrogen addition were cellulose-degrading enzymes, which make carbon available from complex organic molecules. To some extent, this supports our hypothesis that the acquisition of carbon from organic matter will be more important at high levels of mineral nitrogen because of a reduced provision of carbon by the tree and an
53 increased carbon demand by the fungi themselves. As further supporting evidence, other studies also found a stimulation of cellulose degrading enzymes measured in soil after nitrogen addition (Frey et al. 2004, Henry et al. 2005, Stursova et al. 2006,
Zeglin et al. 2007, Keeler et al. 2009).
Leucine aminopeptidase which frees organic nitrogen as amino acids from soil organic matter was not affected by nitrogen addition. However, leucine aminopeptidase and phosphatase activity increased significantly in spring time in comparison to autumn. The observed increases may indicate a higher demand for nutrients of the trees in their starting growth phase or an increased supply for the
ECM fungi themselves to colonize the newly formed root tips. Elsewhere, seasonal changes of ECM fungal enzyme activities were also reported (Courty et al. 2010,
Buée et al. 2005, Mosca et al. 2007). Mosca et al. (2007) showed increases in all enzymes during winter compared to spring in an oak forest, and suggested that the supply of organic carbon from soil is increased because of the photosynthetically inactive oaks. In contrast to the minor change of the enzyme activities of the ECM communities per mm2, total enzyme activities (over all root tips) decreased significantly in the nitrogen-addition plots compared to the control plots. This was due to the substantial reduction to less than half of ECM root tips in the nitrogen-addition plots. In consequence of such a reduction in enzyme activities, the nutrient cycles in this ecosystem might slow down. Since we did not measure the enzyme activities in the soil performed by mycelia and microbes, which have been argued to be much more important than activities at the ECM root surface in a Pinus muricata forest
(Talbot et al. 2013), it is unclear how important the observed reduced enzyme activities are for the ecosystem. Nevertheless, there are several reasons that let us suppose that the enzyme activities are in fact significantly reduced in the root and soil compartment. First, Kjoller et al. (2012) showed that not only fine roots were reduced
54 due to increasing nitrogen deposition but so was the amount of external mycelium.
Second, the high divergence between root-tip and soil enzyme activities observed by
Talbot et al. (2013) might be less significant in ecosystems in which the root density is very high, such as the one that we have studied. Last, nitrogen addition has been repeatedly shown to decrease decomposition rates in spruce forests (Janssens et al.
2010), which is in line with our observation of reduced activities of organic matter decomposing enzymes on the level of total enzyme activities including all ECM root tips.
Tolerant, nitrophilic and nitrophobic ECM morphotypes
We hypothesized that the nitrophobic, nitrophilic and tolerant ECM morphotypes would exhibit distinctive enzyme activity profiles. This could not be substantiated in our study. For example, we hypothesized that tolerant species would be more plastic in their activity profiles allowing them to adapt to the prevalent environmental conditions, as shown by some studies (Courty et al. 2005, Buée et al. 2007, Jones et al. 2010, Jones et al. 2012). Based on our definition of nitrogen sensitivity, the ECM morphotype Cortinarius spp. seems to be tolerant since its abundance in control and nitrogen-addition plots did not differ. As already mentioned, this ECM morphotype consisted of different Cortinarius species in the treatment plots (Table S1). Therefore, the dissimilar enzyme activity profiles of this ECM morphotype in the control and nitrogen-addition plots were likely mainly caused by the changed species composition. However, significant differences - mainly an increase in activities of several enzymes - were observed for Pseudotomentella spp. and Tomentella stuposa. Interestingly, the former was nitrophilic, whereas the latter was nitrophobic.
Our results suggest that these ECM morphotypes are intrinsically good at adapting
55 their enzyme activities, independently of the effect of nitrogen deposition on their population size.
Peter et al. (2001) found slight abundance changes of some species two years after starting the long-term nitrogen-addition experiment. Hygrophorus pustulatus,
Pseudotomentella spp. (T-RFLP Type 6 of Peter et al. 2001) and Tylospora asterophora showed an increasing abundance in the nitrogen-addition plots. In our study, these fungi were the most abundant fungi in the nitrogen-addition plots, while their abundance did not change in the control plots since the start of the treatment.
Therefore, we classify them as nitrophilic. Lilleskov et al. (2011) recently summarized the abundance response of different ECM species to nitrogen addition. Tylospora species appeared to be nitrophilic, whereas Pseudotomentella spp. was reported as potentially nitrophobic, which does not apply to our Pseudotomentella ECM morphotype. Hygrophorus species showed a mixed response according to Lilleskov et al. (2011). Tomentella stuposa, which was the most abundant fungus in all plots at the beginning of the experiment (Peter et al. 2001), exhibited the greatest decrease in abundance due to nitrogen addition. Peter et al. (2001) further found an abundance decrease of Russula integra and Amphinema byssoides (TRFLP-type 2), which was confirmed by our observations and therefore they were classified as nitrophobic. Lilleskov et al. (2011) reported Tomentella spp. and Russula spp. to show a mixed response whereas Amphinema spp. was recorded as potentially nitrophobic.
All ECM morphotypes were able to mine for organic nitrogen, carbon and phosphate, but did not show the enzyme patterns we expected. For instance, the nitrophilic
Pseudotomentella spp. focused in carbon and organic phosphate degradation, but additionally exhibited a specialization in chitin degradation. However, chitin is both a source of nitrogen and carbon. Thus, it is not possible to clearly distinguish whether
56 chitin degradation dominantly helps with nitrogen or carbon supply. Similar uncertainties exist for the activities of leucine aminopeptidase, which is responsible for protein degradation, and of laccase, which helps breaking down phenolic compounds. Like chitin, proteins contain both nitrogen and carbon. The break-down of phenolic compounds by laccase, on the other hand, makes phenolic-bound soil poteins more accessible to fungi (Ramstedt and Söderhäll, 1983).
Lilleskov et al. (2011) suggested that less carbon might be required from the tree when the fungi display small amounts of external mycelium. Therefore, fungi with little mycelium might be favoured in a reduced carbon allocation environment due to nitrogen addition. Two out of three nitrophilic fungi exhibited very little external mycelium, Hygrophorus pustulatus and Tylospora asterophora, complying with the hypothesis of Lilleskov et al. (2011). A characteristic for nitrophobic species might be abundant external mycelium, classified as medium-distance fringe exploration types because most of the nitrophobic fungi observed so far exhibited this structure
(Lilleskov et al. 2011). In our study, only one out of three fungi, Amphinema byssoides, revealed such a structure. The other two fungi were so-called contact exploration types with very little external mycelium. As already mentioned by
Lilleskov et al (2011) generalisations are difficult to make. Pritsch et al. (2011) postulated that fungi with much external mycelium might be specialized in carbon mobilisation from the soil, since parts of the external mycelium are far away from root tips of their host tree and its carbon supply. The observed abundant external mycelium of Pseudotomentella spp. fits the ideas of Pritsch et al. (2011). However, these morphological observations cannot be substantiated with data of enzyme activities, since the external mycelium was not measured in our study. The enzyme activities of the ECM root tips of Pseudotomentella spp. points to a specialization on carbon mobilising abilities. Based on the available data we suggest that both of the
57 mechanisms proposed by Lilleskov et al. (2011) and Pritsch et al. (2011) can co-exist in the same location in different species.
Effects of nitrogen addition on the nutrient status of spruce and soil properties
Phosphate levels were significantly lower in Norway spruce needles from nitrogen addition plots compared to control plots. The threshold values of phosphate contents
(www.luftschadstoffe.at, 2011) indicate that in the nitrogen-addition plots this element was deficient, which was not the case in needles from control plots. The ECM community is not able to supply the trees with enough phosphate, because the number of ECM root tips was 50 % reduced, and consequently total enzyme activities of this community had declined. One could hypothesize that a reduced content of phosphate in the tree would stimulate the phosphatase activity of the ECM community for enhancing the phosphate level in the tree. However, this was not the case. In addition, micro nutrients in the needles indicated a tendency to be lower in nitrogen-treated plots compared to the control plots. The imbalanced nutrient status of the Norway spruces in the fertilized plots might increase their vulnerability to biological (fungal and animal pathogens) and climatic stresses (compare to the review by Braun et al. 2010).
In contrast to the lower phosphate and micro nutrient levels in the nitrogen treated plots, the nitrogen contents in tree needles from both treatments exhibited similar low values (www.luftschadstoffe.at, 2011). The nitrogen levels in the needles of trees from the nitrogen-addition plots increased after starting the experiment (Peter et al.
2001). Similarly, Ingerslev et al. (1999) reported an increase in nitrogen levels of needles in a spruce stand after the beginning of fertilizing. It might be that, through the long-term nitrogen addition, trees produced more biomass compared to control trees, and therefore excess nitrogen is used for maintaining biomass production.
58 Mellert et al. (2004) reported a decline of foliar nitrogen of spruce forests in Europe, although anthropogenic nitrogen deposition was quite high. They interpreted this as a result of dilution processes, since also increases in needle weights were observed.
Soil ph was reduced due to nitrogen addition in our study site. A release of certain heavy metals, such as Al is the consequence (Lilleskov et al. 2001). Gillet et al.
(2010) found in our study site four times higher Al concentrations in the nitrogen- treated plots compared to the controls after 10 years of fertilization. Moreover, we observed a decrease of Ca in the nitrogen-addition plots compared to control plots, whereas other elements were stable. Such factors might additionally shape ECM communities on the structural and functional scale.
Conclusion
Our results show that ecological functions in the nitrogen-treated community were taken over by functional redundant but better adapted ECM morphotypes. However, the reduced species richness may render the forest ecosystem more vulnerable to additional stresses because the available species pool may no longer allow an appropriate adaptation. Additionally, the reduced number of ECM root tips and the consequent reduction of enzyme activities of the fertilized ECM community is likely to influence the forest’s nutrient cycle. We found reduced phosphate levels and micro nutrients in the needles of the nitrogen-treated Norway spruces already indicating an imbalance in their nutrient contents. No clear pattern of the measured functional traits emerged in relation to the tolerant, nitrophobic and nitrophilic behaviour of the fungi.
Other functional traits such as the efficiency of delivering nutrients to the tree might be more important. One strategy might be to measure the natural occurring 15N to obtain insights into the amount of nutrients the fungus delivers to tree (Hobbie et al.
59 2005, Koide et al. 2007). However, another exciting task would be the re-examination of the structure and function of the ECM communities after several years of regeneration without nitrogen addition with the same tools as used in the current study.
Acknowledgements Sincere thanks are due to Rosmarie Eppenberger for support with field work, molecular analysis and enzyme activity measurements. Moreover, for critical reading, discussing and calculation of diversity indices and rarefied ECM morphotypes we are very thankful to Jens M. Turowski. This study was part of FUNDIV grant ANR606-BDIV-06 from the French ANR (Agence Nationale de la Recherche).
