• Vesicular–arbuscular mycorrhiza (VAM) and earthworms are known to affect plant and herbivore performance. However, surprisingly few studies have investigated their interactions.
• In a glasshouse experiment we investigated the effects of earthworms (Aporrectodea caliginosa) and VAM (Glomus intraradices) on the growth and chemistry of Plantago lanceolata and the performance of aphids (Myzus persicae).
• Earthworms did not affect VAM root colonization. Earthworms enhanced shoot biomass, and VAM reduced root biomass. VAM increased plant phosphorus content, but reduced the total amount of N in leaves. Earthworms led to a preferential uptake of soil N compared with 15N from the added grass residues in the absence of VAM. Earthworm presence reduced the concentration of catalpol. Earthworms and VAM combined accelerated the development of M. persicae, while the development tended to be delayed when only VAM or earthworms were present.
• We suggest that earthworms promote plant growth by enhancing soil N availability and may affect herbivores by influencing concentrations of secondary metabolites. VAM enhances the P uptake of plants, but presumably competes with plant roots for N.
Other soil organisms, such as earthworms, enhance nutrient mineralisation in soil (Scheu, 1994) and increase N uptake and plant growth (Haimi et al., 1992). Earthworm-mediated changes in plant chemistry may affect plant interactions with herbivores. Earthworm presence has been documented to increase the reproduction of aphids (Myzus persicae) on Poa annua and Trifolium repens (Scheu et al., 1999) and Cardamine hirsuta (Wurst & Jones, 2003), but to reduce aphid reproduction on Plantago lanceolata (Wurst et al., 2003). Recently, earthworms also have been found to affect the concentration of phytosterols in leaves of P. lanceolata (Wurst et al., 2004).
Studies investigating the combined effects of earthworms and VAM concentrated on earthworms as vectors for the dispersal of VAM propagules (Rabatin & Stinner, 1988, 1989; Reddell & Spain, 1991; Gange, 1993). Pattinson et al. (1997) reported that an increased density of earthworms results in a transient decrease of root colonisation by VAM in Trifolium subterraneum. Since earthworms process large amounts of soil by burrowing and casting (Scheu, 1987) and selectively feed on fungal mycelia (Bonkowski et al., 2000), they might decrease the infectivity of VAM by disruption of the hyphal network. Mechanical soil disturbance has been documented to reduce the infectivity of VAM hyphae (Jasper et al., 1989). By enhancing nutrient availability in soil, earthworms might also alleviate the mycorrhizal dependency of the plants. Surprisingly only one study (Tuffen et al., 2002) has investigated the combined effects of earthworms and VAM on plant growth. Earthworms, but not VAM, enhanced plant growth, and both increased 32P transfer between Allium porrum plants. No significant interaction between earthworms and VAM on plant growth or 32P dynamics was detected. Since the earthworms were separated from the root zone, potential direct effects of earthworms on root colonisation by VAM were precluded.
In the present study we investigated the effects of earthworms (Aporrectodea caliginosa) and VAM (Glomus intraradices) on both P and N uptake from soil and organic residues by P. lanceolata and the subsequent changes in plant growth, plant chemistry (primary and secondary metabolites), and host quality for aphid herbivores (M. persicae). Phytosterols (primary metabolites) and iridoid glycosides (secondary metabolites) were studied as potential determinants of aphid performance. Iridoid glycosides are important secondary metabolites in P. lanceolata; their deterrent effects on generalist herbivores are well documented (Bowers & Puttick, 1988; Puttick & Bowers, 1988). Phytosterols are precursors of insect hormones and must be taken up with diet by insects that are unable to biosynthesize them directly (Svoboda et al., 1994).
Materials and Methods
Experimental containers consisted of PVC tubes (height 25 cm, diameter 10 cm) closed at the bottom by lids. The containers were equipped with ceramic lysimeters, which were connected via a hose system to a vacuum pump to allow drainage of the soil under seminatural conditions (−200 to −500 hPa).
A nutrient-poor mineral soil from a cultivated meadow (Rossberg near Darmstadt, Germany) was used in the experiment (Wurst et al., 2003). The soil was sieved through a 1 cm mesh and autoclaved (121°C; 2 h).
