Arbuscular mycorrhizal colonization, plant chemistry, and aboveground herbivory on Senecio jacobaea

Acta Oecologica 38 (2012) 8e16

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Original article

Arbuscular mycorrhizal colonization, plant chemistry, and aboveground herbivory on Senecio jacobaea Stefan Reidinger a, *, René Eschen a,1, Alan C. Gange a, Paul Finch a, T. Martijn Bezemer b a b

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 50, 6700 AB Wageningen, The Netherlands

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 June 2011 Accepted 11 August 2011 Available online 27 August 2011

Arbuscular mycorrhizal fungi (AMF) can affect insect herbivores by changing plant growth and chemistry. However, many factors can influence the symbiotic relationship between plant and fungus, potentially obscuring experimental treatments and ecosystem impacts. In a field experiment, we assessed AMF colonization levels of individual ragwort (Senecio jacobaea) plants growing in grassland plots that were originally sown with 15 or 4 plant species, or were unsown. We measured the concentrations of carbon, nitrogen and pyrrolizidine alkaloids (PAs), and assessed the presence of aboveground insect herbivores on the sampled plants. Total AMF colonization and colonization by arbuscules was lower in plots sown with 15 species than in plots sown with 4 species and unsown plots. AMF colonization was positively related to the cover of oxeye daisy (Leucanthemum vulgare) and a positive relationship between colonization by arbuscules and the occurrence of a specialist seed-feeding fly (Pegohylemyia seneciella) was found. The occurrence of stem-boring, leaf-mining and sap-sucking insects was not affected by AMF colonization. Total PA concentrations were negatively related to colonization levels by vesicles, but did not differ among the sowing treatments. No single factor explained the observed differences in AMF colonization among the sowing treatments or insect herbivore occurrence on S. jacobaea. However, correlations across the treatments suggest that some of the variation was due to the abundance of one plant species, which is known to stimulate AMF colonization of neighbouring plants, while AMF colonization was related to the occurrence of a specialist insect herbivore. Our results thus illustrate that in natural systems, the ecosystem impact of AMF through their influence on the occurrence of specialist insects can be recognised, but they also highlight the confounding effect of neighbouring plant species identity. Hence, our results emphasise the importance of field studies to elucidate interactions between AMF and organisms of different trophic levels. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Arbuscular mycorrhizal fungi Insect herbivores Plant chemistry Senecio jacobaea Plant diversity

1. Introduction Understanding the mechanisms that determine the abundance of species in ecosystems is one of the most important issues in ecology. While changes in abiotic factors are good predictors of plant community development over large spatial and temporal scales

Abbreviations: AMF, arbuscular mycorrhizal fungi; C, carbon; N, nitrogen; P, phosphorous; PAs, pyrrolizidine alkaloids. * Corresponding author. Present address: Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK. Tel.: þ44 (0) 1904 328590; fax: þ44 (0) 1904 328505. E-mail addresses: [email protected], [email protected] (S. Reidinger), [email protected] (R. Eschen), [email protected] (A.C. Gange), p.fi[email protected] (P. Finch), [email protected] (T. M. Bezemer). 1 Present address: CABI Europe-Switzerland, Rue des Grillons 1, CH-2800 Delémont, Switzerland. 1146-609X/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.actao.2011.08.003

(Wardle et al., 2004), soil organisms can be important drivers of more local and short-term changes (Gange et al., 1990; Van der Putten et al., 1993; De Deyn et al., 2003; Kardol et al., 2006). One of the best-studied groups of soil organisms are the arbuscular mycorrhizal fungi (AMF), which form symbiotic associations with the majority of land plant species (Newman and Reddell, 1987; Smith and Read, 1997). AMF rely on photosynthetically fixed carbon provided by their host plant and in return supply the plant with nutrients that have been taken up from the soil. Due to an increased nutrient uptake, plants forming mycorrhizal associations are often bigger than non-mycorrhizal plants (Smith and Read, 1997). However, plants do not always benefit from AMF and negative plant growth responses to AMF colonization can occur when the net costs of the symbiosis exceed the net benefits (Johnson et al., 1997; Gange and Ayres, 1999). Further, AMF species differ in their effects on plant growth, depending on the identity of both the