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65 Supplemental material
40 Nitrogen-addition Spring 35 Nitrogen-addition Autumn Control Spring b 30 Control Autumn 25
20
15
10 Relative abundance %
5
0 stuposa Tomentella spp. byssoides Tylospora geophilum Amphinema pustulatus Russula-like asterophora Cenococcum Hygrophorus Amanita Russula integra Tomentella sp.3 Cortinarius spp. Tuber puberulum Pseudotomentella Clavulina cristata Clavulina ochraceomaculata
Figure S1. Mean relative abundance of ECM morphotypes in control and nitrogen- addition plots during spring and autumn (n=0-4, + - SE).
160 Acid phosphomonoesterase (Pho) 2 140
120
100
80
60
40
20 enzyme activity pmol/min/mm 0 (Contact) Tylospora (Contact) (Contact) pustulatus (Short) asterophora (Contact) Russula-like (Contact) Hygrophorus byssoides (Contact) Amanita smooth) Amphinema Tomentella (Medium fringe) Cenococcum Russula integra Russula (Medium fringe) spp. (Medium Cortinarius spp. Tomentella sp.3 Tuber puberulum Clavulinacristata geophilum (Short) stuposa (Contact) stuposa (Medium smooth) Pseudotomentella ochraceomaculata Legend is written on page 68;
66 40 Leucine aminopeptidase (Leu) 2 35
30
25
20
15
10
5 enzyme activity pmol/min/mm 0 (Contact) (Contact) (Contact) Tylospora pustulatus (Short) Russula-like asterophora (Contact) (Contact) Hygrophorus byssoides smooth) smooth) Amanita (Contact) Amphinema Tomentella Cenococcum Russula integra (Medium fringe) (Medium (Medium fringe) (Medium Cortinarius spp. Cortinarius sp.3 Tomentella spp. (Medium spp. Tuber puberulum Tuber Clavulina cristata geophilum (Short) stuposa (Contact) stuposa (Medium smooth) (Medium Pseudotomentella ochraceomaculata
140 N-acetylglucosaminidase (Nag) 2
120
100
80
60
40
20
enzyme activity pmol/min/mm activity enzyme 0 (Contact) (Contact) (Contact) Tylospora pustulatus (Short) Russula-like asterophora (Contact) (Contact) Hygrophorus byssoides smooth) smooth) Amanita (Contact) Amphinema Tomentella Cenococcum Russula integra (Medium fringe) (Medium (Medium fringe) (Medium Cortinarius spp. Cortinarius sp.3 Tomentella spp. (Medium spp. Tuber puberulum Tuber Clavulina cristata geophilum (Short) stuposa (Contact) stuposa (Medium smooth) Pseudotomentella ochraceomaculata
2 25 Cellobiohydrolase (Cel)
20
15
10
5
enzyme activity pmol/min/mm activity enzyme 0 (Contact) (Contact) Tylospora (Contact) pustulatus asterophora (Short) Russula-like (Contact) (Contact) Hygrophorus byssoides smooth) smooth) Amanita (Contact) Amphinema Tomentella (Medium fringe) spp. (Medium spp. (Medium fringe) Cenococcum Russula integra Cortinarius spp. Cortinarius Tomentella sp.3 Tuber puberulum Clavulina cristata geophilum (Short) stuposa (Contact) Pseudotomentella (Medium smooth) ochraceomaculata Legend is written on page 68;
67 80 Glucosidase (Gls) 2 70
60
50
40
30
20
10 enzyme activity pmol/min/mm activity enzyme 0 (Contact) (Contact) (Contact) Tylospora pustulatus (Short) asterophora Russula-like (Contact) (Contact) Hygrophorus byssoides smooth) smooth) (Contact) Amanita Amphinema Tomentella Russula integra Cenococcum (Medium fringe) (Medium (Medium fringe) (Medium spp. (Medium spp. Cortinarius spp. Cortinarius sp.3 Tomentella Tuber puberulum Tuber Clavulina cristata Clavulina stuposa (Contact) stuposa geophilum (Short) (Medium smooth) (Medium Pseudotomentella ochraceomaculata
2 Xylosidase (Xyl) 14
12
10
8 6
4 2
0 enzyme activity pmol/min/mm activity enzyme (Contact) (Contact) (Contact) Tylospora pustulatus (Short) asterophora Russula-like (Contact) (Contact) Hygrophorus byssoides smooth) smooth) (Contact) Amanita Amphinema Tomentella Cenococcum Russula integra (Medium fringe) (Medium (Medium fringe) (Medium Cortinarius spp. Cortinarius sp.3 Tomentella spp. (Medium spp. Tuber puberulum Tuber Clavulina cristata geophilum (Short) stuposa (Contact) stuposa (Medium smooth) (Medium Pseudotomentella ochraceomaculata
180 Laccase (Lac) 2 160 140 120 100 80 60 40 20 enzyme activity pmol/min/mm activity enzyme 0 (Contact) (Contact) (Contact) Tylospora pustulatus (Short) asterophora Russula-like (Contact) (Contact) Hygrophorus byssoides smooth) smooth) Amanita (Contact) Amphinema Tomentella Russula integra Cenococcum (Medium fringe) (Medium (Medium fringe) (Medium spp. (Medium spp. Tomentella sp.3 Tomentella Cortinarius spp. Cortinarius Tuber puberulum Tuber Clavulina cristata Clavulina stuposa (Contact) stuposa geophilum (Short) (Medium smooth) (Medium Pseudotomentella ochraceomaculata Figure S2. Enzyme activities among ECM morphotypes, which were taken together from nitrogen-addition and control plots (n=2-16, ± SE). Black bars indicate nitrophobic ECM morphotypes, colourless bars indicate nitrophilic ECM morphotypes, light grey bars indicate tolerant ECM morphotypes and dark grey bars indicate rare ECM morphotypes. The amount and structure of the external mycelium of the ECM morphotypes is specified as exploration types in the brackets.
68
Table S1. ECM morphotypes, their exploration strategy, their morphological features and ITS sequence matches with the respective number of identified ECM root tips from control (C) and nitrogen-addition (N) plots.
ECM morphotype Expl. type Morphological features Sequence matches C N Amanita ochraceomaculata Med. smooth White greyish, woolly with rhizomorphs; Amanita ochraceomaculata 12 Amphinema byssoides Med. fringe Yellowish-white, cottony with abundant Amphinema byssoides 30 5 rhizomorphs; Amphinema sp.1 1 Cortinarius scandens 1 Piloderma sp.1 4 Tomentella sp.1 1 Tylospora asterophora 1 Cenococcum geophilum Short Black with several thick emanating hyphae; Cenococcum geophilum 52 Clavulina cristata Contact Greyish-milky, smooth; Clavulina cristata 17 9 Hygrophorus pustulatus 1 Membranomyces delectabilis 3 Russula sp.1 1 Russula xerampelina 1 Tomentella ellisii 1 Tomentella sp.1 31 Cortinarius spp. Med. fringe White, woolly with abundant rhizomorphs; Cortinarius anomalus 4 Cortinarius decipiens 3 Cortinarius flexipes 1 Cortinarius hinnuleus 2 Cortinarius patibilis 1 Cortinarius scandens 11 Cortinarius sp.1 1 Cortinarius variecolor 1 Cortinarius vernus 1 Amphinema sp.1 5 Amphinema byssoides 1 Piloderma sp.1 1 Tricholoma fulvum 2 Tylospora asterophora 1 Hygrophorus pustulatus Contact Orange brown with brighter tips, smooth; Hygrophorus pustulatus 111 Clavulina cristata 1 Piloderma sp.1 41 Tomentella ellisii 1 Trichophaea gregaria 2 Tylospora asterophora 1 Pseudotomentella spp. Med. smooth Black with silvery parts, few black Pseudotomentella mucidula 6 rhizomorphs; Pseudotomentella sp.1 317 Cortinarius variecolor 1 Thelephora palmata 311 Tricholoma fulvum 1 Russula integra Contact Brown greenish, yellow pustules; Russula integra 22 6 Russula-like Contact Brown, smooth; Russula xerampelina 1 Russula puellaris 3 Russula sapinea 31 Clavulina cristata 12 Inocybe umbratica 2 Thelephora palmata 3 Tomentella sp.1 12 Tuber puberulum 1 Tylospora asterophora 4 Tomentella sp.3 Contact Dark brown, grainy; Tomentella sp.3 1 Tomentella stuposa Contact Dark brown-black, grainy; Tomentella stuposa 27 7 Tomentella sp.1 1 Tomentella sp.2 1 Piloderma sp.1 2 Tuber puberulum Short Light brown orange, densely short-spiny Tuber puberulum 31 Tylospora asterophora Contact Light brown with brighter tips, smooth; Tylospora asterophora 13 17 Amphinema byssoides 2 Clavulina cristata 22 Hygrophorus pustulatus 4 Russula sapinea 24 Tomentella sp.1 32 Tomentella stuposa 1 Tuber puberulum 3
69
Table S2. Soil and needle nutrient contents of control and nitrogen-addition plots. One-way ANOVAs revealed significant differences for needle phosphate levels with P < 0.01.
Ca Mg K Mn P tot N tot C tot C/N BC/Al Ph Soil nutrients g/kg g/kg g/kg mg/kg g/kg g/kg g/kg ratio ratioa (KCl)
Control 17.0 1.4 0.4 0.2 0.2 5.0 77.2 15.6 8.9 3.8
Nitrogen-addition 7.1 1.4 0.3 0.1 0.2 5.0 80.0 15.9 1.0 3.2 Needle nutrients Control tree 1 12.7 1.2 8.2 552.0 1.5 13.2 Control tree 2 17.5 1.0 5.9 438.0 1.1 10.6 Control tree 3 14.2 1.5 6.7 341.0 1.5 11.1 Control tree 4 17.0 1.2 5.8 340.0 1.2 12.0 Mean 15.4 1.2 6.7 417.8 1.3 11.7 SE 1.1 0.1 0.6 50.3 0.1 0.6 Nitrogen-addition tree 5 14.6 1.6 3.6 237.0 0.7 11.8 Nitrogen-addition tree 6 16.6 0.7 6.0 474.0 0.7 11.2 Nitrogen-addition tree 7 13.1 0.7 6.2 353.0 0.9 13.3 Nitrogen-addition tree 8 13.7 0.9 5.4 249.0 0.8 11.6 Mean 14.5 1.0 5.3 328.3 0.8 12.0 SE 0.8 0.2 0.6 55.1 0.1 0.5 P value 0.559 0.285 0.153 0.276 0.005 0.744 a From Gillet et al. 2010
70
Chapter 2
The ectomycorrhizal fungal community of a drought-stressed Scots pine forest is functional resilient to irrigation
Sylvia Hutter, Simon Egli, Renaud Maire, Rosmarie Honegger, Jean Garbaye and
Martina Peter
Summary
In the inner alpine valleys of central Europe Scots pines (Pinus sylvestris) suffer from drought due to elevated temperatures in the course of global warming. Therefore an irrigation experiment was set up in a dry sub alpine forest of a Swiss valley in order to gain information about the growth response of the trees and of their ectomycorrhizal
(ECM) fungal symbionts to changed water relations. ECM community composition was investigated in selected soil cores by 1) quantifying ECM root tips by counting, 2) visual classification by morphotyping and, 3) taxonomic identification by ITS sequencing. Functional aspects were studied by measuring the activities of enzymes involved in the degradation of organic matter on individual ECM root tips. Samples were collected and analysed in autumn 2007 and 2008, and spring 2008 and 2009.
