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Effects of six grassland plant species on soil nematodes: A glasshouse experiment
ARTICLE IN PRESS
Soil Biology & Biochemistry 40 (2008) 906–915 www.elsevier.com/locate/soilbio
Effects of six grassland plant species on soil nematodes: A glasshouse experiment Maria Viketoft Department of Ecology, Swedish University of Agricultural Sciences, P.O. Box 7044, SE-750 07 Uppsala, Sweden Received 26 March 2007; received in revised form 31 October 2007; accepted 5 November 2007 Available online 3 December 2007
Abstract The effects of six individual plant species on the abundance and composition of nematode communities were studied in a glasshouse experiment during 16 weeks. The effect of the presence of plants, the correlation between nematode abundance and plant biomass, the response of plant-feeding nematodes and other nematode groups to different plant species was examined and also whether the effect differed between plant species within a plant functional group. The total number of nematodes increased during the study period in all treatments, although in some treatments, the increase levelled off after 8 or 12 weeks. The identity of the plant species affected both the total abundance of nematodes and the nematode community composition. The number of bacterial-feeding nematodes was greatest under grasses and legumes and was positively correlated with shoot biomass and negatively with root biomass. The response of the plantfeeding nematodes, which differed in abundance under both the investigated legume and the forb species, suggests that the identity of the plant species is more important than the plant functional group. A possible explanation could be related to differences in plant secondary metabolites. Despite some differences in the nematode species pool, the effects of plant species appear quite consistent between the present glasshouse study and previous field experiments. r 2007 Elsevier Ltd. All rights reserved. Keywords: Achillea millefolium; Festuca ovina; Nematode community composition; Nematode diversity; Nematode feeding groups; Phleum pratense; Rumex acetosa; Trifolium hybridum; Trifolium repens
1. Introduction Plant species identity and the composition of the plant community are important determinants of decomposer function and community composition in soil (Wardle, 2005). For microorganisms, this has been shown both in terms of microbial biomass and activity in microcosms with soil from grasslands (Bardgett et al., 1999; Innes et al., 2004) as well as microbial community composition in microcosms (Grayston et al., 1998; Marschner et al., 2001), field experiments (e.g., Smalla et al., 2001; Kowalchuk et al., 2002) and in a naturally mixed spruce-birch stand (Saetre and Ba˚a˚th, 2000). Soil fauna are also affected by individual plant species. In both field and pot experiments, nematodes responded to the identity of different grassland species, with plantTel.: +46 18672526; fax: +46 18672890.
E-mail address: [email protected] 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.11.006
feeding and microbial-feeding nematodes (bacterial and fungal feeders) being the groups most clearly influenced by plant species identity (Wardle et al., 2003; De Deyn et al., 2004; Viketoft et al., 2005). Collembolans have been shown to respond to grass and legume species in pot and field experiments with an assembly of grassland species (Salamon et al., 2004; Milcu et al., 2006), and both mites and collembolans responded to plant species in high Arctic heath vegetation (Coulson et al., 2003). In addition, mites have also been shown to be affected by different plant species used in agroforestry (Badejo and Tian, 1999). Plants affect the belowground community through differences in the amount and quality of resources allocated to the soil, in the extent to which they deplete nutrients and water from the soil, in the chemical composition of the litter produced and through the formation and modification of habitats (Wardle, 2002). For example, many legumes differ from other forbs in their ability to fix atmospheric nitrogen and therefore increase
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the amount of nitrogen in the soil, principally in the form of nitrate (Palmborg et al., 2005). They also produce more high-quality litter that is more easily decomposed (Scherer-Lorenzen et al., 2003). Previous studies on the effect of plant species on nematodes have shown that, in general, plant feeders seem to be most abundant under grasses and have a lower presence under some forbs, while microbial feeders show varying responses to the investigated plant species in different studies (Wardle et al., 2003; De Deyn et al., 2004; Viketoft et al., 2005). In addition, different feeding types of plant feeders, e.g., ecto- and endoparasites, have been shown to respond differently to grasses and forbs (De Deyn et al., 2004). The aim of the present study was to examine, in a glasshouse pot experiment, how six plant species, belonging to three plant functional groups (grasses, legumes and forbs), affect total nematode abundance, nematode community composition and the response of individual nematode genera. A defaunated soil from an experimental grassland that was reinoculated with both microflora and nematodes was used. Thus, the effect of plant species during the establishment phase of nematode communities was evaluated under controlled conditions. In addition, a treatment containing no plants was included, top-down effects by micro- and mesofauna other than nematodes were removed and soil characteristics (e.g., pH, inorganic nitrogen), missing in other studies (Wardle et al., 2003; De Deyn et al., 2004; Viketoft et al., 2005), were determined to relate to the nematode data. Specifically, the following hypotheses are addressed: 1. Low numbers of nematodes should be found in the treatment with no plants, due to the lack of plantderived resources. 2. There is a positive correlation between total nematode abundance and plant productivity (Yeates, 1987). 3. Plant-feeding nematodes should show the strongest response to the identity of plant species because they feed directly on them, although the speed of the response between bacterial- and plant feeders likely differs because of different generation times. Based on previous studies, plant feeders are also expected to be most abundant under grasses. 4. Legumes, due to their N-fixing ability, will affect the nitrogen levels in the soil and hence the bacterial community. This should affect the nematode community composition by promotion of bacterial-feeding nematodes, especially Rhabditis and Panagrolaimus. 5. Different plant species within a functional group will have quite different influences on the nematodes (Viketoft et al., 2005). The division into plant functional groups was based on growth form and N-fixing ability, but the plant species may contain different secondary substances. The nematode fauna under monocultures of the plant species used in this study have previously been investigated
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after seven growing seasons in an experimental grassland in northern Sweden (Viketoft et al., 2005). Although the present study is a 16-week experiment, where plants were getting established and a limited number of nematodes were inoculated, it had higher replication and a treatment containing no plants compared to the field experiment. A comparison between these studies may illustrate interesting aspects of the interactions between plants and soil communities. 2. Materials and methods 2.1. Site and soil In November 2004, soil was collected to 10 cm depth from the experimental field of the BIODEPTH project of the Swedish University of Agricultural Sciences, Umea˚, northern Sweden (631450 N, 201170 E). The soil is classified as silt loam (4.1% clay, 57.9% silt, 38.0% fine sand) and was taken from an area between the experimental plots, which is dominated by Phleum pratense and Trifolium repens. The soil was stored at 4 1C until further processed in February 2005. The vegetation was then removed and the soil was passed through a 4-mm sieve. In order to defaunate the soil, the sieved soil was subjected to freezing (20 1C) for at least 48 h followed by heating in a warming cupboard to 70 1C for 24 h. Then, the soil was subjected to another cycle of freezing and heating. Samples were taken from the defaunated soil to see if the procedure had been successful. A subsample of the defaunated soil was stored frozen until analysed for soil chemistry: pH, dry matter, organic matter content, KCl-extractable NH4–N and NO3–N, and plant-available P (ammonium-lactate extract). The remainder of the soil was stored at room temperature for 2 days until added to the pots. 2.2. Experimental set-up The glasshouse experiment consisted of 75 black plastic pots (height 9.5 cm, diameter 12 cm, drainage holes in the bottom were covered with fibre cloth) that were first filled with 250 g sand and then 700 g of the defaunated soil (moisture content 10%). The sand had been washed through a 0.3-mm sieve with tap water to remove all organic material and then dried at 120 1C for 20 h. Thereafter, the pots were reinoculated with microflora by adding 15 ml of a soil suspension made from the original faunated soil. No specific Rhizobium inoculum was added. The suspension was made by mixing 1 kg of sieved soil in 2 l of MilliQ-water, which was then centrifuged at 2000 rpm for 5 min. The centrifugation was repeated twice. The soil suspension was checked for presence of nematodes but there were none. The pots were then sown with seeds of P. pratense L. (cv. Jonatan), Festuca ovina L. (cv. Fertalia, Dumon, Belgium), Trifolium hybridum L. (cv. Stena), T. repens L. (cv. Undrom) (obtained from Svalo¨f Weibull AB,
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Svalo¨v, Sweden), Achillea millefolium L. or Rumex acetosa L. (obtained from Pratensis AB, Vislanda, Sweden). About 20–25 seeds were sown in each pot and there were 10 pots for each plant species. In addition, there were 10 pots without plants and 5 control pots (without plants and receiving no nematodes), giving a total of 75 pots. The pots were randomly placed in 15 rows in the glasshouse and covered with plastic. After 2 weeks when all plant species had germinated, the plastic was removed and additional seeds were added to pots where less than 15 seeds had germinated. One week later, the pots were thinned to 11 seedlings per pot. However, the seedlings of F. ovina were still very small and were not thinned until two additional weeks had passed (five since the original sowing). In the other pots, seedlings that had emerged since the last thinning were also removed. At time of the first thinning, soil samples were taken from the control pots to ensure that the pots were still nematode free. Then, a nematode inoculum was made from the original faunated soil (i.e. collected in November from a P. pratense/T. repens dominated area, stored till February, sieved over 4-mm sieve) by extraction with the Whitehead tray method (Whitehead and Hemming, 1965) for 24 h. This extraction method was used because of the great amount of soil that needed to be extracted. The suspension was concentrated by decanting and the final nematode concentration was determined to 35 nematodes ml1. All pots except the control pots were watered with 15 ml inoculate, i.e. ca. 525 nematodes ( ¼ week 0, mid-March 2005). The control pots received the same amount of deionised water. To characterise the added nematode fauna, samples from the inoculate were taken and nematodes were identified (see the following text). The conditions in the glasshouse were set at 18 1C for 16 h with supplementary lighting (day, HPI-T lamps, 400 W, Philips) and at 15 1C for 8 h with no supplementary lighting (night). The relative humidity was 60%. To counteract heating of the glasshouse by the sun and keep the soil temperature around 22 1C, the temperature in the glasshouse was lowered to 16 1C (day) and 13 1C (night) after 4 weeks, and the glasshouse chamber was changed in mid-June to avoid soil temperatures above 30 1C. To equalise the amount of radiation for each pot, pots were rearranged weekly by circulating them within and between the rows. The pots were watered as required (usually every second or third day) with deionised water. Twelve weeks after sowing ( ¼ experimental week 8), the plants showed signs of nutrient deficiency and consequently after this the pots received equal amounts of a complete fertiliser (Wallco, N:P:K 51:10:43, Cederroth International AB), with a total of 2 mg NH4 and 3 mg NO3 added per pot and occasion. This was repeated once every second week for the rest of the experiment. Thrips were discovered in connection with the first thinning, and predatory mites, Amblyseius, were added twice to all pots to control them.