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fungus and the plant (Streitwolf-Engel et al., 1997; Van der Heijden et al., 1998). Individuals of the same plant species frequently vary in AMF colonization levels and in the composition of AMF communities (e.g.  Eason et al., 2001; Smilauer, 2001; Carey et al., 2004; Opik et al., 2006, 2009; Wilson et al., 2006; Ishii et al., 2007; Meding and Zasoski, 2008; Uibopuu et al., 2009; Van de Voorde et al., 2010), and the relationship between AMF colonization levels and plant growth responses can range from positive to negative (Gange and Ayres, 1999; Hart and Reader, 2002; Bennett and Bever, 2009; Powell et al., 2009; Garrido et al., 2010; Zaller et al., 2011). Moreover, Garrido et al. (2010) showed that plant biomass allocation can be affected by AMF colonization levels, resulting in increased or decreased biomass allocation to different plant parts. The benefit plants derive from the association with AMF can further depend on the abiotic soil environment (Zaller et al., 2011). Interestingly, plant species typical of disturbed, open habitats often do not form mycorrhizal associations (Janos, 1980), or respond negatively to AMF colonization (Francis and Read, 1995; Rinaudo et al., 2010). Besides influencing plant size, AMF colonization can alter the resistance of plants to insect herbivores. In the majority of manipulative studies investigating the interaction between AMF colonization and insect herbivores, AMF colonization decreased the performance of generalist chewing insects (Koricheva et al., 2009), which may be because of an increase in the production of plant defence compounds (Pozo and Azcón-Aguilar, 2007), to which generalist insects often respond negatively (Manninen,1999). In contrast, the performance of specialist and generalist sucking insects and specialist chewing insects is usually higher on mycorrhizal than on non-mycorrhizal plants (Koricheva et al., 2009), though the mechanism is currently unknown (Gange, 2007). Most AMFeinsect herbivore studies have been conducted by comparing growth of insect herbivores on mycorrhizal and non-mycorrhizal plants under controlled conditions. However, under natural conditions, non-mycorrhizal individuals of plant species capable of forming mycorrhizal associations are hardly ever found (Harley and Harley, 1987). There is a paucity of field studies assessing the complex relationships among naturally occurring variations in AMF colonization levels, plant growth and herbivory, and a lack of consensus as to whether effects observed in the laboratory translate into changes in insect growth and population dynamics in the field (Gehring and Bennett, 2009; Hartley and Gange, 2009). The main reason for the low number of field studies with different levels of AMF colonization is that the abundance of AMF in the field is notoriously difficult to manipulate. For instance, reducing AMF colonization levels through fungicide application also affects soil fungi other than AMF, potentially influencing the abundance of plant pathogens and soil nutrient cycling. Furthermore, soil fungicides can exhibit direct toxic effects on insect herbivores when taken up by the plant (Laird and Addicott, 2008). Therefore, only a few studies have attempted to study AMFeplanteherbivore interactions in the field and it remains unclear whether the effects of AMF on herbivores in controlled greenhouse studies can also be observed under natural, more complex settings. Even though data from non-manipulative field studies are of a correlative nature and do not demonstrate causality, a comparison between AMFeplanteherbivore data obtained from correlative field studies and causal data obtained from manipulative, controlled experiments may unveil whether interactions between AMF and herbivores can also be observed in the field. We studied whether differences in AMF colonization levels of Senecio jacobaea L. (ragwort; Asteraceae) are related to variation in plant size and the number and community structure of insect herbivores feeding on it. S. jacobaea is a biennial plant, spending its first year as a rosette and flowering the following summer. Since it requires gaps in the vegetation for successful establishment,

9

S. jacobaea is most commonly found in disturbed habitats (Harper and Wood, 1957). The plant can contain high levels of pyrrolizidine alkaloids (PAs), which pose a severe threat to grazing livestock and which can influence growth and the community structure of saprophytic soil fungi (Hol and Van Veen, 2002; Kowalchuk et al., 2006). Despite the high toxicity of S. jacobaea, both specialist and generalist insects feed on this plant species (Harper and Wood, 1957). S. jacobaea has previously been described as a facultative mycorrhizal species (Bower, 1997), and soil fungicide application in the field has been found to decrease AMF colonization levels and to increase plant size and it has been suggested that AMF are parasitic in this plant species (Gange et al., 2002). However, it is unknown whether the effect of AMF colonization on S. jacobaea is always negative, or whether it depends on the biotic and abiotic environment of the plant. This study was part of an ongoing field experiment in The Netherlands, with experimental plots that were unsown, or were sown with either 4 or 15 plant species (Bezemer et al., 2006). After sowing, new plant species were allowed to colonize the plots from the soil seed bank and the surrounding vegetation. S. jacobaea, which was not included in any of the seed mixtures, started to establish in the plots soon after the start of the experiment, and initially the cover and biomass of S. jacobaea were highest in the unsown plots, but S. jacobaea cover in these plots sharply declined five years later (Bezemer et al., 2006). The sowing treatments had distinct effects on the community composition of insect herbivores feeding on S. jacobaea: plants in unsown plots were less likely to be infested with leaf miners, seed feeders and stem borers than plants in sown plots, and there was a strong positive relationship between plant size and the occurrence of insect herbivores (Bezemer et al., 2006). The aim of this study was to determine the effects of the sowing treatments on AMF colonization levels of S. jacobaea and on the associated insect herbivore fauna. We hypothesized that AMF colonization of S. jacobaea plants growing in plots with high S. jacobaea cover (unsown plots) would be higher than that in plots with low cover (sown plots), due to accumulations of S. jacobaeaspecific AMF communities, which can reduce the mutualistic benefit received by the plant (Bever, 2002). We further hypothesized that S. jacobaea biomass would be negatively related to AMF colonization levels, because AMF have previously been suggested to negatively affect growth of this plant species (Bower, 1997; Gange et al., 2002), and because species of disturbed habitats often respond negatively to AMF (Francis and Read, 1995; Rinaudo et al., 2010). We tested whether differences in AMF colonization among the sowing treatments were related to the abundance and the community structure of insect herbivores feeding on S. jacobaea. We measured leaf nitrogen and carbon concentrations and the concentration of pyrrolizidine alkaloids (PAs), which are main defence compounds in S. jacobaea (Rice, 1984), in an attempt to elucidate the underlying mechanisms of how AMF and insect herbivores may interact with each other. 2. Materials and methods 2.1. Field experiment The experiment was set up in 1996 on a former arable field of 15 ha that was cultivated until 1995. An area of 0.5 ha was fenced, ploughed and three different sowing treatments were applied to plots of 10  10 m: sowing with 15 plant species, sowing with a subset of 4 species, or not sowing. There were five replicate blocks, with each block containing one plot of each sowing treatment. The five replicate plots of the 4 species treatment were sown with different combinations of plant species (Appendix 1). This