Seeds of Lolium perenne (L.) (Conrad Appel, Darmstadt, Germany) were sown in pots (95 × 11 × 14 cm) in a sandy soil (Flughafen Griesheim, Hessen, Germany) in a glasshouse (16 h light, night/day temperature 18/20°C). Plants were labelled 20 d after sowing by spraying with 143 ml 15N2 urea solution [250 mg 99 atom%15N2 urea (Isotec Inc., Miamisburg, USA) in 500 ml distilled H2O plus 1 ml 30% Brij 35 (Skalar Chemical, Breda, Netherlands)] per day for 21 d (Schmidt & Scrimgeour, 2001; Wurst et al., 2003).
The VAM inoculum consisted of culture substrate mixed with Glomus intraradices (Schenck & Smith) hyphae and spores (Isolat 150, Dr C. Grotkass, Institut für Pflanzenkultur, Schnega, Germany).
Endogeic earthworms, Aporrectodea caliginosa (Savigny), collected at the Jägersburger Wald (Darmstadt, Germany), were transferred three times into fresh autoclaved soil to minimise contamination of the experimental soil with naturally occurring mycorrhizal propagules. This earthworm species is among the most abundant earthworm species of agriculture systems and gardens (Edwards & Bohlen, 1996).
Myzus persicae (Sulzer) were cultured on Brassica oleracea (L.) before they were transferred to the experimental plants. All individuals belonged to one clone (from Brooms Barn, UK) reared on B. oleracea. The aphid culture was kept in a climate chamber (14 h light, 20°C).
Seeds of Plantago lanceolata (L.) (Conrad Appel, Darmstadt, Germany) were sown on wet filter paper in Petri dishes, watered with distilled H2O and placed in the glasshouse. Germinated seedlings were transplanted into seedling trays filled with the autoclaved soil 7 d after sowing.
On 13 March 2002, 28 experimental containers were set up in the glasshouse. The containers were filled with 1000 g (fresh weight) autoclaved soil. Then, 50 ml soil suspension [280 g fresh soil dispensed in 280 ml distilled H2O from which 100 ml was filtered through a 25 µm mesh with 1400 ml distilled H2O] was added to each container to inoculate the autoclaved soil with microorganisms. Four weeks later 400 g autoclaved soil mixed with 0.4 g autoclaved L. perenne roots (unlabelled), 0.1 g autoclaved L. perenne roots (11 atom%15N) and 60 g autoclaved VAM inoculum were added to half of the containers. The other half of the containers was treated in the same way except that the VAM inoculum was not autoclaved (‘mycorrhiza treatment’). One P. lanceolata plant with 2–3 leaves (except the cotyledons) was planted in each container. All containers received 50 ml VAM washing filtrate [140 g VAM inoculum suspended in 280 ml distilled H2O from which 100 ml was filtered through a 25 µm mesh with 1400 ml distilled H2O] to correct for differences in microbial communities (bacteria, fungi, protozoa) between the ‘mycorrhiza’ and the ‘nonmycorrhiza’ treatment. One week later two specimens of A. caliginosa were placed in half of the containers (‘earthworm treatment’). The experimental containers were watered with 50 ml distilled H2O every second day during the first week, then daily. The containers were rearranged randomly every 2 wk.
The performance of the aphid herbivores were tested in a ‘clip cage experiment’. In wk 3 of the experiment two adult aphids from the M. persicae culture were placed each in one clip cage (height 2 cm, diameter 4 cm) on two intermediately aged leaves of each P. lanceolata plant. The number of offspring was counted 7 d later (wk 4). The oldest nymph remained in the clip cage and the time until it started to reproduce was reported.
On 8 July (wk 10), the plants were harvested. Plants were cut at ground level, separated into inflorescences and leaves, freeze dried and weighed. Roots were washed, dried at 100°C for 72 h and weighed. During the root washing procedure earthworms were collected, counted and weighed.
For the assessment of VAM colonisation c. 1.5 g fresh root samples were cleared with 20 ml 1 N KOH (water bath, 80°C, over night). The KOH was decanted and the samples were washed twice with distilled H2O. Then, 10 ml 3.7% HCl and 1–2 drops of ink (Quink, Parker Permanent Blue, Germany) were added. After 2 h the root samples were transferred into Petri dishes and discoloured with lactic acid : H2O (bidest.) (1 : 1). The VAM colonisation was estimated using the gridline intersection method (Giovannetti & Mosse, 1980).