2.3. Nematode sampling Nematode samples were taken at experimental weeks 4, 8, 12 and 16 with a cork borer (diameter 1.5 cm). One sample was taken in each pot at every occasion, avoiding holes from previous samplings. Samples were taken down to the sand and the soil was transferred to plastic tubes, stored at 4 1C and nematodes were extracted within 4 days with a modified Baermann method (Viketoft et al., 2005). This extraction method was used because of the small size of the soil samples and the high recovery of this method (Sohlenius, 1980). Following the extraction, the water content in the soil samples was determined by drying in 60 1C for 24 h. The dried soil was rewetted and returned to the hole in the pot. Total abundance of nematodes was estimated in suspensions from each extraction under low magnification (50 ) and expressed as g1 dry soil weight. In each suspension, the first 100 nematodes were identified to genera and in some cases to species level under higher magnification (200 ). Additional genera in the suspension were marked as present. The nematodes were placed into different feeding groups according to Yeates et al. (1993). Nematodes feeding on epidermal cells or root hairs was represented as plant-associated nematodes. 2.4. Plant growth and soil chemistry Each pot was destructively harvested at experimental week 16 (early July 2005). Plant shoots were first removed, dried at 60 1C for 24 h and weighed. In the shoot biomass for T. repens the weight of stolons was not included. The soil was then divided in halves, one for estimation of root biomass and the other for determination of soil chemistry. Roots were washed over a sieve (0.5 mm), dried overnight at room temperature and then at 60 1C for 24 h and weighed. The soil for determination of soil chemistry was first frozen (20 1C) and after thawing sieved (4 mm) and analysed for pH, dry matter, organic matter content as loss on ignition, and KCl-extractable NH4–N and NO3–N. Soil pH was determined in a 1:5 soil–water slurry. The dry matter was determined by drying the soil at 105 1C, and the soil was thereafter heated to 550 1C to determine the organic matter content. To determine the inorganic N pools, the thawed samples were extracted with 2 M KCl for 2 h and then the extracts were analysed for nitrate and ammonium using flow injection analysis (Tecator FIA 5010, FOSS Tecator, Ho¨gana¨s, Sweden). 2.5. Data treatment and statistical analyses The total nematode abundance was ln(x)-transformed and analysed with a repeated-measures ANOVA (procedure mixed) using autoregressive order 1 covariance, using all four samplings. Effects with po0.0008 were considered significant when accounting for multiple comparisons in a sequential Bonferroni test (21 4 ¼ 84 comparisons).
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The abundance of the different feeding groups and individual genera present in at least half of the plots (Paratylenchus, Aphelenchoides and Acrobeloides) were analysed with a repeated-measures ANOVA using only the second and fourth sampling. The type of covariance structure used was based on the model-fit criteria computed by the statistical program (unstructured for plant feeders, Paratylenchus, bacterial feeders and Acrobeloides; autoregressive order 1 for fungal feeders and Aphelenchoides). Abundance data for the feeding groups and individual genera was ln(x+1)-transformed prior to analysis. Due to a high number of zeros for plant feeders and Paratylenchus, the treatments with no plants and T. hybridum were excluded from the repeated-measures analysis, after confirming that they were significantly different from the other plant treatments with Wilcoxon’s rank test. Effects with po0.002 were significant when accounting for multiple comparisons in sequential Bonferroni tests (21 2 ¼ 42 comparisons, 10 2 ¼ 20 for plant feeders and Paratylenchus). To characterise the nematode fauna under the different plant species, several indices were calculated for the second and fourth sampling: number of taxa (S), Shannon index (H0 ¼ Spi ln pi, where pi is the proportion of the ith species), evenness index (J0 ¼ H0 /(ln S)) and the complement of Simpson’s index (1D, D ¼ Sp2i ) (Magurran, 2004), nematode channel ratio (NCR ¼ B/(B+F), where B and F are abundances of bacterial and fungal feeders, respectively) (Yeates, 2003), maturity index (MI) and plant parasite index (PPI) (Bongers, 1990). However, the MI and PPI indices were exactly 2 for a great majority of the samples, and hence it is not relevant to investigate differences between the plant species, because the values that deviate from 2 are given unproportionally large
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significance for the test statistic. The other indices were analysed with a repeated-measures ANOVA with unstructured covariance, except for S for which autoregressive order 1 covariance was used. NCR was arcsine-transformed prior to analysis and effects were considered significant when po0.0014 (sequential Bonferroni tests applied, 21 2 ¼ 42 comparisons). The plant and soil data was analysed with one-way ANOVA (Tukey’s multiple comparison tests (po0.05)), excepting nitrate and ammonium which were analysed with Kruskal–Wallis tests (pairwise tests: sequential Bonferroni tests applied, po0.008, 21 comparisons). The biomasses were square-root transformed. Relations between plant biomass production (shoot and root biomass) and nematode abundances at the final sampling (total and of the different feeding groups) were tested with Pearson correlations. This was also done when analysing relations between soil characteristics and the nematode abundances (nitrogen data with Spearman rank correlation). As the main interest was in finding variables that might aid in the interpretation of the results rather than testing explicit hypotheses, Bonferroni corrections were not used in these cases. All statistical tests were performed using SAS for Windows 9.1. 3. Results 3.1. Plant growth and soil chemistry Shoot and root biomass differed significantly between the plant treatments (Table 1). In general, the legumes had the highest shoot biomass and forbs the lowest. No such pattern among plant functional groups was found for the root biomass. Instead, R. acetosa clearly had the greatest
Table 1 Plant and soil characteristics for the defaunated soil (before start of the experiment), the control pots (microflora inoculate, no plants, no nematode inoculate) and the plant treatments after 16 weeks Treatment
Shoot biomass (g)
Root biomass (g/kg soil)
Organic matter (%)
pH
Defaunated soil Control
– –
– –
4.65 (0.02) 4.75 (0.005)
5.80 (0.05) 5.63 (0.02)
No plants Phleum pratense Festuca ovina Trifolium hybridum Trifolium repens Achillea millefolium Rumex acetosa
– 4.11 (0.06) c 4.19 (0.08) bc 4.48 (0.06) b
– 9.1 (0.4) cd 10.4 (0.6) bc 11.6 (0.6) b
4.68 4.84 4.85 4.90
5.67 5.77 5.71 5.62
5.70 (0.09) a 2.29 (0.06) d
7.7 (0.4) d 8.6 (0.4) cd
2.01 (0.04) e
F-values/w-values
457.91
(0.02) (0.03) (0.02) (0.02)
d bc bc ab
(0.03) (0.03) (0.02) (0.03)
ab a ab b
NH4 (mg/kg soil)
NO3 (mg/kg soil)
73.8 (0.6) 1.28 (0.10)
0.74 (0.05) 6.13 (1.68)
1.42 0.69 0.78 12.38
(0.09) (0.15) (0.12) (1.05)
b c c a
4.46 0.006 0.018 0.089
(0.49) a (0.002) d (0.006) cd (0.015) b
4.96 (0.03) a 4.88 (0.01) abc
5.49 (0.04) c 5.64 (0.02) b
10.86 (0.52) a 1.12 (0.11) bc
0.003 (0.001) d 0.025 (0.004) c
16.0 (1.0) a
4.80 (0.02) c
5.67 (0.03) ab
0.86 (0.08) c
0.024 (0.005) c
25.08
18.56
10.33
51.67
54.77
Shoot and root biomass (dry weight), organic matter content (loss on ignition), pH (H2O), NH4 (2 M KCl) and NO3 (2 M KCl). Arithmetic means (S.E.), n ¼ 10 (n ¼ 6 for defaunated soil, n ¼ 5 for control). Plant-available P (ammonium-lactate extract) was only determined for the defaunated soil, and was 4.95 mg/100 g soil. F-values are from ANOVAs and w-values from Kruskal–Wallis tests (NH4 and NO3). Different letters indicate significant differences between plant treatments. po0.001.