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approach has been adopted frequently in biodiversity studies to avoid sampling effects (e.g. Van der Putten et al., 2000). After sowing, the plots were not weeded and in all plots the establishment of plants from the soil seed bank and the surroundings was allowed. The sowing treatments resulted in distinct differences in plant and soil community composition and diversity, with the unsown plots having the highest and plots sown with 4 species the lowest plant diversity (Bezemer and Van der Putten, 2007; Bezemer et al., 2010). Details of the experiment and temporal development of the plant and soil community have been described elsewhere (Van der Putten et al., 2000; Hedlund et al., 2003; Fukami et al., 2005; Bezemer et al., 2006; Leps et al., 2007). 2.2. Data collection In early July 2005, we sampled 10 flowering S. jacobaea plants from each of the 15 plots, ensuring that large and small plants were included from each plot. The plants were carefully dug up in soil blocks of ca. 15  15  15 cm, and the plants were kept individually in plastic bags. Since we attempted to perturb the field site as little as possible, we frequently did not succeed in extracting the whole root system of a plant. Therefore, we were not able to collect reliable data on S. jacobaea root biomass. In the laboratory, for each plant we then counted and dissected all flower buds and recorded the percentage of flower heads infested with larvae of seed-feeding flies. We also recorded the presence of aphids, leaf miners and stem borers. Afterwards, the flowers, leaves and stems were oven-dried at 70  C, and weighed. In order to estimate the root colonization levels by AMF, the roots were cleared in 10% KOH for 3 days, followed by acidification in 1% HCl and staining in a 1% HCl containing few drops of Parker Quink dark blue ink (Parker, Boston, Mass., USA) for 1 h (Vierheilig et al., 1998). We randomly selected around thirty 1-cm long root fragments from each plant, placed them onto two microscope slides and recorded the presence of AMF structures for 100 intersections at 800 magnification by the cross-hair eyepiece method of McGonigle et al. (1990). Percentage root length colonization levels by arbuscules, vesicles and total AMF colonization (arbuscules and vesicles) were calculated. Since plants growing in the field are usually colonized by a variety of fungal taxa, and AMF hyphae often lack characteristics that allow distinguishing them from non-AMF hyphae, root colonization levels by fungal hyphae were not included in the analyses. During late Julyeearly August 2005, the plant species cover in each experimental plot of 100 m2 was recorded in 12 permanent quadrats of 1 m2 each. In late August, aboveground biomass was clipped at 2 cm above soil surface in 12 0.25  0.25 m2 subplots adjacent to the permanent quadrats. The plant material was oven-dried at 70  C and weighed to calculate the mean aboveground biomass m2 for each plot. In July 2006, one year after we took the above described plant, mycorrhiza and insect measurements, 24 soil samples (2.5 cm diameter and 15 cm depth) were randomly collected from each plot. The 24 samples from each plot were homogenized, sieved (<0.5 cm), and dried for 3 days at 40  C, and subsequently used for chemical analyses (see below). 2.3. Chemical analyses From the 10 S. jacobaea plants sampled from each of the 15 plots, we randomly selected three plants per plot (45 in total) and analyzed carbon (C), nitrogen (N) and PA concentrations. C and N concentrations were determined using a Flash EA1112 CN analyzer (Interscience, Breda, The Netherlands) according to the manufacturer’s instructions. Total PAs were extracted by acidebase extraction (Hartmann and Zimmer, 1986). To check the percentage recovery following the extraction process, 4,40 -dipyridyl was added to the