While the Scots pines reacted with significantly increased growth rates upon irrigation, only minor differences were noted among the ECM communities in irrigated and dry control plots, only Rhizopogon roseolus and Inocybe spp. being more abundant upon irrigation. The overall ECM species richness did not change belowground upon irrigation, but increased in spring compared to autumn. Moreover,
71
seasonal changes in abundance were observed, e.g. Russula spp., being more abundant in autumn than in spring. Enzyme activities differed among and within ECM morphotypes. At ECM community level the phosphatase and laccase activities were slightly higher in samples derived from dry plots, whereas the leucine aminopeptidase activity was slightly elevated in spring compared to autumn.
The fast positive growth response of drought stressed Scots pines to irrigation might have been facilitated by the structural and functional integrity of the ECM community at this study site.
Key words: biotrophic-saprotrophic continuum, ectomycorrhizal morphotypes, exploration types, global warming, inner alpine dry valley, Pfynwald
72
Introduction
In a changing climate due to global warming higher temperatures and more frequent precipitation deficits during the summer months are predicted for terrestrial ecosystems (IPCC 2007; IPCC 2013). Drought was already shown to affect temperate forests. For instance, high mortality rates for Scots pines were observed in inner alpine valleys in central Europe (Cech and Perny 1998; Dobbertin et al. 2005;
Bigler et al. 2006; Gonthier et al. 2010; Matías and Jump 2012). In a unique data set on tree mortality in an inner alpine Swiss valley (Canton Valais) for the years 1983 to
2003 the highest mortality of Scots pines was recorded at low elevation (below
1000 m a.s.l) and increased mortality on dryer sites with high stand competition
(Rigling et al. 2013). Moreover, pine regeneration, expressed as numbers of saplings per defined area, was low. In this region, the annual precipitation rate was quantitatively quite stable during the last 100 years, but increasing temperatures
(Begert et al. 2005; MeteoSwiss 2014) caused higher evapotranspiration and thus increased drought stress for the plants (Rebetez and Dobbertin 2004).
Drought stress might cause tree death through hydraulic failure within the xylem, i.e. by cavitation and embolism within the water-conducting tissues (Tyree and Sperry
1989; Brodribb and Cochard 2009). Carbon depletion was identified as another potential reason for tree death under drought stress since the stomata are kept closed during daytime in order to reduce transpiratory water loss, resulting in reduced photosynthetic productivity and growth (Borghetti et al. 1998; Bréda et al. 2006;
McDowell 2011; McDowell et al. 2013). Consequently, drought-stressed trees are more vulnerable for detrimental attacks by parasitic insects, fungi, nematodes and mistletoes (Matías and Jump 2012).
73
In 2003 a unique long-term irrigation experiment was established in the well investigated, Scots pine-dominated “Pfynforest” in the main valley of the Swiss canton Valais. Aboveground, an immense increase of needle length, shoot length and tree ring width was measured in Scots pines on irrigated plots (Dobbertin et al.
2010; Eilmann et al. 2010; 2013). An immediate growth response was recorded in drought released trees; this can be interpreted as a resilient reaction of the ecosystem. Interestingly, no significant changes were observed belowground in the fine root biomass (Brunner et al. 2009).
At their root system most trees of temperate to boreal climates, pine species included, associate with a wide range of taxonomically diverse ECM fungi whose external mycelium may expand into soil areas which are not accessible to the fine roots proper. This wide-ranging extension improves the nutrient and water uptake of the trees and facilitates the carbon cycling from the photoautotroph to the heterotrophic microbiome in the soil (Finlay 2008; Smith and Read 2008). In return,
ECM fungi receive photosynthates from their photoautotrophic host. Under drought stress ECM fungi improve the drought tolerance and recovery rates of their tree host
(Garbaye and Churin 1997; Ortega et al. 2004; Lehto and Zwiazek 2011). However, their role in water uptake is poorly understood, especially in large trees whose roots range deep below the soil horizon where no ECM fungi occur (Lehto and Zwiazek
2011).
ECM communities have been shown to be negatively affected by drought, resulting in reduced abundance of fungal biomass and fungal species richness (Shi et al. 2002;
Swaty et al. 2004; Buée et al. 2005; Richard et al. 2011), whereby the reasons for the marked differences in drought tolerance among ECM species are poorly understood.
A strategy of ECM fungi to withstand drought periods could be the shift towards saprophytism in periods of reduced photosynthate availability from the tree host, as
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most ECM taxa are not strictly biotrophic (Koide et al. 2008; Talbot et al. 2008).
Reduced translocation of mobile photosynthates towards the roots by girdling, as carried out in a Fagus sylvatica forest, did not reduce the rate of colonization of beech roots (Pena et al. 2010). But the ECM species richness significantly reduced from approx. 79-90 to 40 species and many rare biotrophic species with strong dependence on photosynthate availability disappeared (Pena et al. 2010).
ECM communities could also respond to drought by selecting ECM species which are well suited to deal with water shortage. ECM taxa of the long-distance exploration type (sensu Agerer 2001) with long, vessel-like external mycelium have been suggested to be more efficient in transporting water than other fungi with little external mycelium such as the contact exploration type (Garbaye 2000; Lehto et al.
2011). In a mature stand of maritime pine (Pinus pinaster) at the Atlantic coast
(Landes) the long and short distance exploration types of ECM taxa were more abundant in a dry site, but ECM fungi with smooth mantle and very little external mycelium predominated in an adjacent humid site (Bakker et al. 2006). However, since nutrients were more abundant in the humid than in the dry area it was difficult do distinguish between nutritional and drought effects in this particular study (Lehto et al. 2011). In addition, it needs to be borne in mind that long-distance exploration types require high amounts of fixed carbon for building up extensive long-distance exploration mycelium. Due to reduced supply by the trees this carbon may not be available in times of drought, which may favour species which produce only small amounts of external mycelium, such as the contact or short-distance exploration types. It is currently unclear which ECM exploration type, or which ECM species and morphotypes, respectively, prevail under drought stress.
ECM fungi, as physiologically facultatively symbiotic heterotrophs, are able to grow on complex carbohydrates and proteins as their only carbon or nitrogen sources.
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Thus ECM fungi produce and secrete a range of enzymes for degrading organic substrates of plant and fungal origin (Courty et al. 2010; Talbot et al. 2008; Talbot et al. 2010; Rineau et al. 2012; 2013). The activities of these enzymes, as released by single ECM root tips collected in the field, can be determined (Pritsch et al. 2004;
2011; Courty et al. 2005). Thus, the activities of enzymes involved in the degradation of cellulose, hemicellulose, lignin, chitin, proteins, and of phosphate containing substrates, as secreted by single ECM species or by whole communities, represent functional traits (Cullings et al. 2009; Jones et al. 2010; Rineau and Courty 2011).
The enzyme activity of ectomycorrhizal root tips depends on taxonomic affiliation, environmental factors such as season and soil, and on human impacts such as fertilization or thinning (Courty et al. 2005; Buée et al. 2007; Mosca et al. 2007;
Rineau and Garbaye 2009a; b; Jones et al. 2012).
Having seen the significant effects of drought and drought release on tree growth
(Dobbertin et al. 2010; Eilmann et al. 2010; 2013), the present study aims at investigating the belowground ECM community structure with and without irrigation at a later stage of this long-term field experiment. Is the decline of Scots pines linked to a decline of mycorrhizal diversity and functioning? These parameters are to be studied by 1) investigating the biodiversity and exploration types among ECM fungi,
2) quantifying the different ECM morphotypes, and 3) measuring the potential activities of enzymes involved in the degradation of organic matter. By studying the seasonality of these parameters, i.e. by sampling in fall and spring, insights into annual changes within the mycorrhizosphere are to be achieved.
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Material and methods
Study area
The experimental site Pfynwald, an Erico-Pinetum sylvestris, is located at an elevation of 615 m a.s.l. in the inner alpine Swiss Rhone valley (Valais), close to a side channel of the river Rhone whose water was used for the irrigation experiments.
The majority of Scots pines (Pinus sylvestris) at the study site are 90-100 years old.
The mean annual precipitation was 598 mm (1961-1990) or 603 mm (1981-2010), respectively, and the mean annual temperature was 9.2°C (1961-1990) or 10.1°C
(1981-2010), respectively, as measured at the weather station in Sion. Between 1961 and 1990 an average of 10.8 heat days per year with temperatures ≥30°C were recorded, but this value increased to 16.0 for the period between 1981 and 2010. In
August 2013 a temperature increase of 1.3°C and a reduction in precipitation of 54 % as compared to the mean values from 1981-2010 was measured (detailed information by MeteoSwiss 2014).
The irrigation experiment was started in 2003 and will last until 2020. Watery years are simulated by approximately doubling the usual precipitation rate by irrigation with sprinklers in the dry months from June to October.
Experimental design
The effects of irrigation were studied in an experimental area of 390 m × 25 m, which was subdivided into eight equally-sized replicate plots of 40 m × 25 m, with a buffer zone of 10 m between each plot. The eight replicates were aligned next to each other in a row. Four of the eight replicates were randomly chosen for irrigation, the other four served as control. For the present study, three irrigated plots and three control plots where randomly chosen for taking soil cores near the pine trees.
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Sampling and processing of soil cores
For studying the ECM community structure, eight soil cores with a volume of 60.8 cm3 were sampled with a corer from each of the three dry control and irrigated plots in October 2007, May 2008, October 2008 and May 2009. The soil cores were transferred to plastic bags and stored at 4°C. The soil core processing started the following day and was finished latest after three weeks. For ECM community analysis, the soil cores were broken, soaked in tap water, the ECM roots collected and washed under tap water.
Classification and identification of ECM root tips
Under a dissection microscope, every ECM root tip per soil core was counted. The morphological differences and different exploration types according to Agerer (1987-
2012; 2001) of each ECM root tip were visually distinguished by morphotyping, parameters being the growth pattern of the ECM root tips, the colouration and surface properties of the fungal mantle, and the structure plus length of the external mycelium. During the washing procedure part of the external mycelium may have been lost from mycorrhizal roots; thus some of the exploration types, as defined in this study, differ from those of Agerer (2001).
For the identification of ECM species, DNA from a minimum of one ECM root tip per
ECM morphotype in a sample was extracted with the DNeasy 96 Plant Kit (Qiagen,
Venlo NL). The internal transcribed spacer (ITS) region was amplified with the universal fungal primer ITS1F and ITS4 using the PCR program of Gardes and Bruns
(1993). The sequencing reaction was carried out with the ITS1F primer and the Big
Dye Kit (Life technologies, Carlsbad CA) for cycle sequencing on an ABI3700 automated sequencer (Life technologies, Carlsbad CA). 334 sequences were successfully identified. The sequences were compared with the database entries of
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UNITE and/or NCBI with a cut off value of 97 % for species identification and 95 %-
97 % for genus identification. A number was added to the name, e.g. Cortinarius sp.1, to differentiate two sequences that shared less then 97 % identity. A morphotype was named after an ECM species if more than half of the species names were identical within the morphotype. In cases where no single species dominated the morphotype, it was named after its most abundant ECM genus (Table S2).