Nitrogen and carbon Freeze dried leaf samples were ground to a powder and c. 1 mg was weighed into tin capsules. Isotope ratio 15N : 14N and total C were measured by an elemental analyser (NA 1500, Carlo Erba, Milan, Italy) coupled with a trapping box (type CN, Finnigan, Bremen, Germany) and a mass spectrometer (MAT 251, Finnigan, Bremen, Germany). Atmospheric nitrogen served as base for δ15N calculation and acetanilide (C8H9NO, Merck, Darmstadt, Germany) as internal standard (Reineking et al., 1993). The 15N atom% excess was calculated by subtracting the natural background (0.365) from the measured 15N atom% in the plants.
Phosphorus After chemical pulping, the phosphorus concentration of the leaves was determined photometrically by a molybdate blue method (Chapman & Pratt, 1961).
Iridoid glycosides Of each ground, freeze-dried green leaf sample (four replicates per treatment; n = 16), 100 mg were extracted overnight in methanol (95%). Phenyl-beta-d-glucopyranoside solution (25 µl; 10 µg µl−1 in 95% methanol) was added as internal standard, then the supernatant was filtered out and discarded and the extract was evaporated to dryness. After partitioning between water and ether, the ether layer was discarded and the water layer (that contains mainly iridoid glycosides and sugars) evaporated to dryness. An aliquot was derivatized with Tri-Sil Z (Pierce Chemical Company, Rockford, IL, USA) and injected into a gas chromatograph (Gardner & Stermitz, 1988; Stamp & Bowers, 2000).
Phytosteroles Green leaves were ground to a powder and 0.5 g of each sample (four replicates per treatment; n = 16) were dissolved in 20 ml of solvent (10 m KOH, 96% ethanol (1 : 5; v/v%) and 0.3% pyrogallol as antioxidant) in a water bath (80°C) for 2.5 h. Cholesterol solution (50 µl; 5 mg ml−1 in chloroform) was added as an internal standard. Phytosterols were extracted by washing twice with 10 ml hexane and evaporated to dryness. After dissolving in 1.5 ml hexane, the extracts were transferred into auto-sampler vials and dried overnight in a thermo-block (50°C). The residual was dissolved with 240 µl N′-N′-dimethylformamide, and 60 µl bistrimethylsilyltrifluoracetamide (BSTFA) was added for methylation (70°C for 10 min). Samples were then injected into a gas chromatograph (Dugassa-Gobena et al., 1996).
Data were analysed by factorial analysis of variance (anova) in a general linear model (GLM; Statistica, Statsoft 2001). The factors were mycorrhiza (‘mycorrhiza’) and presence of earthworms (‘earthworms’). The distribution of errors (Kolmogorov-Smirnov one-sample test) and the homogeneity of variances (Levene test) were inspected and the data were log-transformed if necessary to match the prerequisites for anova.
At the end of the experiment 39% of the roots of plants inoculated with G. intraradices were colonised by mycorrhiza. No mycorrhizal colonisation was detected in roots of the control plants (‘without mycorrhiza’). Earthworms did not affect the colonisation of roots by G. intraradices (F[1,12] = 1.72; P = 0.214).
At harvest 26 of the initially added 28 earthworms were recovered. Total earthworm biomass decreased on average by 34% during the course of the experiment. This decrease was independent of VAM presence (F[1,12] = 2.46; P = 0.142).
Inoculation with G. intraradices caused a decrease in root biomass of P. lanceolata by 16% (F[1,24] = 6.86; P = 0.015; Fig. 1). The amounts of total nitrogen and 15N excess (i.e. 15N uptake from the added litter) in the roots were not affected, but root nitrogen concentration (% w/w) increased from 0.65% to 0.72% in the presence of mycorrhiza. VAM increased root carbon concentration (% w/w) from 39.28% to 40.81%. VAM increased total amount of phosphorus in the roots by 35% and the phosphorus concentration (% w/w) from 1.04% to 1.67% (Fig. 2; Table 1).
Table 1. anova table of F-values on the effects of mycorrhiza (Glomus intraradices) and earthworms (Aporrectodea caliginosa) on total amounts and concentrations of P, N, 15N excess and C in leaves and roots of Plantago lanceolata
VAM did not affect the shoot biomass of P. lanceolata, but reduced the amount of total nitrogen in the leaves by 15% (Fig. 3; Table 1). 15N excess was not affected by VAM. Leaf N concentration was on average 1.05% (w/w) and not affected by VAM. Leaf carbon concentration decreased from 42.15% to 41.00% when only VAM was present, and tended to decrease to 41.59% when only earthworms were present (significant mycorrhiza–earthworm interaction; Table 1). Inoculation with G. intraradices increased the total amount of P in the leaves by 75% (Fig. 2; Table 1) and the P concentration (% w/w) from 0.92% to 1.80%. The phytosterol concentration in the leaves was not affected by VAM (F[1,12] = 0.06; P = 0.811), but correlated positively with leaf nitrogen concentration (F[1,14] = 14.32; P = 0.002; R2 = 0.506; Fig. 4).