plant material as an internal standard at the start of the extraction process. Individual PAs were quantified with GCeMS using a Hewlett Packard HP5890 Series II gas chromatograph coupled to a Hewlett Packard HP5970 Mass Selective Detector (HewlettePackard, Waldbronn, Germany) operated in the electron ionization mode at 70 eV with ion source and quadrupole temperatures of 180  C and 250  C respectively. Chromatography was carried out on an SGE HT5 25 m  0.22 mm fused silica column with 0.1 mm film (SGE UK, Milton Keynes, UK) at an inlet pressure of 10 psi. Caffeine was used as internal standard. Compound identification was carried out by comparison with literature data (Witte et al.,1993) and by matching experimental spectra with NIST 05.2 library (Scientific Instrument Services, Ringoes, New Jersey, USA). Soil pH, plant available P and K were analyzed in 1:10 (w/v) 0.01 M CaCl2. Concentrations of available NHþ 4 eN and NO 3 eN were determined colorimetrically in the CaCl2-extract using a Traacs 800 autoanalyzer (TechniCon Systems Inc.). 2.4. Statistical analysis The sowing treatment effects on AMF colonization levels, S. jacobaea biomass attributes, plant C and N concentrations and plant C/N ratios were analyzed with a general linear mixed model, with sowing treatment as fixed factor, block as a random factor and plant replicate within plots as a random factor nested within plot. Insect herbivore and PA data from samples taken within one plot were averaged prior to the statistical analysis since herbivore attack was generally low, and not all PA groups were present in all plants at a detectable level. Insect and PA response data were subsequently analyzed with a generalized linear model, with sowing treatment as a fixed and block as a random factor. Pairwise comparisons between the sowing treatments were carried out using a Tukey HSD test for parametric data and a Wilcoxon rank sum test for binomial data. Pvalues of the pairwise t-tests were corrected using the Bonferroni method. Plant community composition was compared using a multivariate principle component analysis (PCA). Plant diversity, productivity and ragwort cover, and soil chemistry data per plot was analyzed using ANOVA with sowing treatment as a fixed and block as a random factor. To fulfil assumptions for normality, plant richness data were log transformed. Linear regressions were calculated for relationships between S. jacobaea biomass and AMF colonization, and between AMF colonization and plant chemistry data. Binary logistic regressions were carried out to test for the effects of S. jacobaea biomass attributes, AMF colonization and plant chemistry on the occurrence of individual insect herbivore groups. To test whether variation in the insect community could be explained by the sowing treatments, a canonical correspondence analysis (CCA) was carried out with presence of the individual insect groups as response variables and sowing treatments as explanatory variable. The significance of the analysis was tested using a permutation test with block as randomization stratum. To test whether the level of colonization by arbuscules, vesicles and total AMF colonization was related to the plant community composition we used a linear constrained multivariate analysis (RDA) followed by a permutation test. Percentage and proportion data were arcsine square-root transformed prior to statistical analyses and all statistical analyses were conducted in R (R Development Core Team, 2008). 3. Results 3.1. AMF colonization levels All S. jacobaea plants were colonized by AMF, with average colonization levels being 40.5  1.6% for total colonization, 32.2  1.5% for arbuscules and 21.8  0.9% for vesicles. Total AMF

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colonization levels and colonization by arbuscules were lower in plots sown with 15 plant species than in plots sown with 4 species and unsown plots (AMF total: F2,13 ¼ 11.0, P ¼ 0.005; arbuscules: F2,13 ¼ 5.6, P ¼ 0.030; Fig. 1A and B). Colonization by vesicles tended to be higher in plots sown with 4 species than in plots sown with 15 species and colonization in the unsown plots tended to be in between, but this was not significant (F2,13 ¼ 3.5, P ¼ 0.080; Fig. 1C). Colonization by arbuscules and vesicles and total AMF colonization levels were not related to individual total aboveground plant biomass, or leaf, stem or flower biomass (all P > 0.05). 3.2. Vegetation characteristics and relationship to AMF colonization In 2005, plant species richness was higher in unsown than in sown plots, which was due to a high number of ruderal plant species in the unsown plots. Plant species richness did not differ between the two sown treatments (Table 1). Total aboveground

B

Percentage root length colonized

Percentage root length colonized

A

Total b

50

b 40

a

30 20

0 Arbuscules b

40

b

a 30 20 10 0

C

Vesicles Percentage root length colonized

vegetation biomass was higher in sown than in unsown plots. S. jacobaea cover was more than three times lower in sown than in unsown plots, but did not differ between the two sown treatments. Total aboveground biomass, leaf biomass, stem biomass and total flower biomass of the individual S. jacobaea plants sampled did not differ between the sowing treatments (mean  SE: 3.1  0.2, 0.7  0.1, 1.9  0.1 and 0.5  0.01 g, respectively; all P > 0.05). The plant community composition in plots sown with 15 plant species differed considerably from the composition in unsown plots, whereas the composition in plots sown with 4 plant species, for which the replicates were originally sown with different species combinations, was variable (Fig. 2). More detailed descriptions of the plant community development from 1996 until 2005 in the experimental plots have been published elsewhere (Bezemer et al., 2006; Fukami et al., 2005; Leps et al., 2007). Colonization by arbuscules was positively related to plant species richness and to S. jacobaea cover of the plots (P ¼ 0.043, R2 ¼ 0.53; P ¼ 0.041, R2 ¼ 0.53, respectively). Total AMF colonization levels and colonization by arbuscules were negatively related to total aboveground vegetation biomass (P ¼ 0.016, R2 ¼ 0.61; P ¼ 0.002, R2 ¼ 0.74 respectively). There was a significant relationship between AMF colonization levels and plant community composition (RDA: AMF total: 19.0% explained variation, F ¼ 3.0, P ¼ 0.005; arbuscules: 16.5% explained variation, F ¼ 2.6, P ¼ 0.009 (Fig. 3); vesicles: 14.3% explained variation, F ¼ 2.2, P ¼ 0.03). The RDA analysis showed a strong positive relationship between AMF colonization levels of S. jacobaea and the cover of Leucanthemum vulgare Lam. 3.3. Aboveground insect abundance

10

a

30

a 20

11

a

10

0 15

4 Number of species sown

0

Fig. 1. Sowing treatment effects (means  SE) on total AMF colonization (A), colonization by arbuscules (B) and colonization by vesicles (C) of individual Senecio jacobaea plants. Different letters indicate statistical significant differences at P < 0.05 between treatments, based on a tukey post hoc test.