Enzymatic assay on individual ECM root tips
Different enzyme activities of individual ECM root tips were determined on the same
ECM root tips as described by Pritsch et al. (2011). For the enzyme activity measurements the following fluorogenic substrates were applied: MU–β-d- glucopyranoside (MU–Gls) for β-glucosidase, MU–N-acetyl-β-d-glucosaminide (Mu–
Nag) for N-actetyl-β-d-glucosaminidase, MU–cellobioside (MU–Cel) for cellobiohydrolase, MU–β-d-glucoronide hydrate (MU–Glr) for glucuronidase, xylopyranoside (MU–Xyl) for xylosidase, 7-amino-4-methylcoumarin (AMC-Leu) for leucine aminopeptidase and 2-2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) for laccase activities. All substrates were purchased at Sigma-Aldrich Chemie GmbH
Buchs, Switzerland. The fluorescence yields, as released from the MU (4-methyl- umbelliferone) and AMC (7-amido-4-methylcoumarin) substrates, were analysed at
368 nm excitation and 465 nm emission with a Tecan Infinite M200 microplate reader
(Tecan GmbH, Germany). This microplate reader was also used for photometric measurements of ABTS oxidation products at 425 nm. The automated image analysis software WinRhizo Version 4.1c (Regent Instruments, Canada) was used to determine the projection surface area of each ECM root tip. The enzyme activities were expressed as MU and AMC release or ABTS oxidation in pmol per projection area (mm2) and time (min). Since the MU–β-d-glucuronide hydrate was probably
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degraded throughout our study, the data on glucuronidase activities were omitted from all analyses.
Soil water content
Soil water content from irrigated and the control plots was measured using the soil from 48 soil cores in each sampling campaign (four times). The soil of eight soil cores per replicate plot was pooled, sieved through a 2 mm mesh size sieve and weighed prior to and after drying at 110°C. The weight difference, expressed in percent of the dry matter, represented the water content of the soil (Table S1).
Calculations and statistical analysis
The impact of irrigation and season on 1) taxon diversity indices, 2) number of mycorrhizal root tips per soil core, and 3) enzyme activities of the ECM communities within the eight soil cores of a replicate plot per sampling (n=6) was tested using two- way analysis of variance (ANOVA).
We calculated the rarefied number of species using the analytical equation proposed by Sanders et al. (1968). In addition, we used a stochastic method: each ECM morphotype was assigned a probability of occurrence to by dividing the number of root tips associated with this ECM morphotype by the total number of root tips in the control plot. Then, m ECM morphotypes were randomly selected using these probabilities, where m was the number of observed root tips in the associated irrigated plot. From a total of 10,000 repeats, the average number of species was obtained.
To test the influence of irrigation and season on relative abundance of an ECM morphotype, on relative abundance of an exploration type and on enzyme activities of single ECM morphotypes (data Logn transformed), the values were averaged over
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both samplings of the same season (but separate years) within a replicate plot, because not all species were found in each sampling and the lumping resulted in more stable numbers for the ANOVA. Relative abundance of ECM morphotypes and exploration types were defined as the percentage of ECM root tips of a particular
ECM morphotype or exploration type over all counted mycorrhizal root tips in a replicate plot.
To assess the abundance and enzyme activity differences of treatment, season and
ECM morphotype, principal component analysis (PCA) in Canoco for Windows 4.5
(ter Braak and Šmilauer 2002) was performed. For abundance analysis, a data matrix of 11 columns (ECM morphotypes as species) and 12 rows (control and irrigated plots during spring and autumn as samples) was used. The mean activities of each of the 11 ECM morphotypes were calculated per treatment for both seasons (spring and autumn), resulting in a data matrix of seven columns (7 enzymes) and 44 rows (11
ECM morphotypes × 2 treatments × 2 seasons). To evaluate relationships among enzyme activity profiles, the data on enzyme activities were treated as variables
(species) and on ECM morphotypes as objects (samples). Samples were centred and standardized, species centred by species and data Logn transformed.
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Results
ECM community structure
In total 64,117 ECM root tips were counted and visually classified into 13 ECM morphotypes. Via ITS sequencing 116 ECM species were identified, 68 taxa occurred in the control and 81 in the irrigated plots (Table S2). Irrigation affected neither the number of ECM root tips, nor the number of ECM morphotypes, but the number of ECM morphotypes was significantly higher in spring than in autumn. The
Simpson and Shannon indices of diversity did not reveal significant differences
(Table 1). In spring only marginal differences in the soil water content were recorded between control and irrigated plots (the latter being irrigated in summer to autumn only), but highly significant differences were evident in autumn (Table S1).
The composition of the ECM community was moderately changed by irrigation, as revealed by PCA (Figure 1). The ECM communities in dry control plots tended to have higher scores on the first PCA axis than the ECM communities in irrigated plots.
The most prominent change in response to irrigation was seen in Rhizopogon roseolus, the most abundant ECM morphotype in irrigated plots: in spring it accounted for 16 % of ECM root tips in irrigated versus 2 % in dry control plots, and in autumn for 27 % versus 7 %, respectively (Table 1, Figure 2). Further dominant
ECM morphotypes in all plots were Russula spp., Amphinema byssoides and
Craterellus lutescens (Figure 2). Two-way ANOVAs on abundant ECM morphotypes revealed significant increases for Rhizopogon roseolus and Inocybe spp. due to irrigation, while Russula spp. was more abundant in autumn than in spring (Table 1).
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Table 1. Relative abundances of ECM morphotypes in control and irrigation plots for both seasons (n=0-3, mean ± SE in %), the number of ECM root tips, the rarefied number of ECM morphotypes and diversity indices (n=6, mean ± SE). The amount and structure of the external mycelium of the ECM morphotypes are specified as exploration types. Results of two-way ANOVAs are given as P values. No significant interactions were observed between the factors season and treatment.
Irrigation Irrigation Control Control P value
ECM morphotype Expl. type Spring Autumn Spring Autumn Treatment Season
Boletus edulis Long 3.3 (3.3) 5.7 (0.3) 3.6 (2.8) 6.3 (2.8) 0.886 0.400
Rhizopogon roseolus Long 16.2 (8.4) 27.1 (5.6) 1.9 (1.1) 7.1 (4.1) 0.014 0.179
Amphinema byssoides Med. fringe 14.3 (2.1) 8.9 (0.7) 15.2 (7.2) 14.4 (1.4) 0.436 0.440
Cortinarius spp. Med. fringe 10.2 (6.2) 4.4 (1.8) 11.7 (3.6) 13.4 (3.7) 0.237 0.634
Tricholoma albobrunneum Med. fringe 0.6 ( - ) - 3.3 (1.5) 3.5 (2.0) - -
Cenococcum geophilum Short 6.5 (2.2) 3.4 (1.6) 4.6 (1.1) 2.6 (0.5) 0.406 0.131
Sebacina spp. Short 8.2 (3.8) 5.2 (1.2) 8.4 (4.0) 3.8 (1.8) 0.853 0.236
Craterellus lutescens Contact 4.8 (3.1) 11.7 (7.2) 21.4 (14.5) 11.9 (5.9) 0.364 0.887
Inocybe spp. Contact 6.0 (1.3) 5.0 (1.2) 3.3 (1.8) 0.9 (0.7) 0.030 0.235
Lactarius deliciosus Contact 13.1 (8.1) 0.9 (0.7) 8.9 (6.7) 8.7 (2.5) 0.755 0.291
Peziza michelii Contact 1.0 (0.4) 0.4 (0.1) 0.1 ( - ) 0.2 ( - ) - -
Russula spp. Contact 8.6 (1.7) 18.5 (3.6) 11.1 (3.5) 24.1 (5.6) 0.333 0.019
Tomentella spp. Contact 9.0 (1.8) 10.9 (5.8) 6.7 (1.0) 4.5 (1.3) 0.068 0.936
Number of ECM root tips 3140.5 (353.5) 2481.0 (314.2) 2573.8 (317.5) 2490.8 (166.4) 0.359 0.225
Number of ECM morphotypes 12.0 (0.4) 11.0 (0.4) 12.3 (0.4) 11.3 (0.3) 0.382 0.014
Rarefied number of ECM morphotypes analytic 10.6 (0.4) 10.5 (0.3) 11.0 (0.6) 11.0 (0.4) 0.367 0.873
Rarefied number of ECM morphotypes stochastic 10.6 (0.4) 10.5 (0.2) 11.2 (0.3) 10.9 (0.4) 0.151 0.612
Simpson index of diversity 0.82 (0.0) 0.81 (0.0) 0.78 (0.0) 0.82 (0.0) 0.573 0.606
Shannon index of diversity 2.04 (0.1) 1.92 (0.1) 1.93 (0.1) 1.95 (0.1) 0.711 0.602
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7
8
5 6 7
8 3 5 3
6 4 4
Figure 1. Principle component analysis (axis one: 33.6 %, axis two 23.3 % of variation) indicating the effect of treatment and season on the ECM communities. Each data point corresponds to the ECM community in control plots (4, 5 and 8) and irrigated plots (3, 6 and 7) in spring and autumn based on the relative abundances of the ECM morphotypes. The closer the points are the more similar are the ECM communities in the respective plots. Circles black: irrigated plots autumn, circles grey: irrigated plots spring, squares black: control plots autumn and squares grey: control plots spring;
30
25 Irrigation
% Control 20
15
10 relative abundance relative
5
0
. is ii lus pp um ides s ul eo spp. o cens osus hil chel s a s s e ella spp. te b t arius spp. s lu n u rti Inocy m geop ogon ro Russul ebacina spp. u eziza mi omen o S Boletus ed P op T C a albobrunneum iz occ Lactarius delici oc om Rh Craterell Amphinema bys en C Trichol
Figure 2. Mean relative abundance of ECM morphotypes in irrigation and control plots (n=1-3, ± SE).
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Enzyme activities
The enzyme activities on the level of ECM communities were not markedly different in control and irrigation plots (Figure 3). Three enzymes showed small but significant differences: laccase and phosphatase activities being higher in the dry control than in the irrigated plots, and leucine aminopeptidase activity being higher in spring than in autumn.
The overall enzyme activity profiles were more dependent on the ECM morphotype than on either treatment or season (Figure 4, Figure 5). For example, Boletus edulis and Rhizopogon roseolus exhibiting the highest leucine amino peptidase activities, high sugar degrading activities and very low laccase activities compared to the other
ECM morphotypes (Figure S2). Within single ECM morphotypes, however, the enzyme activities varied significantly in response to treatment and season (Table 2).