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root biomass. The presence of plants affected the soil during the experimental period, which is demonstrated by the pots without plants having the highest nitrate levels and the lowest organic matter content (Table 1). The no-plant treatment also had the highest water content (data not shown). The plant species that differed most from the others were the legumes (Table 1). 3.2. Nematodes The total number of nematodes increased during the study period in all treatments, although in some treatments, the increase levelled off after the second or third sampling (Fig. 1). There was a significant interaction effect between plant and time for the total number of nematodes. The largest differences were found at the first sampling (4 weeks after nematode inoculation) and at the final sampling (16 weeks) (Fig. 1). The abundance of nematodes followed almost the same pattern in P. pratense, R. acetosa and T. repens. At the first sampling, they had low abundances, but at the final sampling, they had the greatest. The opposite pattern was found for A. millefolium and the unplanted treatment with the greatest abundances at the first sampling and lower abundances than most other treatments at the final sampling. T. hybridum and F. ovina both had an intermediate position but diverged at the final sampling when the abundance in T. hybridum decreased. Compared to the inoculate, a number of nematode genera (e.g., Acrobeles and Alaimus) were never recorded in suspensions extracted from the pots. The most abundant of the surviving genera were Paratylenchus, Aphelenchoides and Acrobeloides (Table 2). The abundance of the plantfeeding Paratylenchus was lower in the no-plant treatment and in T. hybridum than in all other treatments at both experimental week 8 and at the final sampling at week 16. In addition, at the final sampling, the abundance in
Ln no. of nematodes / g dw soil
8 aa ab ab bc cd
7 6 5
d a ab ab bc
4 3 2
cde de e
1
A. millefolium was lower than in R. acetosa and T. repens. The abundance of the fungal-feeding Aphelenchoides varied within the treatments, but at the final sampling it was greater in F. ovina than in T. repens. The bacterial-feeding Acrobeloides increased over time in all treatments except no plants. At experimental week 8, the abundance of Acrobeloides was lower in pots planted with R. acetosa and P. pratense than in pots with T. repens, T. hybridum and no plants. At 16 weeks, the treatments formed two groupings: R. acetosa, A. millefolium and the no-plant treatment had lower abundances than the other four plant species. Because the aforementioned genera were so dominant, the responses of the different feeding groups almost mirror the responses of these genera (Table 2). In general, the diversity of the nematode communities was low but affected by plant species (Table 3). At experimental week 8, the only significant difference was recorded with A. millefolium which had more taxa than P. pratense. At the final sampling, the most diverse community was found in pots with A. millefolium, while T. hybridum had the lowest Shannon diversity index and T. repens the lowest number of taxa (Table 3). The community with the most even species-abundance distribution after 16 weeks was found in pots with P. pratense and the least even species-abundance distribution was found in T. hybridum. The NCR showed that there was dominance of bacterial feeders over fungal feeders in soil from all the investigated plant species (Table 3). The community and diversity indices were affected by time (Table 3) and all of them, except NCR, increased during the experimental period. At the final sampling, both shoot and root biomasses were uncorrelated with total abundance of nematodes, plant feeders and fungal feeders. However, there was a strong positive correlation between abundance of bacterial feeders and shoot biomass and a weak negative correlation with root biomass (r ¼ 0.72 and 0.37, respectively, po0.01, n ¼ 60). On the other hand, total nematode abundance was negatively correlated with nitrate and ammonium (r ¼ 0.67 and 0.27, respectively, po0.05, n ¼ 70). Plant feeders and bacterial feeders were negatively correlated with nitrate (r ¼ 0.65 and 0.45, respectively, po0.001), but only plant feeders negatively correlated with ammonium (r ¼ 0.30, po0.05). Fungal feeders were negatively correlated with the amount of ammonium (r ¼ 0.27, po0.05). 4. Discussion
0 -1 0
4
8
12
16
Sampling time (weeks) Fig. 1. Total abundance of nematodes (ln no./g soil dw) over time in soil with no plants ( ), Phleum pratense (J), Festuca ovina (K), Trifolium hybridum (&), Trifolium repens (’), Achillea millefolium (m) and Rumex acetosa (n). ‘‘~’’ represents the inoculate abundance. Different letters indicate significant differences between treatments within sampling dates. Mean arithmetic (S.E.) ¼ 22.27 across treatments.
As hypothesised, the no-plant treatment had the lowest total number of nematodes. However, a correlation between total nematode abundance and plant biomass was not found, but the abundance of bacterial feeders correlated with both shoot and root biomass. Plant-feeding nematodes showed the strongest response to the different plant species but in contrast to the hypothesis they did not have their greatest abundances under grasses. In addition, legumes did not specifically promote bacterial-feeding
Table 2 Abundance (mean no./g soil dw (S.E.), n ¼ 10) of nematode taxa after 8 and 16 experimental weeks of a glasshouse experiment with six different plant species and an unplanted treatment Phleum pratense
Festuca ovina
Trifolium repens
Achillea millefolium
Rumex acetosa
Plant feeders Tylenchorhynchus
8 16 8 16 8 16
x x 0.16 (0.16)
0.14 (0.14) xc 0.14 (0.14) d
x x x x 9.32 (5.02) a 676.45 (127.89) ab
Pratylenchus
10.11 (3.29) a 513.80 (114.70) ab
x x 0.74 (0.34) b 41.36 (25.00) c
x 13.39 (4.61) a 857.26 (140.25) a
0.12 (0.12) x 4.93 (0.66) a 295.90 (65.99) b
x x 2.96 (0.80) a 997.67 (141.94) a
Paratylenchus
Total
8 16
xc 0.28 (0.28) d
9.32 (5.02) a 676.45 (127.89) ab
10.27 (3.34) a 513.80 (114.70) ab
0.74 (0.34) b 41.36 (25.00) c
13.39 (4.61) a 857.26 (140.25) a
5.05 (0.66) a 295.90 (65.99) b
2.96 (0.80) a 997.67 (141.94) a
Plant-associated Filenchus
8 16
0.09 (0.09) 0.60 (0.28)
x 2.73 (2.73)
0.23 (0.15) x
x 1.15 (1.15)
x x
x 5.66 (5.66)
0.12 (0.08) x
Fungal feeders Aphelenchidaea
8 16 8 16 8 16
x 0.34 (0.18) 49.40 (32.76) a 22.03 (7.58) ab
0.97 (0.85) x 3.24 (2.26) a 40.61 (18.54) ab
0.14 (0.14) x 6.80 (3.70) a 35.66 (8.53) a
0.31 1.52 0.84 7.46
0.29 1.61 4.89 4.76
0.75 0.27 4.19 7.36
x x 14.14 (7.15) a 15.54 (4.65) ab
Aphelenchoides Dorylaimellus
Total
8 16
49.40 (32.76) a 22.36 (7.56) a
4.21 (2.26) a 40.61 (18.54) a
6.93 (3.76) a 35.66 (8.53) a
1.15 (0.67) a 8.98 (2.07) a
5.18 (4.62) a 6.37 (2.33) a
4.94 (2.26) a 7.63 (2.06) a
14.14 (7.15) a 15.54 (4.65) a
Bacterial feeders Rhabditis s.l.
8 16 8 16 8 16 8 16 8 16 8 16 8 16 8 16
x 1.29 (0.74) 151.61 (32.51) a 105.94 (11.87) b x 1.76 (0.73)
x x 68.46 (11.24) b 269.98 (31.13) a 0.09 (0.09) 1.44 (0.98)
x
0.80 (0.80) 0.55 (0.55) 141.99 (26.05) a 224.65 (10.37) a 0.07 (0.07) x
x x 146.52 (22.71) a 254.49 (17.34) a x x x
0.48 (0.36) x 89.05 (12.61) ab 115.65 (23.43) b 0.48 (0.21) 1.93 (1.10)
0.20 (0.14) x 51.59 (2.82) b 89.85 (11.35) b 0.31 (0.25) 0.26 (0.26) x
Acrobeloides Cephalobus Eucephalobus Panagrolaimus
x
x
Plectus
x
x x
1.00 (0.54) 8.68 (4.15)
0.10 (0.10) 4.64 (3.09)
Eumonhystera Prismatolaimus
0.93 (0.38) 0.14 (0.14) 2.54 (0.63)
0.11 (0.11) 1.00 (1.00)
x 0.11 (0.11) 2.49 (1.18)
x
0.08 (0.08) x
x x 3.62 (2.00)
x x x
Total
8 16
151.75 (32.51) a 115.31 (12.89) c
68.66 (11.24) b 272.42 (31.55) a
96.12 (7.19) ab 201.18 (31.05) ab
142.86 (25.73) a 225.20 (10.57) a
146.60 (22.68) a 254.49 (17.34) a
91.01 (12.63) ab 129.89 (20.86) bc
52.19 (2.93) b 94.75 (9.93) c
Omnivores Aporcelaimellus
8 16
x x
x
x
x
x
x
x
(0.31) (1.00) (0.58) a (2.14) ab
(0.29) (1.61) (4.33) a (2.13) b
(0.37) (0.27) (2.30) a (1.87) ab
x
93.66 (6.92) ab 198.69 (30.91) a 0.36 (0.36) x
x 1.84 (1.02) x 0.14 (0.14) x
2.84 (1.77)
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x indicates presence of a taxa but at levels too low to be quantified. Different letters indicate significant differences between treatments within sampling dates. Approximately, 525 nematodes were added to each pot at the start of the experiment. a Including Aphelenchus and Paraphelenchus.
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Trifolium hybridum
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Table 3 Nematode indices for the final sampling after 16 weeks H0
S No plants Phleum pratense Festuca ovina Trifolium hybridum Trifolium repens Achillea millefolium Rumex acetosa Repeated-measures ANOVA Plant species Time Plant species time
7.9 6.3 6.5 6.4 6.0 8.0 7.3
(0.5) (0.3) (0.7) (0.5) (0.5) (0.4) (0.4)
F-values 4.44 69.59 1.87
a a a a a a a
(e) (fg) (fg) (fg) (g) (e) (ef)
0.72 0.72 0.74 0.38 0.55 0.83 0.43
J0 (0.07) (0.04) (0.05) (0.07) (0.04) (0.07) (0.07)
6.26 44.88 2.57
a a a b ab a b
0.44 0.72 0.61 0.35 0.66 0.59 0.38
1D (0.02) (0.06) (0.04) (0.06) (0.07) (0.03) (0.03)
4.10 21.73 1.97
bcd a abc d ab abc cd
0.35 0.45 0.44 0.20 0.36 0.47 0.22
NCR (0.04) (0.03) (0.03) (0.05) (0.03) (0.03) (0.04)
6.10 39.19 2.12
ab a a b ab a b
0.86 0.89 0.85 0.96 0.97 0.93 0.87
(0.03) (0.04) (0.03) (0.01) (0.01) (0.02) (0.04)
a a a a a a a
(g) (efg) (g) (ef) (e) (efg) (fg)
3.69 6.21 0.62
S (no. taxa), H0 (Shannon diversity index), J0 (evenness), 1D (Simpson’s index), NCR (nematode channel ratio). Arithmetic means (S.E.), n ¼ 10. Different letters indicate significant differences among plant treatments, po0.0014, sequential Bonferroni tests applied. Different letters in parenthesis for S and NCR indicate significant differences when not accounting for multiple comparisons. po0.05. po0.01. po0.001.