One species of seed-feeding insect was found (Pegohylemyia seneciella, Diptera, Anthomyiidae) and the proportion of flower heads infested with this species tended to be higher in unsown than in sown plots, but this was not significant (F2,8 ¼ 3.67, P ¼ 0.074; Fig. 4A). The sowing treatments did not influence the proportion of plants that were infested with an un-identified species of stem borer (F2,8 ¼ 2.90, P ¼ 0.11, Fig. 4B), a leaf-mining insect (Lyriomya synchenesiae, Diptera, Agromyzidae; F2,8 ¼ 0.71, P ¼ 0.52, Fig. 4C) or an un-identified aphid species (F2,8 ¼ 1.20, P ¼ 0.35, Fig. 4D). Irrespective of the sowing treatment, the probability of seed-feeding insect occurrence was positively related to colonization by arbuscules (Z ¼ 2.33, P ¼ 0.020), but not to colonization by vesicles (Z ¼ 0.49, P ¼ 0.626) or total AMF colonization (Z ¼ 1.46, P ¼ 0.144). There were no significant relationships between AMF colonization levels and infestation rates by stem borers (arbuscules: Z ¼ 0.93, P ¼ 0.355; vesicles: Z ¼ 0.72, P ¼ 0.471; total AMF colonization: Z ¼ 1.28, P ¼ 0.201), leaf miners (arbuscules: Z ¼ 0.97, P ¼ 0.332; vesicles: Z ¼ 0.90, P ¼ 0.369; total AMF colonization: Z ¼ 0.72, P ¼ 0.473), or aphids (arbuscules: Z ¼ 0.68, P ¼ 0.496; vesicles: Z ¼ 0.58, P ¼ 0.560; total AMF colonization: Z ¼ 0.35, P ¼ 0.728). The probability of plants being infested with aphids was positively related to the leaf biomass of the plant (Z ¼ 2.21, P ¼ 0.027), and the occurrence of stem borers was positively related to stem and total plant biomass (stem: Z ¼ 2.21, P ¼ 0.027; total: Z ¼ 3.74, P < 0.001). The occurrence of seed feeders was positively related to the total flower biomass (Z ¼ 3.42, P ¼ 0.001) and the total aboveground biomass (Z ¼ 3.20, P ¼ 0.001). The probability of a plant being infested with leaf miners was unrelated to both total plant biomass (Z ¼ 0.69, P ¼ 0.492) and leaf biomass (Z ¼ 0.85, P ¼ 0.394). 3.4. Plant and soil chemistry Foliar N concentrations were around 17% higher in plots sown with 15 plant species than in plots sown with 4 species and unsown

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Table 1 Characteristics of the plant community and cover of Senecio jacobaea in the different sowing treatments (means  1 SE), and results of an ANOVA test. Within rows, means followed by different letters are significantly different (P < 0.05) based on a Tukey HSD test. Measurement

Number of species sown

Species richness Total aboveground biomass (g m2) Senecio jacobaea cover (%)

Sowing treatment

15 species

4 species

0 species

F2,8

P

12.9  0.1b 770.1  74.9a 3.2  0.7b

12.7  0.9b 543. 1  58.1a 4.0  0.8b

15.8  0.3a 434.0  41.8b 12.9  0.8a

9.73 8.26 37.61

0.007 0.011 <0.001

plots (F2,8 ¼ 5.66, P ¼ 0.029; 15 species: 2.08  0.17, 4 species: 1.75  0.10, unsown: 1.79  0.08). Leaf C concentrations were not affected by the sowing treatments (F2,8 ¼ 1.47, P ¼ 0.29). There was no significant relationship between the concentration of leaf C and N and the probability that a plant was infested with seed feeders stem borers, leaf miners or aphids (all P > 0.05). Foliar C and N concentrations and C/N ratios were also unrelated to AMF colonization levels (all P > 0.05). The PAs senecionine, seneciophylline, jacobine, jacozine and jacoline were detected. Concentrations of these compounds and total PA concentrations did not significantly differ between the sowing treatments (Table 2). Neither concentrations of the single PA compounds, nor total PA concentrations were related to the probability of plants being infested with seed feeders, stem borers, leaf miners or aphids (all P > 0.05), and did not explain any variance in insect community structure (P > 0.05). However, there was a negative correlation between both jacoline and total PA concentration and colonization levels by vesicles (P < 0.05, R2 ¼ 0.13 and P < 0.05, R2 ¼ 0.12, respectively). The sowing treatments had no effect on soil pH or plant available soil P, K, NHþ 4 and NO3 concentrations in 2006 (Appendix 2).

be higher than that of plants on plots with low S. jacobaea cover (plots sown with 4 or 15 species). To an extent this was upheld, as colonization by arbuscules was positively related to S. jacobaea cover, irrespective of the plots from which the plants came from. However, AMF colonization in plots sown with 4 species was not different from that in unsown plots despite large differences in S. jacobaea cover between these two treatments. It is therefore unlikely that the cover of S. jacobaea alone determined AMF colonization levels of this plant species and other factors need to be taken into account. Plant diversity can impact on soil nutrient content (Wedin and Tilman, 1990; Spehn et al., 2002) and AMF colonization can be influenced by soil nutrient levels (Johnson et al., 2003; Boddington and Dodd, 2000; Treseder, 2004). Even though the initial sowing treatments influenced the composition of the plant communities, for instance by affecting legume abundance, we did not detect any significant differences among the treatments in any of the soil characteristics that we measured the following year. Thus, although we cannot exclude possible changes in soil chemistry between the year this study was conducted and the following year when the soil chemistry measurements were taken, it appears unlikely that differences in AMF colonization levels between sowing treatments were mediated by differences in soil chemistry. Root colonization by AMF can also be positively related to the availability of infective AMF units (e.g. spores) in the soil (Garrido et al., 2010), and AMF colonization levels can decrease with increasing vegetation biomass because of increased competition for infective AMF units (“dilution effect”) (Koide, 1991; Facelli et al., 1999). Vegetation biomass was highest in unsown plots and, irrespective of the sowing treatments, total aboveground biomass of the vegetation was negatively related to AMF colonization levels. This suggests that competition for AMF between S. jacobaea and other plant species in the community influenced its colonization