Presence of earthworms neither affected root biomass (Fig. 1), nor the amount of total N, 15N excess and the concentration of N in the roots. However, earthworms decreased the proportion of 15N (atom% excess) in roots by 20%, but only in the absence of mycorrhiza (significant mycorrhiza–earthworm interaction; Fig. 5; Table 1). Carbon concentration, total amount of phosphorus and P concentration in roots were not affected by earthworms.
By contrast to the roots, earthworms increased total shoot biomass by 15% (F[1,24] = 4.69; P = 0.041) (Fig. 1). In the leaves, earthworm presence decreased the proportion of 15N (atom% excess) by 14%, and this effect tended to be stronger in the absence of mycorrhiza (−24%; Fig. 4; Table 1). In the presence of earthworms the concentration of catalpol in the leaves was reduced (F[1,12] = 33.15; P < 0.001; Fig. 6). The phytosterol concentration in the leaves was not affected by earthworms (F[1,12] = 1.85; P = 0.199).
The reproduction of M. persicae was not affected by VAM (F[1,24] = 0.55; P = 0.467) or earthworms (F[1,24] = 0.55; P = 0.467). The development time of the nymphs tended to be delayed when only VAM or earthworms were present, but was accelerated in the presence of both VAM and earthworms (F[1,21] = 6.84; P = 0.016 for the interaction mycorrhiza × earthworms; Fig. 7). The development time of aphids decreased with increasing carbon concentration in the leaves (F[1,23] = 4.63; P = 0.042; R2 = 0.167).
No direct interaction between earthworms and VAM was found. Earthworm activity did not reduce VAM root colonisation. Since the effects of earthworms on VAM root colonisation were reported to be density-dependent (Pattinson et al., 1997), a higher number of earthworms might have decreased the infectivity of VAM.
Similar to the study of Tuffen et al. (2002) earthworms, but not VAM, enhanced plant growth. Earthworms increased shoot biomass of P. lanceolata and led to a preferential plant uptake of soil N compared with litter N in the absence of VAM. Scheu (1994) proposed that there is an earthworm mobilizable pool of N in soil. Earthworms break up soil aggregates during the gut passage and thus physically protected N is mobilized. The results of the present study indicate that A. caliginosa promoted plant growth mainly via mobilisation of soil N. However, the mycorrhizal plants took up less of the earthworm-mobilized N and thus their 15N : 14N isotope ratio remained unchanged. The total amount of N in the leaves was also decreased in the mycorrhizal plants. By contrast, plant phosphorus concentration was doubled in mycorrhizal plants compared with nonmycorrhizal plants.
VAM are known to increase plant phosphorus content (Smith & Read, 1997; Goverde et al., 2000), and decreased plant N concentration in association with VAM has been documented (Gange & West, 1994; Gange & Nice, 1997). In the present study leaf N concentration remained unchanged, but the total amount of N in the leaves decreased in symbiosis with G. intraradices. The smaller root system of the mycorrhizal plants presumably hampered plant N uptake. Although G. intraradices has been reported to absorb N and effectively deplete soil for inorganic N, it transports only part of it to the host plant (Johansen et al., 1992). Generally, uptake of N by VAM hyphae does not lead to an increased plant N content (Smith & Read, 1997). In many studies on N uptake by VAM, roots were separated physically from the hyphal compartment where the 15N-labelled source was supplied (Ames et al., 1983; Frey & Schüepp, 1992; Johansen et al., 1992; Tobar et al., 1994; Hodge et al., 2001) and thus potential competition between roots and VAM hyphae for the 15N was precluded. When both roots and VAM hyphae had access to the 15N source (Hodge et al., 2000), the mycorrhizal symbiosis did not result in an increase of plant 15N uptake. Since the mycorrhizal plants were more depleted in N than the nonmycorrhizal plants in the present study, we assume that G. intraradices competed with the roots for N. Consistently, Hawkins et al. (2000) documented that hyphae of G. intraradices took up less 15N when carrot roots were present than in the hyphal compartment without roots.