nematodes, but they affected the nitrogen levels in the soil. As hypothesised, the identity of the plant species was more important than the plant functional group for the nematode communities. Plants have been shown to affect the water content, and the amounts of mineral nutrients and organic compounds in the soil. Therefore, it was not surprising to find higher water content and low organic matter content in the noplant treatment. The high concentration of nitrate in the no-plant treatment could be a result of the application of nutrient solution for the last part of the experiment in combination with no uptake by plants. There was possibly also net mineralisation of soil nitrogen. A high concentration of inorganic nitrate was also found in unplanted soil in another pot experiment (Khalid et al., 2007) and in soil solution from bare ground plots in a field experiment (Scherer-Lorenzen et al., 2003). As hypothesised, the pots without plants had the lowest abundance of nematodes at the final sampling. This was mainly due to a very low abundance of plant feeders, while the other feeding groups were more similar to the plant treatments. However, the number of bacterial-feeding nematodes was lower in the no-plant treatment than in grasses and legumes. This is in agreement with Wardle et al. (1999) who found that their removal of all seedlings from created gaps in a perennial grassland resulted in lower numbers of bacterial-feeding nematodes. There was no correlation between total nematode abundance and plant biomass production, but the abundance of bacterial-feeding nematodes correlated with both shoot and root biomass. The positive correlation with shoot biomass may depend on the fact that grasses and legumes had the greatest shoot biomasses in this study and they invest more in short-term C availability in the rhizosphere through root respiration and exudation than forbs (Warembourg et al., 2003). This higher availability of
carbon affects bacteria and hence bacterial-feeding nematodes positively. There is also a possible feedback back to the plants because microbial grazing by nematodes mineralises nutrients for plant uptake (Ingham et al., 1985). The negative correlation between bacterial feeders and root biomass was due to the much greater root biomass of R. acetosa. Excluding this plant species from the analysis rendered the correlation insignificant. Both R. acetosa and A. millefolium had low abundances of bacterial feeders but very different root biomass, and therefore one possible explanation for the low numbers is instead plant chemical substances. The plant species with the lowest total nematode abundances at the first sampling were those with the greatest at the final sampling. All of these plant species (P. pratense, R. acetosa and T. repens) had high abundances of the ectoparasitic plant feeder Paratylenchus at the last sampling. Paratylenchus has the ability to increase to enormous numbers in the rhizosphere of host plants (Bernard et al., 1998), and for P. nanus grasses were the only hosts (Bell and Watson, 2001b). The abundance of P. nanus has also been shown to correlate positively with soil temperature and negatively with soil moisture (Bell and Watson, 2001a). The generation time for Paratylenchus is about 1 month (Rhoades and Linford, 1961), and therefore the difference between the plant species could not be seen until the final sampling. High abundances of Paratylenchus under P. pratense, T. repens and R. acetosa were also found in the experimental grassland from which soil and nematodes were taken (Viketoft et al., 2005). The two legumes had very different abundances of Paratylenchus. This is also in correspondence to results from the field experiment (Viketoft et al., 2005). A likely explanation could be that T. hybridum contains more and higher levels of phyto-oestrogen (isoflavones) than T. repens (Wu et al., 2003). A variety of T. pratense with
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high phyto-oestrogen levels was less attractive to both adults and larvae of the clover root weevil (Sitona lepidus) (Murray et al., 2007). Different plant secondary metabolites have been shown to have antiparasitic effects against gastrointestinal nematodes (Athanasiadou and Kyriazakis, 2004). Other plant species containing plant-defensive compounds may also negatively affect the abundance of plant-feeding nematodes, e.g., Plantago lanceolata (De Deyn et al., 2004). In addition, there was also a great difference in the abundance of Paratylenchus between R. acetosa and A. millefolium. It is possible that this also depends on plant secondary metabolites, but this needs to be studied further. The effect of legumes on nematodes was not as obvious as in the monocultures in the experimental grassland (Viketoft et al., 2005). In particular, in the present study there were very low abundances of the bacterial feeders Rhabditis, Panagrolaimus, Eucephalobus and Chiloplacus, genera that showed the strongest responses to legumes in the experimental grassland. Instead, it was found that the dominant bacterial feeder, Acrobeloides, did not differ in abundance between legumes and grasses, but was less abundant under forbs and the unplanted treatment. In the field experiment, this genus was most common under F. ovina (Viketoft et al., 2005). The nitrogen fixation by legumes resulted in large amounts of ammonium in the soil of these plants. In other studies, the nitrate concentration has usually been affected by legumes (Gastine et al., 2003; Palmborg et al., 2005). Among the plant treatments, T. hybridum had greater amounts of nitrate than the other investigated plant species. The overall greater amounts of ammonium in the present study could be a result of nitrate being readily used by the plants (Miller and Cramer, 2005). The soil from pots with legumes, in particular T. repens, also had lower pH than the other plant treatments. This acidification by legumes is in agreement with other studies (Yan et al., 1996). This difference in pH is probably not harmful to the nematodes (Nicholas, 1984; Khanna et al., 1997), and is within the pH range at the BIODEPTH site in Umea˚ where the soil was originally collected (C. Palmborg, personal communication). Forbs in the experimental grassland (Viketoft et al., 2005) had high abundances of fungal feeders but this was not found in the present study. Instead fungal feeders seemed to be promoted by grasses, but in general they had low and very varying abundances within the different plant treatments. However, the decrease in NCR with time could be an indication for an increased importance of fungal decomposition in most plant treatments. It is possible that the centrifugation during preparation of the inoculate for microflora disfavoured fungi, so that it was mostly bacteria that were indeed inoculated. The results from the present glasshouse study do not contradict the results found in monocultures of the same plant species in an experimental grassland (Viketoft et al., 2005), but there are some differences (Fig. 2). For example,
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100%
80%
60%
Omn Bf Ff Pa Pf
40%
20%
0% Pp
Fo
Th
Tr
Am
Rua
Fig. 2. Comparison between the nematode fauna (at final sampling) under six plant species in this glasshouse study (left bar) and in monocultures in an experimental grassland (means for the autumn sampling) (right bar) (Viketoft et al., 2005). Proportional contribution of nematode-feeding groups: Omn, omnivores (black); Bf, bacterial feeders (light grey); Ff, fungal feeders (white); Pa, plant-associated (striped); Pf, plant feeders (dark grey). No, no plants; Pp, Phleum pratense; Fo, Festuca ovina; Th, Trifolium hybridum; Tr, Trifolium repens; Am, Achillea millefolium; Rua, Rumex acetosa.
there were only occasional occurrences of omnivores in the glasshouse and plant-associated nematodes were far more common in the experimental grassland. There were also some differences for individual plant species, e.g., T. hybridum had a lower proportion of plant feeders in the glasshouse than in the field experiment, while F. ovina had a higher proportion of plant feeders in the glasshouse. Due to the defaunation, the pots in the glasshouse represent a more artificial environment with a different competitive situation than the soil in the field experiment. It is likely that some of the nematodes that were added to the pots were negatively affected by the handling during the preparation and application of the inoculate. Also, the inoculation procedure favours nematode species with short generation times and high population growth rates, and the few species that do reproduce have the possibility to reach unnaturally high abundances. The number of nematodes of Paratylenchus and Acrobeloides (per gram soil dry weight) in the glasshouse clearly exceed the numbers found in the experimental grassland for all the investigated plant species (Viketoft et al., 2005). A comparison of the number of taxa and the Shannon diversity index between the glasshouse and the field reveals that the fauna indeed is depauperate in the glasshouse (6–8 and 15–24 taxa, respectively, H0 0.38–0.83 and 1.10–2.17, respectively). Naturally, the experimental grassland is more heterogeneous than the pots in the glasshouse and this can partly explain the higher diversity in the field, but a major point is that it was a limited number of nematodes that were inoculated. Another important factor is time because the experimental grassland has been established for 7 years while the present study lasted 16 weeks. It takes several years for the nematode populations to come to dynamic equilibrium