4. Discussion 4.1. Relationship between AMF colonization levels, plant community and soil characteristics Plants can accumulate species-specific mycorrhizal communities that comprise AMF species that are best adapted to colonize that host plant species, and accumulations of such communities can result in increased AMF population growth (Bever, 2002). Thus, we hypothesized that AMF colonization levels of S. jacobaea growing on plots with high S. jacobaea cover (unsown plots) would

A

B Tanacetum vulgare

15 4 3

0

Leucanthemum vulgare

4 1

3

5 2 4

2 5 2

1

PCA-2 (14.8%)

PCA-2 (14.8%)

3 Achillea millefolium Poa pratense

Festuca rubra

4 5

1

PCA-1 (33.2%)

Trifolium repens Bromus spp Phleum pratense Cerastium Agrostis fontanum capillaris Crepis capillaris Vicia hirsuta Taraxacum officinale

Lotus corniculatus

PCA-1 (33.2%)

Fig. 2. Principal component analysis of the plant community composition in 2005 in plots sown originally with 15 or 4 species or that were not sown. Shown are the biplots of (A) sample, and (B) species scores for the first and second axes. In (A) the numbers beside the samples indicate field blocks. In (B) the 15 plant species that show the highest correlation with AMF colonization are shown. The eigen values for both axes are also presented.

S. Reidinger et al. / Acta Oecologica 38 (2012) 8e16

Table 2 Pyrrolizidine alkaloid concentrations (mg per gram dry weight, mean  SE) for Senecio jacobaea plants in response to the sowing treatments, and results of a generalized linear model.

Agrostis capillaris

Phleum pratense

Plantago lanceolata

PA compound

Number of species sown

Senecionine Seneciophylline Jacobine Jacozine Jacoline PA total

0.10 0.04 0.57 0.23 0.56 1.50

Trifolium repens Taraxacum officinale Crepis capillaris

Vicia hirsuta Cerastium fontanum

RDA 2

15 species Linaria vulgaris

AMF colonization Hypochaeris radicata Trifolium repens Leucanthemum vulgare

Lotus corniculatus

Festuca rubra Tanacetum vulgare

RDA 1 Fig. 3. Multivariate Redundancy analysis (RDA; 16.5% explained variation) for the relationship between plant community composition of the 15 plots and the percentage AMF colonization by arbuscules of individual Senecio jacobaea plants growing in those plots. The 15 plant species that show the highest correlation with AMF colonization are shown.

levels. However, total aboveground vegetation biomass did not significantly differ between the two sown treatments, whereas AMF colonization levels were lower in the plots sown with 15 species than in plots sown with 4 species, indicating that competition for AMF spores can only partly explain the observed treatment differences in AMF colonization levels. Increased species richness of a plant community can result in an increased diversity and spore density of the associated AMF community, which in turn may affect AMF colonization levels (Burrows and Pfleger, 2002). Irrespective of the sowing treatments, plant species richness was positively related to AMF colonization of S. jacobaea. However, colonization in plots sown with 4 plant species was not different from colonization in unsown plots whereas species richness

A

0.4 0.3 0.2 0.1

0.04 0.02 0.13 0.06 0.19 0.27

4 species 0.18 0.12 1.02 0.29 0.37 1.95

     

0.08 0.09 0.46 0.08 0.17 0.70

0 species 0.05 0.05 0.30 0.13 0.21 0.70

     

0.03 0.01 0.21 0.08 0.09 0.31

F2,8

P

0.60 1.05 1.25 1.41 1.35 1.75

0.57 0.40 0.34 0.30 0.31 0.23

Stem borers

0.6 Proportion of plants infested

Proportion of plants infested

B

0.5

     

Sowing treatment

was significantly lower in plots sown with 4 species than in unsown plots, indicating that differences in plant species richness can only explain part of the treatment differences in AMF colonization. These results are supported by a recent study carried out at the same field site but one year later, which showed that there was no relationship between the AMF community composition of individual S. jacobaea plants and plant community composition (Van de Voorde et al., 2010). Yet, in the present study, plant community composition explained a significant amount of variation in AMF colonization, with AMF colonization levels being positively related to the cover of L. vulgare. Remarkably, this species has previously been reported to increase the mycorrhizal potential of soils (Bharadwaj et al., 2007). Plant species of disturbed habitats often show negative growth responses to AMF (Francis and Read, 1995; Rinaudo et al., 2010), and the size of S. jacobaea can increase after soil fungicide application (Gange et al., 2002). Further, previous work has shown that the performance of S. jacobaea in this long-term field experiment was related to differences in soil fungal communities (Bezemer et al., 2006). Therefore, we hypothesized that AMF colonization levels can explain the performance of this plant species, but we found no relationship between AMF colonization levels and any of the recorded plant biomass attributes. Plants can accumulate species-specific soil microbial communities, which in turn can feed

Seed feeders

0.6

0

0.5 0.4 0.3 0.2 0.1 0

C

D

Leaf miners

Aphids

0.3

Proportion of plants infested

0.2 Proportion of plants infested

13

0.15

0.1

0.05

0.2

0.1

0

0 15

4 Number of species sown

0

15

4

0

Number of species sown

Fig. 4. Sowing treatment effects (means  SE) on the proportion of Senecio jacobaea plants being infested with seed feeders (A), stem borers (B), leaf miners (C) and aphids (D).