5 Irrigation Spring * Irrigation Autumn 4.5 Control Spring
2 4 Control Autumn
3.5
3 * 2.5
2 * 1.5
enzyme activity pmol/min/mm activity enzyme 1
0.5
0 Pho Leu Nag Cel Gls Xyl Lac
Figure 3. Enzyme activities per mm2 root tip surface of ECM communities (n=6, ±SE) over all ECM morphotypes in irrigated and control plots of both seasons. The values are given Logn transformed for a better overview. Enzyme abbreviations: Pho: acid phosphomonoesterase; Leu: leucine aminopeptidase; Nag: N- acetylglucosaminidase; Cel: cellobiohydrolase; Gls: glucosidase; Xyl: xylosidase; Lac: laccase; A two-factor ANOVA revealed significant treatment (Pho and Lac) and season (Leu) effects. Significant differences are reported as asterisks:*:P < 0.05.
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Leu
Pho
Lac
Xyl
Cel
Gls Nag
-1.0 1.5
-1.5 2.0 Figure 4. Principle component analysis (axis one: 58.4 %, axis two 22.6 % of variation) showing that the seven enzyme activities of each ECM morphotype are mostly specific to the corresponding ECM morphotype and not to the treatment or season. Each data point indicates the mean enzyme activities of the ECM morphotypes in control (squares and diamonds) and irrigation (triangles) plots during spring (diamonds and right triangle) and autumn (squares and left triangle). black: Amphinema byssoides, lilac: Craterellus lutescens, red: Boletus edulis, grey: Inocybe spp., light green: Cenococcum geophilum, blue: Cortinarius spp., violet: Sebacina spp., dark green: Rhizopogon roseolus, brown: Tomentella spp., dark red: Lactarius deliciosus, dark blue: Russula spp., Enzyme abbreviations: Pho: acid phosphomonoesterase; Leu: leucine aminopeptidase; Nag: N- acetylglucosaminidase; Cel: cellobiohydrolase; Gls: glucosidase; Xyl: xylosidase; Lac: laccase;
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Table 2. Effects of irrigation and season on individual and overall enzyme activities of dominant ECM morphotypes (n=2-3). Two-way ANOVAs revealed significant differences and are reported as follows: ++ or - -: P < 0.01; + or -: P < 0.05. +: higher activity in irrigation (above) or spring (below); -: higher activity in control (above) or autumn (below). No significant interactions were observed between the factors season and treatment. The exploration types are specified by the amount and structure of the external mycelium of the ECM morphotypes. Enzyme abbreviations: Pho: acid phosphomonoesterase; Gls: glucosidase; Xyl: xylosidase; Nag: N- acetylglucosaminidase; Leu: leucine aminopeptidase; Lac: laccase; Cel: cellobiohydrolase;
Overall enzyme Factor treatment Expl. type Pho Leu Nag Cel Gls Xyl Lac activity Boletus edulis Long - - - - Rhizopogon roseolus Long - Amphinema byssoides Med. fringe - - Cortinarius spp. Med. fringe Cenococcum geophilum Short Sebacina spp. Short + Craterellus lutescens Contact - ++ Inocybe spp. Contact Lactarius deliciosus Contact Russula spp. Contact - Tomentella spp. Contact - Factor season Boletus edulis Long Rhizopogon roseolus Long + Amphinema byssoides Med. fringe Cortinarius spp. Med. fringe Cenococcum geophilum Short + + Sebacina spp. Short + Craterellus lutescens Contact Inocybe spp. Contact Lactarius deliciosus Contact Russula spp. Contact Tomentella spp. Contact
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Irrigation Spring Pho Pho Leu Irrigation Spring Pho Leu Irrigation Spring Leu Nag Nag Cel Nag Cel Gls Cel Gls Irrigation Autumn Xyl Gls Xyl Lac Irrigation Autumn Irrigation Autumn Xyl Lac Lac
Control Spring Control Spring Control Spring Russula spp. (Contact) spp. Russula Tomentella spp. (Contact) (Contact) spp. Tomentella Craterellus lutescensCraterellus (Contact) Control Autumn Control Autumn Control Autumn
0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 enzyme activity pmol/min/mm2 enzyme activity pmol/min/mm2 enzyme activity pmol/min/mm2
Irrigation Spring Pho Leu Irrigation Spring Pho Irrigation Spring Pho Nag Leu Leu Cel Nag Nag Gls Cel Cel Irrigation Autumn Xyl Gls Gls Lac Irrigation Autumn Xyl Irrigation Autumn Xyl Lac Lac Control Spring Control Spring Control Spring Sebacina spp. (Short) spp. Sebacina
Cenococcum geophilum (Short) Control Autumn Control Autumn Control Autumn Amphinema byssoides (Med. fringe) fringe) byssoides (Med. Amphinema 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 enzyme activity pmol/min/mm2 0 50 100 150 200 250 300 350 400 450 2 enzyme activity pmol/min/mm2 enzyme activity pmol/min/mm
Irrigation Spring Pho Irrigation Spring Pho Irrigation Spring Pho Leu Leu Leu Nag Nag Nag Cel Cel Cel Gls Irrigation Autumn Gls Gls Irrigation Autumn Xyl Xyl Irrigation Autumn Xyl Lac Lac Lac
Control Spring Control Spring Control Spring Boletus edulis (Long) Rhizopogon roseolus (Long) roseolus Rhizopogon
Cortinarius spp. (Med. fringe) spp. (Med. Cortinarius Control Autumn Control Autumn Control Autumn
0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 2 2 enzyme activity pmol/min/mm enzyme activity pmol/min/mm enzyme activity pmol/min/mm2
Figure 5. Overall enzyme activity profiles of selected ECM morphotypes (n=2-3). The exploration types are specified by their amount and structure of the external mycelium. Enzyme abbreviations: Pho: acid phosphomonoesterase; Leu: leucine aminopeptidase; Nag: N-acetylglucosaminidase; Cel: cellobiohydrolase; Gls: glucosidase; Xyl: xylosidase; Lac: laccase;
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Exploration types
The exploration strategies of the different ECM morphotypes (sensu Agerer 2001) did neither correlate with their abundance changes to treatment factors nor with their enzyme activity patterns. For instance, the two long-distance exploration types,
Boletus edulis and Rhizopogon roseolus, responded differently to the treatment factors in terms of abundance changes and of enzyme activities within the morphotype.
In addition to treatment-impact on individual ECM morphotypes, we investigated impact on exploration types, each of which may consist of several morphotypes. Only the long-distance exploration type increased significantly in abundance in the irrigated as compared to the dry control plots (% values for spring irrigation: 18.4 ±
10.6, control: 5.5 ± 2.6, autumn irrigation: 31.0 ± 4.9, control: 13.3 ± 3.5, p-value
0.04). No other exploration types revealed significant changes.
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Discussion
ECM community structure and exploration types
Irrigation influenced the belowground ECM community structure in the dry Scots pine forest Pfynwald. In both seasons (spring and autumn) Inocybe spp. and Rhizopogon roseolus increased significantly in the irrigated compared to the dry control plots. This is surprising since Rhizopogon roseolus has been shown to grow even under severe drought and thus was assumed to preferentially colonize dry ecosystems (Sanchez et al. 2001). Moreover, Rhizopogon roseolus exhibits the long-distance exploration type and its observed abundance change is in contradiction to the hypothesis that this exploration type is favoured by drought conditions, as it is more efficient in transporting water to the host than other ECM exploration types (Garbaye 2000;
Lehto et al. 2011). We thus suggest that Rhizopogon roseolus benefits from an enhanced flow of tree-derived photosynthates in irrigated plots while building up its extensive external mycelium. In addition, the two most dominant ECM morphotypes in the dry control plots belong to the contact exploration type with very little external mycelium. Both observations support the hypothesis that exploration types with little external mycelium perform better under drought-induced shortage of mobile photosynthate supply than exploration types with much external mycelium.
The significant increase of the belowground abundance of Inocybe spp. due to irrigation correlated with increased numbers of fruiting bodies aboveground, as recorded from 2003 until 2007 at the beginning of the irrigation experiment (Egli et al. unpublished data). Craterellus lutescens also responded to irrigation with a dramatic increase of fruiting body production in fall, although its belowground abundance was equal in control and irrigated plots; in spring it was even lower in the irrigated than in the control plots. Other studies have reported discrepancies between above- and
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belowground abundance among several ECM taxa on diverse hosts, irrespective of particular treatments (e.g., Gardes and Bruns 1996; Peter et al. 2001). A positive impact of rainfall or a negative effect of drought, respectively, on ECM fruiting body formation was documented in a long-term study on the fruiting phenology of ECM and saprotrophic fungi (Sato et al. 2012) and in Lactarius deliciosus in a pine forest in
Spain, but no correlations were found between Boletus edulis and weather parameters at this study site (De la Varga et al. 2013). In fall, when the tree has largely completed its annual biomass production (needles, twigs and tree rings having been formed), the flux of photosynthates from the tree to the fungus seems to be crucial for fruiting body formation by ECM taxa, as was shown with girdling experiments (Högberg et al. 2001).
None of the ECM morphotypes significantly decreased in abundance in response to irrigation. Still, Cortinarius spp. was slightly less abundant in autumn in irrigated than in dry control plots. Similar results were obtained in a rainfall exclusion experiment in a Mediterranean evergreen oak (Quercus ilex) ecosystem, where Cortinariaceae were more abundant under dry conditions (Richard et al. 2011). Furthermore, in the present study no differences in ECM species diversity were recorded in drought- stressed and irrigated plots. Comparable results were obtained in a water-exclusion experiment in a temperate beech forest (Shi et al. 2002) and in the rainfall exclusion experiment in the Mediterranean Oak ecosystem (Richard et al. 2011). In contrast a decrease of ECM species diversity was recorded in a pinyon pine forest (Pinus edulis) in northern Arizona with drought-induced high tree mortality (up to 70% mortality among mature pinyon pines) compared to a low mortality pine forest nearby
(Swaty et al. 2004). However, this ecosystem was more severely drought-stressed than our study area.
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Higher ECM species richness was recorded at our study site in spring than in autumn. In contrast, in an oak forest (Q. petraea, Q. robur) in the temperate climate of North Eastern France the highest belowground ECM richness was documented in autumn (Courty et al. 2008). These discrepancies might be due to the marked climatic differences between both ecosystems.
Seasonal impacts on the abundance of Russula spp. were observed, the latter being more abundant in autumn than in spring at our study site, but more prominent in spring than in autumn in a Mediterranean evergreen oak (Quercus ilex) forest
(Richard et al. 2011). Hardly any seasonal variation in abundance of a Russula species was recorded in a red pine (Pinus resinosa) plantation in Pennsylvania
(USA), while other taxa revealed marked seasonal variation at this study site (Koide et al. 2007a). In general, ECM fungi with temporary variation in abundance were assumed to reduce the interspecific competition between ECM taxa (Koide et al.
2007b). Another reason for these discrepancies might be the fact that we lumped different ECM species within a particular ECM morphotype. Only from a comparatively small number of ECM root tips the ECM species were successfully identified by sequencing, and many of them were recovered only a few times; therefore we relied on the classification after their morphological appearance into
ECM morphotypes. Some morphotypes were very distinct and comprised only one species, while others included diverse ECM species, usually belonging to the same genus. Due to this limited resolution some ECM species with seasonally variable abundance might not have been recognized in our study.