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with the management regime (Yeates et al., 1999) and after cessation of agriculture to recover from the stress associated with cropping (Ha´neˇl, 2003). The maintenance of the experimental grassland for 7 years has lead to the development of a nematode fauna requiring more stabilised conditions (Viketoft et al., unpublished data). In conclusion, this and several other recent studies demonstrate that plants affect the soil nematode community and that the identity of plant species is of importance for the nematode abundance and community composition. This applies for both field experiments and under more controlled glasshouse conditions as in this study. Despite some differences in the nematode species pool, the effects of plant species appear quite consistent between field experiments and the present glasshouse study. This implies that future results found in the glasshouse can be relevant for field conditions. Acknowledgements The author thanks Cecilia Palmborg for collecting the soil, Karin O¨nneby for help with the set-up of the experiment and Birgitta Vegerfors-Persson for statistical advice. Author also thanks Jan Bengtsson, Bjo¨rn Sohlenius and Cecilia Palmborg for valuable comments on previous versions of the manuscript and the anonymous reviewers for constructive comments on the manuscript. This study was funded by the Oscar and Lili Lamm Foundation and the Swedish Research Council (grant to Jan Bengtsson). References Athanasiadou, S., Kyriazakis, I., 2004. Plant secondary metabolites: antiparasitic effects and their role in ruminant production systems. Proceedings of the Nutrition Society 63, 631–639. Badejo, M.A., Tian, G., 1999. Abundance of soil mites under four agroforestry tree species with contrasting litter quality. Biology and Fertility of Soils 30, 107–112. Bardgett, R.D., Mawdsley, J.L., Edwards, S., Hobbs, P.J., Rodwell, J.S., Davies, W.J., 1999. Plant species and nitrogen effects on soil biological properties of temperate upland grasslands. Functional Ecology 13, 650–660. Bell, N.L., Watson, R.N., 2001a. Population dynamics of Paratylenchus nanus in soil under pasture: 1. Aggregation and abiotic factors. Nematology 3, 187–197. Bell, N.L., Watson, R.N., 2001b. Identification and host range assessment of Paratylenchus nanus (Tylenchida: Tylenchulidae) and Paratrichodorus minor (Triplonchida: Trichodoridae). Nematology 3, 483–490. Bernard, E.C., Gwinn, K.D., Griffin, G.D., 1998. Forage grasses. In: Barker, K.R., Pederson, G.A., Windham, G.L. (Eds.), Plant and Nematode Interactions. Agronomy Monograph 36, Madison, pp. 427–454. Bongers, T., 1990. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14–19. Coulson, S.J., Hodkinson, I.D., Webb, N.R., 2003. Microscale distribution patterns in high Arctic soil microarthropod communities: the influence of plant species within the vegetation mosaic. Ecography 26, 801–809. De Deyn, G.B., Raaijmakers, C.E., Van Ruijven, J., Berendse, F., Van der Putten, W.H., 2004. Plant species identity and diversity effects on
different trophic levels of nematodes in the soil food web. Oikos 106, 576–586. Gastine, A., Scherer-Lorenzen, M., Leadley, P.W., 2003. No consistent effects of plant diversity on root biomass, soil biota and soil abiotic conditions in temperate grassland communities. Applied Soil Ecology 24, 101–111. Grayston, S.J., Wang, S., Campbell, C.D., Edwards, A.C., 1998. Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biology & Biochemistry 30, 369–378. Ha´neˇl, L., 2003. Recovery of soil nematode populations from cropping stress by natural secondary succession to meadow land. Applied Soil Ecology 22, 255–270. Ingham, R.E., Trofymow, J.A., Ingham, E.R., Coleman, D.C., 1985. Interactions of bacteria, fungi, and their nematode grazers: effects on nutrient cycling and plant growth. Ecological Monographs 55, 119–140. Innes, L., Hobbs, P.J., Bardgett, R.D., 2004. The impacts of individual plant species on rhizosphere microbial communities in soils of different fertility. Biology and Fertility of Soils 40, 7–13. Khalid, M., Soleman, N., Jones, D.L., 2007. Grassland plants affect dissolved organic carbon and nitrogen dynamics in soil. Soil Biology & Biochemistry 39, 378–381. Khanna, N., Cressman, C.P., Tatara, C.P., Williams, P.L., 1997. Tolerance of the nematode Caenorhabditis elegans to pH, salinity, and hardness in aquatic media. Archives of Environmental Contamination and Toxicology 32, 110–114. Kowalchuk, G.A., Buma, D.S., de Boer, W., Klinkhamer, P.G.L., van Veen, J.A., 2002. Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms. Antonie Van Leeuwenhoek 81, 509–520. Magurran, A.E., 2004. Measuring Biological Diversity. Blackwell Publishing, Oxford, UK, 256pp. Marschner, P., Yang, C.-H., Lieberei, R., Crowley, D.E., 2001. Soil and plant specific effects on bacterial community composition in the rhizosphere. Soil Biology & Biochemistry 33, 1437–1445. Milcu, A., Partsch, S., Langel, R., Scheu, S., 2006. The response of decomposers (earthworms, springtails and microorganisms) to variations in species and functional group diversity of plants. Oikos 112, 513–524. Miller, A.J., Cramer, M.D., 2005. Root nitrogen acquisition and assimilation. Plant and Soil 274, 1–36. Murray, P.J., Cheung, A.K.M., Abberton, M.T., 2007. Intraspecific variation in Trifolium pratense: impact on feeding and host location by Sitona lepidus (Coleoptera, Curculionidae). Journal of Pest Science 80, 51–57. Nicholas, W.L., 1984. The Biology of Free-Living Nematodes. Clarendon Press, Oxford, 251pp. Palmborg, C., Scherer-Lorenzen, M., Jumpponen, A., Carlsson, G., HussDanell, K., Ho¨gberg, P., 2005. Inorganic soil nitrogen under grassland plant communities of different species composition and diversity. Oikos 110, 271–282. Rhoades, H.L., Linford, M.B., 1961. Biological studies on some members of the genus Paratylenchus. Proceedings of the Helminthological Society of Washington 28, 51–59. Saetre, P., Ba˚a˚th, E., 2000. Spatial variation and patterns of soil microbial community structure in a mixed spruce-birch stand. Soil Biology & Biochemistry 32, 909–917. Salamon, J.-A., Schaefer, M., Alphei, J., Schmid, B., Scheu, S., 2004. Effects of plant diversity on Collembola in an experimental grassland ecosystem. Oikos 106, 51–60. Scherer-Lorenzen, M., Palmborg, C., Prinz, A., Schulze, E.D., 2003. The role of plant diversity and composition for nitrate leaching in grasslands. Ecology 84, 1539–1552. Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H., Berg, G., 2001. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Applied and Environmental Microbiology 67, 4742–4751.
ARTICLE IN PRESS M. Viketoft / Soil Biology & Biochemistry 40 (2008) 906–915 Sohlenius, B., 1980. Abundance, biomass and contribution to energy flow by soil nematodes in terrestrial ecosystems. Oikos 34, 186–194. Viketoft, M., Palmborg, C., Sohlenius, B., Huss-Danell, K., Bengtsson, J., 2005. Plant species effects on soil nematode communities in experimental grasslands. Applied Soil Ecology 30, 90–103. Wardle, D.A., 2002. Communities and Ecosystems: Linking the Aboveground and Belowground Components. Princeton University Press, Princeton, NJ, 392pp. Wardle, D.A., 2005. How plant communities influence decomposer communities. In: Bardgett, R.D., Usher, M.B., Hopkins, D.W. (Eds.), Biological Diversity and Function in Soils. Cambridge University Press, Cambridge, pp. 119–138. Wardle, D.A., Bonner, K.I., Barker, G.M., Yeates, G.W., Nicholson, K.S., Bardgett, R.D., Watson, R.N., Ghani, A., 1999. Plant removals in perennial grassland: vegetation dynamics, decomposers, soil biodiversity, and ecosystem properties. Ecological Monographs 69, 535–568. Wardle, D.A., Yeates, G.W., Williamson, W., Bonner, K.I., 2003. The response of a three trophic level soil food web to the identity and diversity of plant species and functional groups. Oikos 102, 45–56. Warembourg, F.R., Roumet, C., Lafont, F., 2003. Differences in rhizosphere carbon-partitioning among plant species of different families. Plant and Soil 256, 347–357.
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Soil Biology & Biochemistry 40 (2008) 906–915 www.elsevier.com/locate/soilbio
Effects of six grassland plant species on soil nematodes: A glasshouse experiment Maria Viketoft Department of Ecology, Swedish University of Agricultural Sciences, P.O. Box 7044, SE-750 07 Uppsala, Sweden Received 26 March 2007; received in revised form 31 October 2007; accepted 5 November 2007 Available online 3 December 2007
Abstract The effects of six individual plant species on the abundance and composition of nematode communities were studied in a glasshouse experiment during 16 weeks. The effect of the presence of plants, the correlation between nematode abundance and plant biomass, the response of plant-feeding nematodes and other nematode groups to different plant species was examined and also whether the effect differed between plant species within a plant functional group. The total number of nematodes increased during the study period in all treatments, although in some treatments, the increase levelled off after 8 or 12 weeks. The identity of the plant species affected both the total abundance of nematodes and the nematode community composition. The number of bacterial-feeding nematodes was greatest under grasses and legumes and was positively correlated with shoot biomass and negatively with root biomass. The response of the plantfeeding nematodes, which differed in abundance under both the investigated legume and the forb species, suggests that the identity of the plant species is more important than the plant functional group. A possible explanation could be related to differences in plant secondary metabolites. Despite some differences in the nematode species pool, the effects of plant species appear quite consistent between the present glasshouse study and previous field experiments. r 2007 Elsevier Ltd. All rights reserved. Keywords: Achillea millefolium; Festuca ovina; Nematode community composition; Nematode diversity; Nematode feeding groups; Phleum pratense; Rumex acetosa; Trifolium hybridum; Trifolium repens
1. Introduction Plant species identity and the composition of the plant community are important determinants of decomposer function and community composition in soil (Wardle, 2005). For microorganisms, this has been shown both in terms of microbial biomass and activity in microcosms with soil from grasslands (Bardgett et al., 1999; Innes et al., 2004) as well as microbial community composition in microcosms (Grayston et al., 1998; Marschner et al., 2001), field experiments (e.g., Smalla et al., 2001; Kowalchuk et al., 2002) and in a naturally mixed spruce-birch stand (Saetre and Ba˚a˚th, 2000). Soil fauna are also affected by individual plant species. In both field and pot experiments, nematodes responded to the identity of different grassland species, with plantTel.: +46 18672526; fax: +46 18672890.