14

S. Reidinger et al. / Acta Oecologica 38 (2012) 8e16

back on growth of the same or other plants (Bever, 1994; Bever et al., 1997; Ehrenfeld et al., 2005). Negative plant-soil feedbacks are often caused by an accumulation of soil pathogens (Mills and Bever, 1998; Van der Putten, 2003), but can also be caused by accumulations of soil organisms that are usually regarded as plant mutualists like AMF (Bever, 2002). Positive feedbacks can result from accumulations of plant-growth promoting soil organisms, such as AMF (Klironomos, 2002). We therefore suggest that the sowing treatments also influenced soil fungal groups other than AMF, which consequently determined the performance of S. jacobaea. The positive relationship between the abundance of L. vulgare and AMF colonization levels of S. jacobaea in our study is striking and appears to be an example of species-specific soil feedback involving AMF. Clearly, more work is needed to disentangle the role of the identity of neighbouring plant species on AMF colonization levels (Hausmann and Hawkes, 2009; Van de Voorde et al., 2010). 4.2. Relationship between AMF colonization levels and S. jacobaea characteristics A number of studies reported AMF colonization levels of plants that are colonized by a single AMF species to decrease following foliar herbivory (e.g. Gehring and Whitham, 1994; Gange, 2007). However, plants growing in natural environments are usually not colonized by a single AMF species and Bennett and Bever (2009) showed that the negative effect of herbivory on AMF colonization levels can disappear when the plants are colonized by an AMF species mixture (but see Gange et al. (2002) for a study with S. jacobaea). Even though the design of this study does not allow us to rule out possible effects of the herbivores on AMF colonization levels, this seems to be unlikely in view of the low degree of damage caused by the herbivores. Furthermore, non-reproductive S. jacobaea rosettes sampled in the early summer of the previous year showed similar differences in colonization levels between treatments as found in the present study (S.R., unpublished), but herbivore damage on these plants was almost absent, since insect herbivores predominantly attack reproductive S. jacobaea plants. This suggests that the interactions between AMF and insect herbivores in our study were bottom-up rather than top-down driven. Average total AMF colonization levels of S. jacobaea were around 41%, which is surprisingly high, taking into account that AMF colonization levels of this plant species have previously been described to rarely exceed 4% (Bower, 1997; Gange et al., 2002). The difference in AMF colonization levels between previous studies (Bower, 1997; Gange et al., 2002) and the present study might be due to the fact that the studies were conducted on geographically different field sites. Variation in AMF colonization of a plant species from one geographic location to another has been reported before (e.g. Rodríguez-Echeverría et al., 2007). Indeed, in another study carried out at the same field site as the present study, AMF colonization levels comparable to that in the present study were reported (Van de Voorde et al., 2010). Differences in soil nutrient availability are unlikely to explain the low colonization levels reported in previous studies and the much higher levels described in this study, because in the present study, soil P levels were around 50% higher than the values reported in Bower (1997) and Gange et al. (2002), and AMF colonization is usually negatively related to soil P concentrations (Treseder, 2004). S. jacobaea often forms distinct geographical populations which exhibit differences in the quantity and composition of PAs (Ma cel et al., 2004). However, the amount of variation within the study population was can be substantial, indicating that other, environmental factors may also affect the concentration of individual PAs. The quantity and composition of PAs has been shown to influence