Impacts of irrigation and season on enzyme activities of ECM communities
The fungal enzyme activities of the well watered and dry ECM communities differed, but only to a minor extent. Small but significant increases in the phosphatase and
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laccase activities in the dry control plots were evident. The adaptation to drought includes the production of compatible solutes such as sugar alcohols (Shi et al.
2002), and the biosynthesis of the relevant enzyme systems demands enhanced phosphate and nitrogen inputs for the fungal partner and also for the tree. Laccases contribute to lignin degradation, since they reduce oxygen to water by using hydrogen from phenols, the latter being abundant components of lignin (Cullen and
Kersten 2004). Fine roots might increase their lignin production in response to drought stress, as has been observed in herbs colonized by arbuscular mycorrhizal fungi (Yang et al. 2006; Yoshimura et al. 2008). The increase of laccase activity, as observed in the dry ECM community, could therefore indicate an enhanced degradation of a high lignin level in this soil. However, neither the lignin content of fine roots nor the total lignin content of the soil of irrigated and control plots have been determined in the present study.
It is important to note that the enzyme activities, as determined in this study, are potential enzyme activities measured in vitro under controlled laboratory conditions.
The differences in enzyme activities between drought-stressed and irrigated ECM fungi under field conditions during the dry season are presumably higher with lower activities in dry control plots than measured in the laboratory. With declining water availability the mobility of solutes and the metabolic activities of soil organisms are reduced (Manzoni et al. 2012), and consequently the fungal enzyme activities decrease. The increase in the potential phosphatase and laccase activities in the dry controls should therefore allow decomposing more efficiently the substrates once water becomes available.
Leucine aminopeptidase activity was significantly higher in spring than in autumn.
The nitrogen level in the needles of the pine trees at this site is close to deficiency
(Dobbertin et al. 2010). Therefore, an increased supply of amino acids for satisfying
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the demand of nitrogen at the start of the growth phase of the trees appears desirable. But also many ECM taxa proper may have their main period of vegetative mycelial growth and biomass production in spring, as shown by real-time PCR of the external mycelium of Boletus edulis and Lactarius deliciosus in a Scotch pine forest in Spain (De la Varga et al. 2013). Seasonal changes of enzyme activities were also found in other ECM ecosystems (Courty et al. 2006; Buée et al. 2005; Mosca et al.
2007). Higher activities of all enzymes of the dominant ECM taxa of a deciduous oak forest were recorded in winter as compared to spring (Mosca et al. 2007). These results were partly explained as an increased saprotrophytic acquisition of organic carbon from the soil than from the photosynthetically inactive oak trees during winter.
Enzyme activities within and among ECM morphotypes
Some ECM taxa show a high ability of adaptation of their enzyme activities to changing climatic and environmental conditions (Jones et al. 2010; 2012; Buée et al.
2007; Courty et al. 2005). At our study site drought tolerant ECM species with no abundance changes upon irrigation were speculated to have a high ability of adaptation; this turned out to be correct for a few enzymes of some drought tolerant taxa. However, also Rhizopogon roseolus, whose abundance increased upon irrigation, revealed a high ability of adaptation in one enzyme.
Cenoccocum geophilum showed a significant increase of leucine aminopeptidase and glucosidase activities in spring compared to autumn. These enzymes mobilize amino acids and sugars from organic debris in the soil and might facilitate a higher supply of these molecules to the fungi themselves and to the tree in spring. In mean viability scores of ectomycorrhizae formed on red pine (Pinus resinosa), as kept in mycorrhizotrons, spring was the season of lowest and autumn of highest viability in
C. geophilum, but the opposite situation was found in other ECM taxa (Fernandez et
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al. 2013). Thus spring is most likely the time when decaying C. geophilum together with dead mycorrhizal root tips of other taxa and with dead external mycelia and plant litter is degraded and new mycorrhizal root tips are being formed. Cenococcum geophilum was assumed to be fairly drought-resistant, because of its high abundance in the mycorrhizospere under dry conditions, its remarkable viability after desiccation, and its thick cell walls (Pigott et al. 1982; Jany et al. 2003; Buée et al.
2005; di Pietro et al. 2007). In particular, the strong melanisation of the latter has been experimentally recognized as an important functional trait in desiccation tolerance (Fernandez and Koide 2013; Fernandez et al. 2013). At our study site, however, this fungus was neither particularly abundant in the dry control plots nor were its abundance or enzyme activities changed after irrigation. Similarly C. geophilum revealed either no reaction to water exclusion in a Q. ilex stand (Richard et al. 2011) or even lower abundance and enzyme activities on experimentally drought stressed oak seedlings (Herzog et al. 2013). However, C. geophilum represents a cryptic species complex (Douhan and Rizzo 2005), a possible explanation for its inconsistent abundance and function under different water regimes.
As none of the ECM morphotypes at our study site were distinctly more abundant due to drought than others we could not test whether this would be linked to higher enzyme activities compared to other ECM morphotypes. Nevertheless, the enzyme activity profiles of individual ECM morphotypes were mostly specific, which is in line with other studies (Jones et al. 2010; 2012; Rineau and Garbaye 2009a; Courty et al.
2010; Diedhiou et al. 2010). However, Boletus edulis and Rhizopogon roseolus revealed the highest leucine amino peptidase activities and high activities of the carbohydrate and phosphate degrading enzymes. In contrast, their laccase acitivitiy was among the lowest compared to the other ECM morphotypes. This refers to a
95
high specialization on protein, carbohydrate and phosphate degradation and to an inferior ability for lignin degradation. These functional similarities might be based on their phylogenetic relationship, both species belonging to the Boletales. Our results contrast to those of Rineau et al. (2011), who found no relationship of functional traits among related ECM fungi and support the findings of Tedersoo et al. (2012) who showed that phylogenetic lineage played an important role in determining the potential activities of some enzymes such as acid phosphatase, leucine aminopeptidase and cellobiohydrolase in an afrotropical rainforest.
Combining results of drought stressed and irrigated Scots pine trees and their ECM communities of this study site
Our study revealed that the ECM community structure and function was not significantly altered by irrigation. The still intact ECM community at the drought stressed plots in this inner alpine Erico-Pinetum sylvestris might be one among other reasons why the Scots pines recovered well in terms of growth upon improved water supply (Dobbertin et al. 2010; Eilmann et al. 2010). Periodic drought stress events, as experienced at the Pfynwald study site, seem to be tolerable for most members of this ECM community as long as tree recovery is possible in periods of sufficient rainfall and lower evapotranspiratory losses. However, episodic, long-lasting severe drought stress situations with irreversible xylem cavitation and thus significantly increased tree mortality, as occurred in pinyon pine forests in Arizona (Swaty et al.
2004), changed the ECM community structure irreversibly.
Carbon depletion due to drought-induced reduction of photosynthetic activity, as observed in Scots pines at the Pfynwald study site (Eilmann et al. 2010; 2013), might lead to the mobilization of stored carbon pools in the roots, such as starch, by the tree. Such a mobilization benefits both the tree and their ECM partners, as has been
96
observed in girdling experiments (Druebert et al. 2009). Fine roots were shown to have higher priority for carbon allocation within a tree than the foliage (Waring et al.
1987), and this might explain the small differences in fine root but marked differences in needle biomass of drought stressed and drought released Scots pines, as observed at the Pfynwald site (Brunner et al. 2009; Dobbertin et al. 2010). The fact that the activity of only one enzyme involved in carbon degradation increased in the
ECM community of the dry plots also implies a sufficient carbon supply by the trees.
Very little is known about life spans and turnover rates of non-mycorrhizal fine roots and ECM root tips. Mycorrhization was shown to slow the degradation of the root down (Langley et al. 2006), and mycorrhizal root tips of some ECM species grew older or degraded more rapidly, respectively, than others (Ekblad et al. 2013;
Fernandez et al. 2013). Therefore, part of the intraspecific or intramorphotypic differences in enzyme activities, as found in the present study (Figure 5), might be due to differences in viability among the mycorrhizal root tips, no viability tests having been made.
It will be an interesting task to re-examine the ECM community structure and its functional traits at the end of the irrigation experiment in 2020 by using the same techniques as in the present study, but also refined ones, tools being available for studying the metabolomics of ECM taxa in association with their hosts (Larsen et al.
2011). Ideally, fluxomic studies (Sachar-Hill 2007) carried out on model systems will improve our knowledge of qualitative and quantitative aspects of fluxes in the tree- ectomycorrhiza-soil microbe network.
Acknowledgements Sincere thanks are due to Rosmarie Eppenberger for support with field work, molecular analysis and enzyme activity measurements. Moreover, for critical reading, discussing and calculation of diversity indices and rarefied ECM morphotypes we are very thankful to Jens M. Turowski.This study was part of FUNDIV grant ANR606-BDIV-06 from the French ANR (Agence Nationale de la Recherche).
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Supplemental material
Pho Irrigation Spring Irrigation Spring Pho Leu Leu Nag Nag Cel Cel Gls Gls Irrigation Autumn Xyl Irrigation Autumn Xyl Lac Lac
Control Spring Control Spring 794 Inocybe spp. (Contact) Inocybe spp. Lactarius deliciosus (Contact) deliciosus Lactarius Control Autumn Control Autumn
0 100 200 300 400 500 0 100 200 300 400 500 2 2 enzyme activity pmol/min/mm enzyme activity pmol/min/mm
Irrigation Spring Pho Irrigation Spring Pho Leu Leu Nag Nag Cel Cel Gls Gls Irrigation Autumn Xyl Irrigation Autumn Xyl Lac Lac
Control Spring Control Spring Peziza micheliiPeziza (Contact)
Control Autumn Control Autumn Tricholoma albobrunneum (Med. fringe) (Med. albobrunneum Tricholoma
0 100 200 300 400 500 0 100 200 300 400 500 enzyme activity pmol/min/mm2 enzyme activity pmol/min/mm2
Figure S 1. Overall enzyme activity profiles of selected ECM morphotypes (n=0-3). The amount and structure of the external mycelium of the ECM morphotypes are specified as exploration types. Enzyme abbreviations: Pho: acid phosphomonoesterase; Leu: leucine aminopeptidase; Nag: N-acetylglucosaminidase; Cel: cellobiohydrolase; Gls: glucosidase; Xyl: xylosidase; Lac: laccase;
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Phosphatase (Pho)
2 180 160 140 120 100 80 60 40 20 0 enzyme activity pmol/min/mm Peziza michelii (Long) (Contact) fringe) fringe) Lactarius (Contact) fringe) (Short) deliciosus roseolus fringe) (Long) (Medium lutescens (Contact) (Short) Craterellus (Contact) byssoides (Contact) (Medium geophilum Rhizopogon Amphinema Cortinarius Tricholoma Tomentella Inocybe spp. Cenococcum Russula spp. Russula spp. (Medium albobrunneum Boletus edulis spp. (Contact) Sebacina spp.