E-mail address: [email protected] 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.11.006
feeding and microbial-feeding nematodes (bacterial and fungal feeders) being the groups most clearly influenced by plant species identity (Wardle et al., 2003; De Deyn et al., 2004; Viketoft et al., 2005). Collembolans have been shown to respond to grass and legume species in pot and field experiments with an assembly of grassland species (Salamon et al., 2004; Milcu et al., 2006), and both mites and collembolans responded to plant species in high Arctic heath vegetation (Coulson et al., 2003). In addition, mites have also been shown to be affected by different plant species used in agroforestry (Badejo and Tian, 1999). Plants affect the belowground community through differences in the amount and quality of resources allocated to the soil, in the extent to which they deplete nutrients and water from the soil, in the chemical composition of the litter produced and through the formation and modification of habitats (Wardle, 2002). For example, many legumes differ from other forbs in their ability to fix atmospheric nitrogen and therefore increase
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the amount of nitrogen in the soil, principally in the form of nitrate (Palmborg et al., 2005). They also produce more high-quality litter that is more easily decomposed (Scherer-Lorenzen et al., 2003). Previous studies on the effect of plant species on nematodes have shown that, in general, plant feeders seem to be most abundant under grasses and have a lower presence under some forbs, while microbial feeders show varying responses to the investigated plant species in different studies (Wardle et al., 2003; De Deyn et al., 2004; Viketoft et al., 2005). In addition, different feeding types of plant feeders, e.g., ecto- and endoparasites, have been shown to respond differently to grasses and forbs (De Deyn et al., 2004). The aim of the present study was to examine, in a glasshouse pot experiment, how six plant species, belonging to three plant functional groups (grasses, legumes and forbs), affect total nematode abundance, nematode community composition and the response of individual nematode genera. A defaunated soil from an experimental grassland that was reinoculated with both microflora and nematodes was used. Thus, the effect of plant species during the establishment phase of nematode communities was evaluated under controlled conditions. In addition, a treatment containing no plants was included, top-down effects by micro- and mesofauna other than nematodes were removed and soil characteristics (e.g., pH, inorganic nitrogen), missing in other studies (Wardle et al., 2003; De Deyn et al., 2004; Viketoft et al., 2005), were determined to relate to the nematode data. Specifically, the following hypotheses are addressed: 1. Low numbers of nematodes should be found in the treatment with no plants, due to the lack of plantderived resources. 2. There is a positive correlation between total nematode abundance and plant productivity (Yeates, 1987). 3. Plant-feeding nematodes should show the strongest response to the identity of plant species because they feed directly on them, although the speed of the response between bacterial- and plant feeders likely differs because of different generation times. Based on previous studies, plant feeders are also expected to be most abundant under grasses. 4. Legumes, due to their N-fixing ability, will affect the nitrogen levels in the soil and hence the bacterial community. This should affect the nematode community composition by promotion of bacterial-feeding nematodes, especially Rhabditis and Panagrolaimus. 5. Different plant species within a functional group will have quite different influences on the nematodes (Viketoft et al., 2005). The division into plant functional groups was based on growth form and N-fixing ability, but the plant species may contain different secondary substances. The nematode fauna under monocultures of the plant species used in this study have previously been investigated
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after seven growing seasons in an experimental grassland in northern Sweden (Viketoft et al., 2005). Although the present study is a 16-week experiment, where plants were getting established and a limited number of nematodes were inoculated, it had higher replication and a treatment containing no plants compared to the field experiment. A comparison between these studies may illustrate interesting aspects of the interactions between plants and soil communities. 2. Materials and methods 2.1. Site and soil In November 2004, soil was collected to 10 cm depth from the experimental field of the BIODEPTH project of the Swedish University of Agricultural Sciences, Umea˚, northern Sweden (631450 N, 201170 E). The soil is classified as silt loam (4.1% clay, 57.9% silt, 38.0% fine sand) and was taken from an area between the experimental plots, which is dominated by Phleum pratense and Trifolium repens. The soil was stored at 4 1C until further processed in February 2005. The vegetation was then removed and the soil was passed through a 4-mm sieve. In order to defaunate the soil, the sieved soil was subjected to freezing (20 1C) for at least 48 h followed by heating in a warming cupboard to 70 1C for 24 h. Then, the soil was subjected to another cycle of freezing and heating. Samples were taken from the defaunated soil to see if the procedure had been successful. A subsample of the defaunated soil was stored frozen until analysed for soil chemistry: pH, dry matter, organic matter content, KCl-extractable NH4–N and NO3–N, and plant-available P (ammonium-lactate extract). The remainder of the soil was stored at room temperature for 2 days until added to the pots. 2.2. Experimental set-up The glasshouse experiment consisted of 75 black plastic pots (height 9.5 cm, diameter 12 cm, drainage holes in the bottom were covered with fibre cloth) that were first filled with 250 g sand and then 700 g of the defaunated soil (moisture content 10%). The sand had been washed through a 0.3-mm sieve with tap water to remove all organic material and then dried at 120 1C for 20 h. Thereafter, the pots were reinoculated with microflora by adding 15 ml of a soil suspension made from the original faunated soil. No specific Rhizobium inoculum was added. The suspension was made by mixing 1 kg of sieved soil in 2 l of MilliQ-water, which was then centrifuged at 2000 rpm for 5 min. The centrifugation was repeated twice. The soil suspension was checked for presence of nematodes but there were none. The pots were then sown with seeds of P. pratense L. (cv. Jonatan), Festuca ovina L. (cv. Fertalia, Dumon, Belgium), Trifolium hybridum L. (cv. Stena), T. repens L. (cv. Undrom) (obtained from Svalo¨f Weibull AB,
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Svalo¨v, Sweden), Achillea millefolium L. or Rumex acetosa L. (obtained from Pratensis AB, Vislanda, Sweden). About 20–25 seeds were sown in each pot and there were 10 pots for each plant species. In addition, there were 10 pots without plants and 5 control pots (without plants and receiving no nematodes), giving a total of 75 pots. The pots were randomly placed in 15 rows in the glasshouse and covered with plastic. After 2 weeks when all plant species had germinated, the plastic was removed and additional seeds were added to pots where less than 15 seeds had germinated. One week later, the pots were thinned to 11 seedlings per pot. However, the seedlings of F. ovina were still very small and were not thinned until two additional weeks had passed (five since the original sowing). In the other pots, seedlings that had emerged since the last thinning were also removed. At time of the first thinning, soil samples were taken from the control pots to ensure that the pots were still nematode free. Then, a nematode inoculum was made from the original faunated soil (i.e. collected in November from a P. pratense/T. repens dominated area, stored till February, sieved over 4-mm sieve) by extraction with the Whitehead tray method (Whitehead and Hemming, 1965) for 24 h. This extraction method was used because of the great amount of soil that needed to be extracted. The suspension was concentrated by decanting and the final nematode concentration was determined to 35 nematodes ml1. All pots except the control pots were watered with 15 ml inoculate, i.e. ca. 525 nematodes ( ¼ week 0, mid-March 2005). The control pots received the same amount of deionised water. To characterise the added nematode fauna, samples from the inoculate were taken and nematodes were identified (see the following text). The conditions in the glasshouse were set at 18 1C for 16 h with supplementary lighting (day, HPI-T lamps, 400 W, Philips) and at 15 1C for 8 h with no supplementary lighting (night). The relative humidity was 60%. To counteract heating of the glasshouse by the sun and keep the soil temperature around 22 1C, the temperature in the glasshouse was lowered to 16 1C (day) and 13 1C (night) after 4 weeks, and the glasshouse chamber was changed in mid-June to avoid soil temperatures above 30 1C. To equalise the amount of radiation for each pot, pots were rearranged weekly by circulating them within and between the rows. The pots were watered as required (usually every second or third day) with deionised water. Twelve weeks after sowing ( ¼ experimental week 8), the plants showed signs of nutrient deficiency and consequently after this the pots received equal amounts of a complete fertiliser (Wallco, N:P:K 51:10:43, Cederroth International AB), with a total of 2 mg NH4 and 3 mg NO3 added per pot and occasion. This was repeated once every second week for the rest of the experiment. Thrips were discovered in connection with the first thinning, and predatory mites, Amblyseius, were added twice to all pots to control them.
2.3. Nematode sampling Nematode samples were taken at experimental weeks 4, 8, 12 and 16 with a cork borer (diameter 1.5 cm). One sample was taken in each pot at every occasion, avoiding holes from previous samplings. Samples were taken down to the sand and the soil was transferred to plastic tubes, stored at 4 1C and nematodes were extracted within 4 days with a modified Baermann method (Viketoft et al., 2005). This extraction method was used because of the small size of the soil samples and the high recovery of this method (Sohlenius, 1980). Following the extraction, the water content in the soil samples was determined by drying in 60 1C for 24 h. The dried soil was rewetted and returned to the hole in the pot. Total abundance of nematodes was estimated in suspensions from each extraction under low magnification (50 ) and expressed as g1 dry soil weight. In each suspension, the first 100 nematodes were identified to genera and in some cases to species level under higher magnification (200 ). Additional genera in the suspension were marked as present. The nematodes were placed into different feeding groups according to Yeates et al. (1993). Nematodes feeding on epidermal cells or root hairs was represented as plant-associated nematodes. 2.4. Plant growth and soil chemistry Each pot was destructively harvested at experimental week 16 (early July 2005). Plant shoots were first removed, dried at 60 1C for 24 h and weighed. In the shoot biomass for T. repens the weight of stolons was not included. The soil was then divided in halves, one for estimation of root biomass and the other for determination of soil chemistry. Roots were washed over a sieve (0.5 mm), dried overnight at room temperature and then at 60 1C for 24 h and weighed. The soil for determination of soil chemistry was first frozen (20 1C) and after thawing sieved (4 mm) and analysed for pH, dry matter, organic matter content as loss on ignition, and KCl-extractable NH4–N and NO3–N. Soil pH was determined in a 1:5 soil–water slurry. The dry matter was determined by drying the soil at 105 1C, and the soil was thereafter heated to 550 1C to determine the organic matter content. To determine the inorganic N pools, the thawed samples were extracted with 2 M KCl for 2 h and then the extracts were analysed for nitrate and ammonium using flow injection analysis (Tecator FIA 5010, FOSS Tecator, Ho¨gana¨s, Sweden). 2.5. Data treatment and statistical analyses The total nematode abundance was ln(x)-transformed and analysed with a repeated-measures ANOVA (procedure mixed) using autoregressive order 1 covariance, using all four samplings. Effects with po0.0008 were considered significant when accounting for multiple comparisons in a sequential Bonferroni test (21 4 ¼ 84 comparisons).