growth of saprophytic fungi and the structure of non-AM rhizosphere fungal communities (Hol and Van Veen, 2002; Kowalchuk et al., 2006), but whether PAs influence AMF is unknown. A relationship between concentrations of chemical defences and AMF colonization levels has been reported for the forb Plantago lanceolata (De Deyn et al., 2009). In that study, a negative correlation was found between the iridoid glycoside aucubin concentrations in plants of three genotypes that differed in the production of this compound and the number of AMF arbuscules in the roots of those plants. Inoculation of the plants with AMF did not affect the concentrations of any of the measured defence compounds, indicating that differences in AMF colonization levels were the result of a plant factor. In the present study, jacoline and total PA concentrations were negatively related to root colonization by vesicles and we suggest that the concentration of defence compounds in S. jacobaea also affected AMF colonization. We found no effect of the sowing treatments on PA concentrations of the plants, or a relationship with attack by any of the herbivores, and it is unclear what caused the variation in PA content of the plants in our study. Bezemer et al. (2006) showed that the size of S. jacobaea plants was positively related to infestation rates by seed feeders, leaf miners and stem borers. In the present study, with samples taken from the same experimental plots three years later, we also found positive relationships between plant biomass attributes and the occurrence of aphids, stem borers and seed feeders. Moreover, AMF colonization levels explained a significant amount of variation in the occurrence of seed-feeding insects: the probability of plants being infested with P. seneciella was positively related to AMF colonization levels. P. seneciella is a specialist seed feeder of S. jacobaea and closely related species (Crawley and Pattrasudhi, 1998), hence these findings support the theory that specialist insect herbivores benefit from AM fungi (Gange, 2007; Koricheva et al., 2009). They also corroborate previous reports of AMF increasing infestation rates by seedfeeding insects (Gange et al., 2005). However, this study does not provide evidence for an influence of AMF colonization levels on the occurrence of other, perhaps more generalist herbivores, whose occurrence correlated with the size of the plants, or parts of those plants. We found no relationship between PA concentrations and the occurrence of insect herbivores. Our results thus appear to confirm the previously reported absence of PA effects on specialist herbivores on S. jacobaea (Rothschild et al., 1979; Hartmann, 1999; Ma cel et al., 2002; Naumann et al., 2002; Macel and Vrieling, 2003). However, the other insects associated with the plants in our study, some of which may have been generalists, were also not affected. Leaf N concentrations were highest in plots sown with 15 plant species and these plots also had the highest legume cover (Bezemer et al., 2006), but the sowing treatments had no effect on plant available N concentrations in the soil. It has been shown that AMF can transfer substantial amounts of N and other nutrients, such as P, between host plants (e.g. Smith and Read, 1997; He et al., 2009; Simard and Durall, 2004; Wilson et al., 2006; Meding and Zasoski, 2008), and we suggest that this may explain some of our results. The performance and abundance of most insect herbivores is usually limited by the amount of N in their diet (Mattson, 1980), but in our study we did not find a significant relationship between plant N concentrations and insect occurrence. This is a similar result to that of Koricheva et al. (2009), who found no evidence for a role of N availability in insect responses to AMF in a meta-analysis. However, arbuscules are considered to be the part of the AM symbiosis where most of nutrient exchange between plant and fungus occurs (Smith and Read, 1997), and the positive relationship between occurrence of seed feeders and AM arbuscules suggests nutritional benefits of AMF for herbivorous insects.

S. Reidinger et al. / Acta Oecologica 38 (2012) 8e16

5. Conclusions

Appendix 2

As part of a long-term field study, we investigated the effect of vegetation diversity on AMF colonization levels of S. jacobaea, a natural invader of the experimental field plots, and the interactions of the plants with insect herbivores. Although the integrity of the sown diversity treatments was not maintained, treatment effects on the vegetation persisted and AMF colonization levels of S. jacobaea differed. We did not identify one single factor that explained these differences, but correlations across the three treatments suggest that some of the variation in AMF colonization levels was due to the abundance of one plant species, Linaria vulgaris, which is known to stimulate AMF colonization levels of plants growing in its surroundings. The abundance of arbuscules was correlated with the occurrence of a specialist seed-feeding insect. By contrast, the occurrence of most identified insect groups on the S. jacobaea plants was influenced by the biomass of different plant parts, which was not affected by the sowing treatments or AMF colonization levels. Most of the ecosystem effects of AMF have been studied in experiments that were often carried out in controlled conditions, usually involving only a few species. One reason for this simplification is that AMF are difficult to manipulate under field conditions and the symbiosis is affected by many environmental factors that can interfere with the experimental treatments or obscure their effects. Our results illustrate that even in natural systems, the ecosystem impact of AMF through their influence on the degree of plant attack by specialist insect herbivores can be recognised. In this case, some of the effects may even have been caused by the presence of a different plant species. Hence, our results emphasise the importance of field studies to elucidate complex interaction between AMF and organisms of different trophic levels. Acknowledgements We are grateful to the European Union for funding this work within the BIORHIZ programme (Biotic interactions in the rhizosphere of plants as structuring forces for plant communities MRTNCT-2003-505090). T.M.B. is funded by the Netherlands Organization for Scientific Research (NWO, VIDI grant no 864.07.009). Publication 5086 of the Netherlands Institute of Ecology (NIOOKNAW). Appendix 1

Plant species sown at the start of the experiment in 1996 in plots sown with 15 or 4 species in each block (B). Functional group

Grasses

Legumes

Other forbs

15

Plant species

Festuca rubra Phleum pratense Poa pratensis Agrostis capillaris Anthoxanthum odoratum Lotus corniculatus Trifolium pratense Trifolium dubium Trifolium arvense Vicia cracca Plantago lanceolata Tanacetum vulgare Hypericum perforatum Hypochaeris radicata Linaria vulgaris

15 species

4 species

B1eB5

B1

B2

X X X X X

X X

X

X X X X X X X X X X

X

B3

B4

B5 X

X

X X

X X

X

X X X X X X X X X

Soil nutrient concentrations and soil pH in response to the sowing treatments, and results of an ANOVA. Number of species sown 15 species P (mg kg1) K (mg kg1) NO3 (mg kg1) NH4 (mg kg1) pH CaCl2

4.77 42.25 1.57 0.17 5.19

    

0.37 5.65 0.50 0.08 0.07

Sowing treatment

4 species 5.28 49.01 0.70 1.09 5.13

    

0.56 7.67 0.35 0.73 0.06

0 species 4.01 60.53 1.56 1.17 5.26

    

0.33 5.16 0.12 0.78 0.03

F2,8

P

3.42 1.69 2.36 1.65 1.73

0.09 0.25 0.16 0.25 0.24

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