Leucine aminopeptidase (Leu)
45
40 2 m 35
30
25
20
15
10 enzyme activity pmol/min/m 5
0 Lactarius (Contact) deliciosus (Short) lutescens (Long) (Contact) (Short) Craterellus (Contact) (Contact) geophilum (Contact) Inocybe spp. (Contact) (Contact) Cenococcum Russula spp. byssoides Tricholoma Rhizopogon Boletus edulis Boletus Amphinema Sebacina spp. Sebacina Peziza michelii albobrunneum roseolus (Long) (Medium fringe) (Medium fringe) (Medium Cortinarius spp. Cortinarius Tomentella spp. (Medium fringe)(Medium
N-acetyl glucosaminidase (Nag) 2 160 140 120 100 80 60 40 20 0 enzyme activitypmol/min/mm (Long) fringe) Lactarius (Contact) (Short) deliciosus fringe) lutescens (Long) roseolus (Contact) (Contact) (Medium fringe) (Short) Craterellus (Contact) byssoides (Contact) (Medium geophilum Amphinema Rhizopogon (Contact) Cortinarius Inocybe spp. Inocybe Tricholoma Tomentella Cenococcum Russula spp. spp. (Medium Boletus edulis Boletus Sebacina spp. albobrunneum spp. (Contact) (Contact) spp. Peziza michelii
Legend is written on page 109;
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Cellobiohydrolase (Cel)
25 2
20
15
10
5 enzyme activity pmol/min/mm 0 Peziza michelii (Long) (Contact) fringe) fringe) Lactarius (Contact) fringe) (Short) roseolus fringe) deliciosus lutescens (Contact) (Long) (Medium (Short) Craterellus (Contact) (Contact) byssoides (Medium geophilum Rhizopogon Amphinema Cortinarius Tricholoma Tomentella Inocybe spp. Cenococcum Russula spp. spp. (Medium albobrunneum Boletus edulis spp. (Contact) (Contact) spp. Sebacina spp. Sebacina
Glucosidase (Gls)
90 2 80
70
60
50
40
30
20
10 enzyme activity pmol/min/mm 0 (Long) fringe) Lactarius (Contact) deliciosus (Short) fringe) lutescens (Medium roseolus (Long) (Contact) fringe) (Short) byssoides (Contact) Craterellus (Contact) (Medium geophilum (Contact) Amphinema Rhizopogon Cortinarius Inocybe spp. Tricholoma Russula spp. Tomentella Cenococcum spp. (Medium Boletus edulisBoletus Sebacina spp. Sebacina micheliiPeziza albobrunneum spp. (Contact)
Xylosidase (Xyl)
7 2
6
5
4
3
2
1
enzyme activity pmol/min/mm activity enzyme 0 (Long) Lactarius fringe) (Contact) deliciosus (Short) fringe) (Medium lutescens roseolus (Contact) (Contact) (Long) fringe) (Short) byssoides Craterellus (Contact) (Contact) (Medium geophilum Amphinema (Contact) Rhizopogon Cortinarius Tricholoma Inocybe spp. Tomentella Cenococcum Russula spp. spp. (Medium Boletus edulis Sebacina spp. albobrunneum Peziza michelii Peziza spp. (Contact) Legend is written on page 109;
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Laccase (Lac)
140
120 2
100
80
60
40 enzyme activityenzyme pmol/min/mm 20
0 Lactarius (Contact) deliciosus (Short) lutescens (Long) (Contact) (Contact) (Short) Craterellus (Contact) (Contact) geophilum (Contact) Inocybe spp. Inocybe (Contact) (Contact) Cenococcum byssoides Russula spp. Russula Tricholoma Rhizopogon Boletus edulis Boletus Amphinema Sebacina spp. Peziza michelii Peziza albobrunneum roseolus (Long) (Medium fringe) (Medium fringe) (Medium Cortinarius spp. Cortinarius Tomentella spp. (Medium fringe) fringe) (Medium Figure S2. Enzyme activities of ECM morphotypes, averaged over irrigation and control plots (n=6-12, ± SE). Grey bars indicate ECM morphotypes with much external mycelium, whereas colourless bars indicate ECM morphotypes with little external mycelium. The amount and structure of the external mycelium of the ECM morphotypes is specified as exploration types in the brackets.
Table S1: Soil water content in % of control and irrigated plots in autumn and spring. One-way ANOVA revealed significant differences between irrigated and control plots in autumn.
Season Treatment Water content % P value Autumn Control (4) 13.1 0.002 Autumn Control (5) 11.1 Autumn Control (8) 10.8 Autumn Irrigated (3) 21.0 Autumn Irrigated (6) 21.2 Autumn Irrigated (7) 25.1 Season Treatment Water content % P value Spring Control (4) 23.0 0.275 Spring Control (5) 19.7 Spring Control (8) 19.2 Spring Irrigated (3) 21.5 Spring Irrigated (6) 21.7 Spring Irrigated (7) 28.4
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Table S2: ECM morphotypes, exploration types, morphological characteristics, species and the number of species resulting from ITS sequencing and subsequent comparisons with database entries in control (C) and irrigated (I) plots.
ECM morphotypes Expl. types Morphological characterization Species C I Amphinema byssoides Med. fringe Yellowish-white, woolly Amphinema byssoides 519 with abundant rhizomorphs; Amphinema sp. 1 64 Amphinema sp. 2 1 Atheliaceae sp. 1 1 Helotiales sp. 1 1 Inocybe sp. 2 2 Sebacina sp. 3 11 Sebacina sp. 4 1 Sebacina sp. 6 1 Sistotrema sp. 1 1 Boletus edulis Long White-silvery, with few thick Boletus edulis 81 rhizomorphs; Rhizopogon roseolus 1 Cenococcum geophilum Short Black with several thick Cenococcum geophilum 24 emanating hyphae; Cenococcum sp. 2 1 Tomentella lapida 11 Cortinarius spp. Med. fringe White, woolly with abundant Amphinema sp. 2 1 rhizomorphs; Cortinarius badiolaevis 11 Cortinarius caesiocanescens 53 Cortinarius corrosus 1 Cortinarius decipiens 11 Cortinarius dionysae 1 Cortinarius glaucopus 3 Cortinarius ochrophyllus 2 Cortinarius sp. 1 2 Cortinarius sp. 2 1 Cortinarius sp. 3 1 Cortinarius venetus 22 Piloderma fallax 2 1 Piloderma olivaceum 10 Piloderma sp. 1 1 Rhizopogon roseolus 1 Russula sanguinea 1 Tomentellopsis sp. 1 1 Tricholoma albobrunneum 2 Craterellus lutescens Contact Salmon with brighter tips, Cantharellus lutescens 83 slight cottony; Rhizopogon roseolus 2 Inocybe spp. Contact Greyish, smooth, gelatinous; Amphinema sp. 1 1 Cenococcum sp. 1 1 Clavulina rugosa 3 Inocybe leiocephala 1 Inocybe nitidiuscula 22 Inocybe pseudodestricta 1 Inocybe sp. 1 1 Inocybe sp. 2 211 Inocybe sp. 3 1 Pseudotomentella rhizopunctata 1 Rhizopogon roseolus 2 Russula cessans 1 Russula sanguinea 12 Lactarius deliciosus Contact orange-brown with brighter Amphinema byssoides 1 tips, older parts greenish, Lactarius deliciosus 52 white and orange laticifers Pyronemataceae sp. 1 1 visible; Tomentella sp. 5 3 Trichophaea woolhopeia 1 Peziza michelii Contact Brown-greenish, warty; Peziza michelii 14 Rhizopogon roseolus Long White - salmon, cottony with Amphinema sp. 1 1 few thick rhizomorphs; Hebeloma edurum 1 Rhizopogon roseolus 10 9 Russula badia 1 Suillus luteus 1 continued
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Table S2 continued ECM morphotypes Expl. types Morphological characterization Species name results C I Russula spp. Contact Brown, smooth; Inocybe nitidiuscula 3 Inocybe sp. 2 3 Pseudotomentella sp. 2 1 Russula acrifolia 32 Russula cessans 52 Russula postiana 3 Russula roseipes 21 Russula sanguinea 74 Russula sp. 1 1 Sebacina dimitica 1 Sebacina epigaea 1 Sebacina sp. 5 11 Tomentella sp. 5 11 Tomentella sp. 7 8 Tomentella stuposa 1 Tomentella terrestris 1 Tomentellopsis sp. 1 3 Tricholoma terreum 1 Sebacina spp. Short Brown, smooth with cottony Elaphomyces muricatus 1 emanating hyphae; Inocybe nitidiuscula 1 Peziza sp. 1 1 Phialocephala sp. 1 1 Russula sanguinea 1 Sebacina dimitica 6 Sebacina epigaea 2 Sebacina sp. 1 13 Sebacina sp. 2 1 Sebacina sp. 3 1 Sebacina sp. 4 4 Tomentella lapida 1 Tomentella sp. 1 1 Tomentella sp. 11 1 Tomentella spp. Contact Dark brown, Geopora cervina 2 smooth – granular; Humaria hemisphaerica 1 Meliniomyces bicolor 1 Peziza succosa 1 Pseudotomentella rhizopunctata 1 Pseudotomentella sp. 1 1 1 Russula badia 1 Sphaerosporella sp. 1 1 Suillus granulatus 4 Tomentella fuscocinerea 1 Tomentella lapida 41 Tomentella lilacinogrisea 58 Tomentella punicea 1 Tomentella sp. 2 5 Tomentella sp. 3 11 Tomentella sp. 4 12 Tomentella sp. 6 1 Tomentella sp. 8 21 Tomentella sp. 9 2 Tomentella sp. 10 1 Tomentella sp. 11 1 Tomentella stuposa 2 Tomentella umbrinospora 15 Tricholoma albobrunneum Med. fringe White, fringy with abundant Tricholoma albobrunneum 91 rhizomorphs;
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General Discussion
ECM community structure
We found reactions of the ectomycorrhizal (ECM) community structure to long-term nitrogen addition in a spruce forest, to irrigation in a dry pine forest and to different seasons such as spring and autumn in both types of environment. A change of the dominant ECM morphotypes, a diversity decrease and a strong decline in the number of ECM root tips was detected as a response to nitrogen addition. In contrast, in the irrigation experiment, the ECM communities did not show such a drastic response. Only two ECM morphotypes exhibited abundance increases to irrigation, whereas no ECM morphotypes where favoured by drought. Moreover, no change in diversity and root tip number was observed to irrigation, but species richness increased in spring compared to autumn. The results from the nitrogen study are congruent with results reported in the literature (Frey et al. 2004, Cox et al.
2010, Kjoller et al. 2012), whereas drought exhibited a more drastic response of ECM communities in other studies. For instance, an increase in abundance of ECM species was observed due to drought (Shi et al. 2002, Richard et al. 2011).
Moreover, in a pinyon pine forest in northern Arizona with drought-induced high tree mortality compared to a low mortality pine forest nearby, a decrease of ECM species diversity was observed (Swaty et al. 2004). However, this ecosystem was more drought-stressed than our study area.
Individual ECM species may have exhibited a reaction, but it was not possible to observe it due to the low resolution in species classification achieved by the methodology used. In both studies, all ECM root tips were classified into different
ECM morphotypes via morphological features. From each morphotype per soil core sample, DNA from a single individual was extracted for molecular identification. Only
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about one third of these samples resulted in species names due to methodical problems. Therefore, we named the ECM morphotypes after the most abundant species or genus within the ECM morphotype.