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The abundance of the different feeding groups and individual genera present in at least half of the plots (Paratylenchus, Aphelenchoides and Acrobeloides) were analysed with a repeated-measures ANOVA using only the second and fourth sampling. The type of covariance structure used was based on the model-fit criteria computed by the statistical program (unstructured for plant feeders, Paratylenchus, bacterial feeders and Acrobeloides; autoregressive order 1 for fungal feeders and Aphelenchoides). Abundance data for the feeding groups and individual genera was ln(x+1)-transformed prior to analysis. Due to a high number of zeros for plant feeders and Paratylenchus, the treatments with no plants and T. hybridum were excluded from the repeated-measures analysis, after confirming that they were significantly different from the other plant treatments with Wilcoxon’s rank test. Effects with po0.002 were significant when accounting for multiple comparisons in sequential Bonferroni tests (21 2 ¼ 42 comparisons, 10 2 ¼ 20 for plant feeders and Paratylenchus). To characterise the nematode fauna under the different plant species, several indices were calculated for the second and fourth sampling: number of taxa (S), Shannon index (H0 ¼ Spi ln pi, where pi is the proportion of the ith species), evenness index (J0 ¼ H0 /(ln S)) and the complement of Simpson’s index (1D, D ¼ Sp2i ) (Magurran, 2004), nematode channel ratio (NCR ¼ B/(B+F), where B and F are abundances of bacterial and fungal feeders, respectively) (Yeates, 2003), maturity index (MI) and plant parasite index (PPI) (Bongers, 1990). However, the MI and PPI indices were exactly 2 for a great majority of the samples, and hence it is not relevant to investigate differences between the plant species, because the values that deviate from 2 are given unproportionally large
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significance for the test statistic. The other indices were analysed with a repeated-measures ANOVA with unstructured covariance, except for S for which autoregressive order 1 covariance was used. NCR was arcsine-transformed prior to analysis and effects were considered significant when po0.0014 (sequential Bonferroni tests applied, 21 2 ¼ 42 comparisons). The plant and soil data was analysed with one-way ANOVA (Tukey’s multiple comparison tests (po0.05)), excepting nitrate and ammonium which were analysed with Kruskal–Wallis tests (pairwise tests: sequential Bonferroni tests applied, po0.008, 21 comparisons). The biomasses were square-root transformed. Relations between plant biomass production (shoot and root biomass) and nematode abundances at the final sampling (total and of the different feeding groups) were tested with Pearson correlations. This was also done when analysing relations between soil characteristics and the nematode abundances (nitrogen data with Spearman rank correlation). As the main interest was in finding variables that might aid in the interpretation of the results rather than testing explicit hypotheses, Bonferroni corrections were not used in these cases. All statistical tests were performed using SAS for Windows 9.1. 3. Results 3.1. Plant growth and soil chemistry Shoot and root biomass differed significantly between the plant treatments (Table 1). In general, the legumes had the highest shoot biomass and forbs the lowest. No such pattern among plant functional groups was found for the root biomass. Instead, R. acetosa clearly had the greatest
Table 1 Plant and soil characteristics for the defaunated soil (before start of the experiment), the control pots (microflora inoculate, no plants, no nematode inoculate) and the plant treatments after 16 weeks Treatment
Shoot biomass (g)
Root biomass (g/kg soil)
Organic matter (%)
pH
Defaunated soil Control
– –
– –
4.65 (0.02) 4.75 (0.005)
5.80 (0.05) 5.63 (0.02)
No plants Phleum pratense Festuca ovina Trifolium hybridum Trifolium repens Achillea millefolium Rumex acetosa
– 4.11 (0.06) c 4.19 (0.08) bc 4.48 (0.06) b
– 9.1 (0.4) cd 10.4 (0.6) bc 11.6 (0.6) b
4.68 4.84 4.85 4.90
5.67 5.77 5.71 5.62
5.70 (0.09) a 2.29 (0.06) d
7.7 (0.4) d 8.6 (0.4) cd
2.01 (0.04) e
F-values/w-values
457.91
(0.02) (0.03) (0.02) (0.02)
d bc bc ab
(0.03) (0.03) (0.02) (0.03)
ab a ab b
NH4 (mg/kg soil)
NO3 (mg/kg soil)
73.8 (0.6) 1.28 (0.10)
0.74 (0.05) 6.13 (1.68)
1.42 0.69 0.78 12.38
(0.09) (0.15) (0.12) (1.05)
b c c a
4.46 0.006 0.018 0.089
(0.49) a (0.002) d (0.006) cd (0.015) b
4.96 (0.03) a 4.88 (0.01) abc
5.49 (0.04) c 5.64 (0.02) b
10.86 (0.52) a 1.12 (0.11) bc
0.003 (0.001) d 0.025 (0.004) c
16.0 (1.0) a
4.80 (0.02) c
5.67 (0.03) ab
0.86 (0.08) c
0.024 (0.005) c
25.08
18.56
10.33
51.67
54.77
Shoot and root biomass (dry weight), organic matter content (loss on ignition), pH (H2O), NH4 (2 M KCl) and NO3 (2 M KCl). Arithmetic means (S.E.), n ¼ 10 (n ¼ 6 for defaunated soil, n ¼ 5 for control). Plant-available P (ammonium-lactate extract) was only determined for the defaunated soil, and was 4.95 mg/100 g soil. F-values are from ANOVAs and w-values from Kruskal–Wallis tests (NH4 and NO3). Different letters indicate significant differences between plant treatments. po0.001.
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root biomass. The presence of plants affected the soil during the experimental period, which is demonstrated by the pots without plants having the highest nitrate levels and the lowest organic matter content (Table 1). The no-plant treatment also had the highest water content (data not shown). The plant species that differed most from the others were the legumes (Table 1). 3.2. Nematodes The total number of nematodes increased during the study period in all treatments, although in some treatments, the increase levelled off after the second or third sampling (Fig. 1). There was a significant interaction effect between plant and time for the total number of nematodes. The largest differences were found at the first sampling (4 weeks after nematode inoculation) and at the final sampling (16 weeks) (Fig. 1). The abundance of nematodes followed almost the same pattern in P. pratense, R. acetosa and T. repens. At the first sampling, they had low abundances, but at the final sampling, they had the greatest. The opposite pattern was found for A. millefolium and the unplanted treatment with the greatest abundances at the first sampling and lower abundances than most other treatments at the final sampling. T. hybridum and F. ovina both had an intermediate position but diverged at the final sampling when the abundance in T. hybridum decreased. Compared to the inoculate, a number of nematode genera (e.g., Acrobeles and Alaimus) were never recorded in suspensions extracted from the pots. The most abundant of the surviving genera were Paratylenchus, Aphelenchoides and Acrobeloides (Table 2). The abundance of the plantfeeding Paratylenchus was lower in the no-plant treatment and in T. hybridum than in all other treatments at both experimental week 8 and at the final sampling at week 16. In addition, at the final sampling, the abundance in
Ln no. of nematodes / g dw soil
8 aa ab ab bc cd
7 6 5
d a ab ab bc
4 3 2
cde de e
1
A. millefolium was lower than in R. acetosa and T. repens. The abundance of the fungal-feeding Aphelenchoides varied within the treatments, but at the final sampling it was greater in F. ovina than in T. repens. The bacterial-feeding Acrobeloides increased over time in all treatments except no plants. At experimental week 8, the abundance of Acrobeloides was lower in pots planted with R. acetosa and P. pratense than in pots with T. repens, T. hybridum and no plants. At 16 weeks, the treatments formed two groupings: R. acetosa, A. millefolium and the no-plant treatment had lower abundances than the other four plant species. Because the aforementioned genera were so dominant, the responses of the different feeding groups almost mirror the responses of these genera (Table 2). In general, the diversity of the nematode communities was low but affected by plant species (Table 3). At experimental week 8, the only significant difference was recorded with A. millefolium which had more taxa than P. pratense. At the final sampling, the most diverse community was found in pots with A. millefolium, while T. hybridum had the lowest Shannon diversity index and T. repens the lowest number of taxa (Table 3). The community with the most even species-abundance distribution after 16 weeks was found in pots with P. pratense and the least even species-abundance distribution was found in T. hybridum. The NCR showed that there was dominance of bacterial feeders over fungal feeders in soil from all the investigated plant species (Table 3). The community and diversity indices were affected by time (Table 3) and all of them, except NCR, increased during the experimental period. At the final sampling, both shoot and root biomasses were uncorrelated with total abundance of nematodes, plant feeders and fungal feeders. However, there was a strong positive correlation between abundance of bacterial feeders and shoot biomass and a weak negative correlation with root biomass (r ¼ 0.72 and 0.37, respectively, po0.01, n ¼ 60). On the other hand, total nematode abundance was negatively correlated with nitrate and ammonium (r ¼ 0.67 and 0.27, respectively, po0.05, n ¼ 70). Plant feeders and bacterial feeders were negatively correlated with nitrate (r ¼ 0.65 and 0.45, respectively, po0.001), but only plant feeders negatively correlated with ammonium (r ¼ 0.30, po0.05). Fungal feeders were negatively correlated with the amount of ammonium (r ¼ 0.27, po0.05). 4. Discussion
0 -1 0
4
8
12
16
Sampling time (weeks) Fig. 1. Total abundance of nematodes (ln no./g soil dw) over time in soil with no plants ( ), Phleum pratense (J), Festuca ovina (K), Trifolium hybridum (&), Trifolium repens (’), Achillea millefolium (m) and Rumex acetosa (n). ‘‘~’’ represents the inoculate abundance. Different letters indicate significant differences between treatments within sampling dates. Mean arithmetic (S.E.) ¼ 22.27 across treatments.
As hypothesised, the no-plant treatment had the lowest total number of nematodes. However, a correlation between total nematode abundance and plant biomass was not found, but the abundance of bacterial feeders correlated with both shoot and root biomass. Plant-feeding nematodes showed the strongest response to the different plant species but in contrast to the hypothesis they did not have their greatest abundances under grasses. In addition, legumes did not specifically promote bacterial-feeding
Table 2 Abundance (mean no./g soil dw (S.E.), n ¼ 10) of nematode taxa after 8 and 16 experimental weeks of a glasshouse experiment with six different plant species and an unplanted treatment Phleum pratense
Festuca ovina
Trifolium repens
Achillea millefolium
Rumex acetosa
Plant feeders Tylenchorhynchus
8 16 8 16 8 16
x x 0.16 (0.16)
0.14 (0.14) xc 0.14 (0.14) d
x x x x 9.32 (5.02) a 676.45 (127.89) ab
Pratylenchus
10.11 (3.29) a 513.80 (114.70) ab
x x 0.74 (0.34) b 41.36 (25.00) c
x 13.39 (4.61) a 857.26 (140.25) a
0.12 (0.12) x 4.93 (0.66) a 295.90 (65.99) b
x x 2.96 (0.80) a 997.67 (141.94) a
Paratylenchus
Total
8 16
xc 0.28 (0.28) d
9.32 (5.02) a 676.45 (127.89) ab
10.27 (3.34) a 513.80 (114.70) ab
0.74 (0.34) b 41.36 (25.00) c
13.39 (4.61) a 857.26 (140.25) a
5.05 (0.66) a 295.90 (65.99) b
2.96 (0.80) a 997.67 (141.94) a
Plant-associated Filenchus
8 16
0.09 (0.09) 0.60 (0.28)
x 2.73 (2.73)
0.23 (0.15) x
x 1.15 (1.15)
x x
x 5.66 (5.66)
0.12 (0.08) x
Fungal feeders Aphelenchidaea
8 16 8 16 8 16
x 0.34 (0.18) 49.40 (32.76) a 22.03 (7.58) ab
0.97 (0.85) x 3.24 (2.26) a 40.61 (18.54) ab
0.14 (0.14) x 6.80 (3.70) a 35.66 (8.53) a
0.31 1.52 0.84 7.46
0.29 1.61 4.89 4.76
0.75 0.27 4.19 7.36
x x 14.14 (7.15) a 15.54 (4.65) ab
Aphelenchoides Dorylaimellus
Total
8 16
49.40 (32.76) a 22.36 (7.56) a
4.21 (2.26) a 40.61 (18.54) a
6.93 (3.76) a 35.66 (8.53) a
1.15 (0.67) a 8.98 (2.07) a
5.18 (4.62) a 6.37 (2.33) a
4.94 (2.26) a 7.63 (2.06) a
14.14 (7.15) a 15.54 (4.65) a
Bacterial feeders Rhabditis s.l.