Earthworm presence reduced the catalpol concentration in P. lanceolata leaves. As far as we are aware, this is the first documentation that decomposers can affect plant secondary metabolites. Increasing fertilizer has been reported to decrease concentrations of iridoid glycosides in P. lanceolata (Fajer et al., 1992; Jarzomski et al., 2000). By enhancing N availability and plant growth, earthworms might have produced physiological responses comparable with fertilization. However, in a former study earthworms did not affect iridoid glycoside concentrations in P. lanceolata, despite increasing plant growth and N uptake (Wurst et al., 2003; Wurst et al., 2004). In the present study, inoculation with G. intraradices did not affect the iridoid glycosides, although higher aucubin and catalpol levels in mycorrhizal compared with fungicide-treated P. lanceolata plants have been documented (Gange & West, 1994). The inconsistency of the results indicates that effects of earthworms and VAM on plant chemistry depend on soil characteristics and experimental organisms. The sterilisation of the soil in the present study changed soil characteristics (Alphei & Scheu, 1993), and likely affected interactions between soil organisms and the plants. Since mycorrhizal effects depend on the genotypes of the organisms involved (Klironomos, 2003), it is not surprising that effects of a single VAM isolate differ from the effects of a whole mycorrhizal community. Both earthworms and VAM have the potential to change plant defensive chemistry (Gange & West, 1994; Dugassa-Gobena et al., 1996; Wurst et al., 2004) and herbivore performance. However, more investigations are necessary to uncover the mechanisms responsible for these effects and the environmental conditions under which they occur.
The phytosterol concentration in P. lanceolata was not affected by earthworms or VAM, but increased with increasing N concentration of the leaves. This correlation has been documented before (Wurst et al., 2004) though in the latter study earthworms increased the phytosterol concentration of P. lanceolata when grass residues were distributed homogeneously into the soil. Note that we only investigated effects of earthworms and VAM on plants with aphids (there were no control plants without an aphid treatment). Possible combined effects of soil organisms and aphids on plant performance can thus not be excluded. However, since a maximum of two aphids were clip-caged per plant, the effect of aphids was probably small.
Earthworms and VAM combined accelerated aphid development. Nymphs of M. persicae reached earlier maturity when both earthworms and VAM were present, but the development time tended to be delayed when only VAM or earthworms were present. Since the phytosterol concentration was not affected by earthworms or VAM in the present study, it is unlikely that the accelerated development of M. persicae was caused by changes in plant phytosterol concentration. Presumably, the earthworm-mediated increase in plant growth and N uptake and the VAM-mediated increase in plant P uptake synergistically increased host quality for the aphids. Gange et al. (1999) reported a reduced development time and increased fecundity of M. persicae on P. lanceolata in symbiosis with G. intraradices at low P levels. In the present study the reproduction of M. persicae was not affected by the presence of earthworms or VAM. This is in contrast to former studies where earthworms (Scheu et al., 1999; Wurst & Jones, 2003; Wurst et al., 2003) and VAM (Gange & West, 1994; Gange et al., 1999) affected the reproduction of M. persicae. Since the soil was autoclaved in the present study which is known to mobilize nutrients (Alphei & Scheu, 1993), nutrients may not have limited plant growth. Consequently, the effects of earthworms and mycorrhiza on plant and herbivore performance may have been less pronounced than in nonautoclaved soil.
In conclusion, earthworms and VAM significantly affected the performance of P. lanceolata, but these effects were mainly independent of each other. Earthworms increased shoot biomass, and VAM reduced root biomass. As expected, the content of P in leaves and roots was enhanced in the presence of mycorrhiza. However, the mycorrhizal association reduced the amount of N in the leaves suggesting that G. intraradices competed with the roots for available N. Earthworms reduced the catalpol concentration of P. lanceolata documenting the potential of decomposers to influence concentrations of plant secondary metabolites. Earthworms and mycorrhiza combined accelerated the development of M. persicae. Further investigations are necessary to understand under which soil conditions earthworms and VAM change plant chemistry (primary and secondary metabolites) and affect herbivore performance.
We thank Dr C. Storm and U. Lebong (Technische Universität Darmstadt) for help with the phosphorus analysis. This study was sponsored in part by a grant to S. Wurst from the ‘Hessisches Ministerium für Wissenschaft und Kunst’.