Enzyme activities of ECM communities
The functional abilities on the level of the ECM communities of both studies were relatively stable, although a change in the community composition was found, which shows a redundant functional behaviour among ECM fungi. This can be seen as a resilient reaction of these ecosystems due to the investigated factors. However, small but significant increases were observed for some sugar degrading enzymes to nitrogen-addition and to drought stress, which indicates increased carbon degradation from the soil. The hypothesis that the trees reduce their carbon allocation to their associated symbionts when nitrogen is added to the soil (chapter 1) and drought stress occurs (chapter 2) can therefore be supported. Moreover, in both studies the leucine aminopeptidase activity (releases amino acids) increased in spring time compared to autumn. A possible explanation is that trees and fungi need an extra amount of protein in their starting growth phase during spring. Courty et al.
(2007) also reported an increase of leucine aminopeptidase in spring at the beginning of the bud burst of oak in North-eastern France and so supports our findings.
When including the number of ECM root tips in the calculation of the enzyme activities on the level of the ECM communities, a partly different picture emerges. In the irrigation study, no change of ECM root tip number and consequently no change in the total enzyme activities were observed. In contrast, the number of root tips in the nitrogen-addition plots was reduced by more then 50 %, and correspondingly the enzyme activities were drastically reduced. Nitrogen addition therefore might
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influence the nutrient cycles of this forest ecosystem negatively. First hints to this were already found in the reduced phosphate levels and other elements in the needles of the nitrogen-addition trees. The resulting nutrient deficiency might increase the vulnerability of these trees to further stresses (Braun et al. 2010).
The importance of these observed changes in enzyme activities is currently a matter of debate. It has been shown that the enzyme activities of mycelia and microbes in the soil dominate, rather than enzyme activities at the root surface (Talbot et al.
2013). Yet, Kjoller et al. (2012) showed that both the amount of fine roots and the external mycelium were reduced in response to nitrogen addition. Moreover, it is well known that decomposition rates reduce as a response to nitrogen addition in spruce forests (Janssens et al. 2010), which is in line with the reduced total enzyme activities in chapter 1.
Enzyme activities of ECM morphotypes
All ECM morphotypes were able to produce the studied enzymes and the enzyme activities differed between ECM morphotypes. Other studies using this method reported similar results (Courty et al. 2005, Courty et al. 2006, Buée et al. 2007,
Mosca et al. 2007, Rineau et al. 2009, Jones et al. 2010, Jones et al. 2012).
Comparing the ECM morphotypes which occur in both study sites such as
Amphinema byssoides, Russula or Cortinarius, a similar enzyme profile but higher values in the irrigation study was observed for Amphinema byssoides and Russula.
In the case of Cortinarius, the enzyme activities were much higher in the nitrogen- addition plots compared to the control plots but the profiles were similar. In contrast, the enzyme profiles and values were quite constant for Cortinarius in the irrigation study and the enzyme profiles looked similar in both studies. This suggests a high
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ability for the ECM morphotypes to adapt in enzyme values but a low ability to change the enzyme activity ratios.
We found very low activities of the laccase and leucine aminopeptidase enzymes for some ECM morphotypes. This suggests a restriction for ECM fungi in specific functions such as lignin degradation in the case of laccase enzyme and protein degradation in the case of leucine aminopeptidase. In contrast, essential functions such as phosphatase showed high activities throughout all ECM morphotypes.
Similar interpretations on the aforementioned enzymes were put forward by Pritsch and Garbaye (2011).
ECM morphotypes whose abundance does not change due to irrigation, fertilization or season could have a higher ability to adapt in enzyme activities to changing conditions compared to ECM fungi which decreased or increased in abundance. Our results show that ECM morphotypes not altering in abundance exhibited similar changes in enzyme activities as ECM morphotypes that differed in abundance.
Therefore, the abundance of ECM fungi is independent of the plasticity of enzyme activities.
We expected that ECM morphotypes with a high ability to mobilize carbon from soil would be favoured due to nitrogen addition, drought and spring. This would be reflected as an abundance increase in the nitrogen-addition plots, in the dry controls of the irrigation experiment and in spring time. Although several ECM morphotypes exhibited high activities of enzymes involved in carbon mobilisation, these ECM morphotypes were not particularly more abundant in the nitrogen-addition plots, in the dry controls or in spring, in which we infer a reduced carbon flow. Other functional traits not measured in our studies might therefore be more important in influencing the abundances of ECM morphotypes. These other traits might include the efficieny of nutrient and or water delivery to the tree (Koide et al. 2007).
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Exploration types
The amount and structure of external mycelium of ECM species classified into exploration types (Agerer 2001) was expected to correlate with abundance changes due to the investigated factors of ECM morphotypes and their enzyme activities. It has been suggested that ECM morphotypes with little external mycelium, like contact and short-distance exploration types, would be favoured when carbon allocation from trees is limited (Lilleskov et al. 2011). Many ECM morphotypes of both studies exhibited little external mycelium and did not show a preference for conditions in either the control or the treatment plots or for a season.
Furthermore, ECM fungi with much external mycelium such as long-distance exploration types may be specialized in carbon mobilisation from the soil, since they exploit regions far from the root tips of their host trees and its carbon supply (Pritsch and Garbaye 2011). This notion was supported by a study in an afro tropical rainforest (Tedersoo et al. 2012). There, exploration types with much mycelium have higher activities compared to exploration types with little mycelium, except for laccase. Again, we did not find support for the hypothesis in our studies. No distinct enzyme activity patterns were observed for ECM morphotypes with much or little external mycelium.
It has previously been proposed that several ECM fungi belonging to the medium- distance fringe exploration type are sensitive to nitrogen addition (Lilleskov et al.
2011). In our study, only one nitrogen-sensitive ECM morphotype was classified as medium-distance fringe exploration type. Garbaye (2000) and Lehto et al. (2011) suggested that long-distance exploration types feature better water uptake compared to other fungi and therefore such types would be favoured in dry environments. Our results do not support this point of view. Based on our data, general conclusions concerning exploration types and their relation to function and structure of ECM
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communities are difficult to make. However, a relation of δ15N fungal patterns with different soil depths was found by Hobbie et al. (2014). These authors observed that on average ECM fungi with much external mycelium assimilated their nitrogen from deeper soil horizons, whereas fungi with little external mycelium mobilised their nitrogen from shallower soil. Nevertheless, some fungi with little external mycelium were found which also took their nitrogen from deeper soil.
Conclusion
Even though a shift in the ECM composition was observed to a high level in the nitrogen-addition experiment and to a moderate level in the irrigation experiment and due to season, both studies indicate that the functional traits of the ECM communities remained relatively stable. This indicates redundant functional abilities among ECM fungi and therefore these results can be interpreted as a resilient reaction of these ecosystems. However, due to the reduction of the ECM root tips in the nitrogen treated plots, also the functional abilities of this ECM community were reduced, which may influence the nutrient cycle of this ecosystem negatively. In contrast, in the irrigation experiment the ECM community of the dry controls was still intact concerning structural and functional aspects which might be one reason for the previously observed fast recovery of the drought stressed trees to irrigation
(Dobbertin et al. 2010, Eilmann et al. 2010, 2013).
Several ECM morphotypes exhibited a high degree of adaptation in their functional traits, independently of the ECM morphotype abundance reaction due to the different investigated factors, which is not consistent with our hypotheses. ECM morphotypes with abundance changes are therefore also able to adapt to altering environmental conditions with their varying functional abilities not only ECM morphotypes with
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stable abundances. Thus, the plasticity of enzyme activities does not depend on the abundance of ECM fungi.
No general conclusions can be put forward regarding the possible decline in carbon acquisition from the tree to the fungus due to the studied factors. Changed abundances were not related in any systematic way to patterns in enzyme activities, as we expected from the assumption of reduced carbon supply as in our hypotheses.
Experiments with stable isotopes such as 13C and 15N could provide additional information on the functional variation among ECM fungi (Koide et al. 2007). It also might be that other factors such as the efficiency of fungi to deliver nutrients to their hosts might be more important in shaping the ECM communities. For instance, the amount of nitrogen which the fungus delivers to its host tree could be measured by using naturally occurring 15N in the fungi and their hosts (Hobbie et al. 2005, Koide et al. 2007).
Another exciting task would be the re-examination of the structure and function of the
ECM communities at the former nitrogen-addition experiment and after the end of the irrigation experiment with the same approach as used in this thesis. But in general, further studies investigating additional functional traits are needed to elucidate a deeper understanding of the structure - function relation of ECM communities.
Measuring other enzymes involved in the degradation of soil organic matter through the method used in this dissertation might be an approach. Moreover, this method could be combined with measurements of resistance to desiccation on excised ECM root tips by measuring the loss of volume and electrolytes as in the study of Di Pietro et al. (2007).
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Danksagung
Dr. Martina Peter und Dr. Simon Egli möchte ich meinen grossen Dank aussprechen.
Sie haben mir ermöglicht, diese Doktorarbeit im Rahmen des FUNDIV Projektes zu schreiben. Martina Peter hat mich betreut, während Simon Egli als Gruppenleiter tätig war. Grossen Dank gilt meinem Dissertations-Komitee Prof. Markus Aebi, Prof.
Rosmarie Honegger, Dr. Jean Garbaye und Dr. Martina Peter für die Unterstützung und Beurteilung dieser Dissertation.
Ausserdem bedanke ich mich bei den Menschen, die beim FUNDIV Projekt mitgewirkt, und die Projekt Meetings jeweils zu einem interessanten und lustigen
Ereignis werden liessen. Des Weiteren bedanke ich mich herzlich bei allen
Menschen, mit denen ich im Laufe der letzten Jahre an der WSL zutun hatte: Käthi
Liechti, Rosmarie Eppenberger, Renaud Maire, Ivano Brunner, Stefan Rieder, Anita
Zumsteg, Benjamin Lange, Eva Maria Stimm, Beat Frey, Claude Herzog, Jan
Steffen, Andreas von der Dunk, James Glover, Beat Stierli, Rene Graf, Tina Endrulat,
Nicole Regier, Nadine Grisel, Barbara Meier und Tabea Kipfer.
Bei folgenden Menschen die mich die letzten Jahre begleitet haben, und nicht von der WSL sind, bedanke ich mich ebenfalls sehr herzlich: Uwe Franz, Brigitte Suter,
Karin Winter, Albert Noll, Nicole Blaser, Claudia Straub, Barbara Barco, Heidi und
Ruedi Gloor, Robert Stallmach, Ursi Leu und Anja Schwyzer.
Meine ganz besondere Dankbarkeit drücke ich meiner Familie aus, die mich immer unterstützt, egal was kommt. Jens Turowski gebührt ebenfalls grossen Dank. Vor allem in der Zeit vor der Fertigstellung meiner Dissertation war er für mich da und hat mir sehr geholfen.
Finanziert wurde meine Doktorarbeit im Rahmen des FUNDIV Projekts durch den französischen Nationalfond ANR, wofür ich ebenfalls sehr dankbar bin.
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