8 16 8 16 8 16 8 16 8 16 8 16 8 16 8 16
x 1.29 (0.74) 151.61 (32.51) a 105.94 (11.87) b x 1.76 (0.73)
x x 68.46 (11.24) b 269.98 (31.13) a 0.09 (0.09) 1.44 (0.98)
x
0.80 (0.80) 0.55 (0.55) 141.99 (26.05) a 224.65 (10.37) a 0.07 (0.07) x
x x 146.52 (22.71) a 254.49 (17.34) a x x x
0.48 (0.36) x 89.05 (12.61) ab 115.65 (23.43) b 0.48 (0.21) 1.93 (1.10)
0.20 (0.14) x 51.59 (2.82) b 89.85 (11.35) b 0.31 (0.25) 0.26 (0.26) x
Acrobeloides Cephalobus Eucephalobus Panagrolaimus
x
x
Plectus
x
x x
1.00 (0.54) 8.68 (4.15)
0.10 (0.10) 4.64 (3.09)
Eumonhystera Prismatolaimus
0.93 (0.38) 0.14 (0.14) 2.54 (0.63)
0.11 (0.11) 1.00 (1.00)
x 0.11 (0.11) 2.49 (1.18)
x
0.08 (0.08) x
x x 3.62 (2.00)
x x x
Total
8 16
151.75 (32.51) a 115.31 (12.89) c
68.66 (11.24) b 272.42 (31.55) a
96.12 (7.19) ab 201.18 (31.05) ab
142.86 (25.73) a 225.20 (10.57) a
146.60 (22.68) a 254.49 (17.34) a
91.01 (12.63) ab 129.89 (20.86) bc
52.19 (2.93) b 94.75 (9.93) c
Omnivores Aporcelaimellus
8 16
x x
x
x
x
x
x
x
(0.31) (1.00) (0.58) a (2.14) ab
(0.29) (1.61) (4.33) a (2.13) b
(0.37) (0.27) (2.30) a (1.87) ab
x
93.66 (6.92) ab 198.69 (30.91) a 0.36 (0.36) x
x 1.84 (1.02) x 0.14 (0.14) x
2.84 (1.77)
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x indicates presence of a taxa but at levels too low to be quantified. Different letters indicate significant differences between treatments within sampling dates. Approximately, 525 nematodes were added to each pot at the start of the experiment. a Including Aphelenchus and Paraphelenchus.
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No plants
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Table 3 Nematode indices for the final sampling after 16 weeks H0
S No plants Phleum pratense Festuca ovina Trifolium hybridum Trifolium repens Achillea millefolium Rumex acetosa Repeated-measures ANOVA Plant species Time Plant species time
7.9 6.3 6.5 6.4 6.0 8.0 7.3
(0.5) (0.3) (0.7) (0.5) (0.5) (0.4) (0.4)
F-values 4.44 69.59 1.87
a a a a a a a
(e) (fg) (fg) (fg) (g) (e) (ef)
0.72 0.72 0.74 0.38 0.55 0.83 0.43
J0 (0.07) (0.04) (0.05) (0.07) (0.04) (0.07) (0.07)
6.26 44.88 2.57
a a a b ab a b
0.44 0.72 0.61 0.35 0.66 0.59 0.38
1D (0.02) (0.06) (0.04) (0.06) (0.07) (0.03) (0.03)
4.10 21.73 1.97
bcd a abc d ab abc cd
0.35 0.45 0.44 0.20 0.36 0.47 0.22
NCR (0.04) (0.03) (0.03) (0.05) (0.03) (0.03) (0.04)
6.10 39.19 2.12
ab a a b ab a b
0.86 0.89 0.85 0.96 0.97 0.93 0.87
(0.03) (0.04) (0.03) (0.01) (0.01) (0.02) (0.04)
a a a a a a a
(g) (efg) (g) (ef) (e) (efg) (fg)
3.69 6.21 0.62
S (no. taxa), H0 (Shannon diversity index), J0 (evenness), 1D (Simpson’s index), NCR (nematode channel ratio). Arithmetic means (S.E.), n ¼ 10. Different letters indicate significant differences among plant treatments, po0.0014, sequential Bonferroni tests applied. Different letters in parenthesis for S and NCR indicate significant differences when not accounting for multiple comparisons. po0.05. po0.01. po0.001.
nematodes, but they affected the nitrogen levels in the soil. As hypothesised, the identity of the plant species was more important than the plant functional group for the nematode communities. Plants have been shown to affect the water content, and the amounts of mineral nutrients and organic compounds in the soil. Therefore, it was not surprising to find higher water content and low organic matter content in the noplant treatment. The high concentration of nitrate in the no-plant treatment could be a result of the application of nutrient solution for the last part of the experiment in combination with no uptake by plants. There was possibly also net mineralisation of soil nitrogen. A high concentration of inorganic nitrate was also found in unplanted soil in another pot experiment (Khalid et al., 2007) and in soil solution from bare ground plots in a field experiment (Scherer-Lorenzen et al., 2003). As hypothesised, the pots without plants had the lowest abundance of nematodes at the final sampling. This was mainly due to a very low abundance of plant feeders, while the other feeding groups were more similar to the plant treatments. However, the number of bacterial-feeding nematodes was lower in the no-plant treatment than in grasses and legumes. This is in agreement with Wardle et al. (1999) who found that their removal of all seedlings from created gaps in a perennial grassland resulted in lower numbers of bacterial-feeding nematodes. There was no correlation between total nematode abundance and plant biomass production, but the abundance of bacterial-feeding nematodes correlated with both shoot and root biomass. The positive correlation with shoot biomass may depend on the fact that grasses and legumes had the greatest shoot biomasses in this study and they invest more in short-term C availability in the rhizosphere through root respiration and exudation than forbs (Warembourg et al., 2003). This higher availability of
carbon affects bacteria and hence bacterial-feeding nematodes positively. There is also a possible feedback back to the plants because microbial grazing by nematodes mineralises nutrients for plant uptake (Ingham et al., 1985). The negative correlation between bacterial feeders and root biomass was due to the much greater root biomass of R. acetosa. Excluding this plant species from the analysis rendered the correlation insignificant. Both R. acetosa and A. millefolium had low abundances of bacterial feeders but very different root biomass, and therefore one possible explanation for the low numbers is instead plant chemical substances. The plant species with the lowest total nematode abundances at the first sampling were those with the greatest at the final sampling. All of these plant species (P. pratense, R. acetosa and T. repens) had high abundances of the ectoparasitic plant feeder Paratylenchus at the last sampling. Paratylenchus has the ability to increase to enormous numbers in the rhizosphere of host plants (Bernard et al., 1998), and for P. nanus grasses were the only hosts (Bell and Watson, 2001b). The abundance of P. nanus has also been shown to correlate positively with soil temperature and negatively with soil moisture (Bell and Watson, 2001a). The generation time for Paratylenchus is about 1 month (Rhoades and Linford, 1961), and therefore the difference between the plant species could not be seen until the final sampling. High abundances of Paratylenchus under P. pratense, T. repens and R. acetosa were also found in the experimental grassland from which soil and nematodes were taken (Viketoft et al., 2005). The two legumes had very different abundances of Paratylenchus. This is also in correspondence to results from the field experiment (Viketoft et al., 2005). A likely explanation could be that T. hybridum contains more and higher levels of phyto-oestrogen (isoflavones) than T. repens (Wu et al., 2003). A variety of T. pratense with
ARTICLE IN PRESS M. Viketoft / Soil Biology & Biochemistry 40 (2008) 906–915
high phyto-oestrogen levels was less attractive to both adults and larvae of the clover root weevil (Sitona lepidus) (Murray et al., 2007). Different plant secondary metabolites have been shown to have antiparasitic effects against gastrointestinal nematodes (Athanasiadou and Kyriazakis, 2004). Other plant species containing plant-defensive compounds may also negatively affect the abundance of plant-feeding nematodes, e.g., Plantago lanceolata (De Deyn et al., 2004). In addition, there was also a great difference in the abundance of Paratylenchus between R. acetosa and A. millefolium. It is possible that this also depends on plant secondary metabolites, but this needs to be studied further. The effect of legumes on nematodes was not as obvious as in the monocultures in the experimental grassland (Viketoft et al., 2005). In particular, in the present study there were very low abundances of the bacterial feeders Rhabditis, Panagrolaimus, Eucephalobus and Chiloplacus, genera that showed the strongest responses to legumes in the experimental grassland. Instead, it was found that the dominant bacterial feeder, Acrobeloides, did not differ in abundance between legumes and grasses, but was less abundant under forbs and the unplanted treatment. In the field experiment, this genus was most common under F. ovina (Viketoft et al., 2005). The nitrogen fixation by legumes resulted in large amounts of ammonium in the soil of these plants. In other studies, the nitrate concentration has usually been affected by legumes (Gastine et al., 2003; Palmborg et al., 2005). Among the plant treatments, T. hybridum had greater amounts of nitrate than the other investigated plant species. The overall greater amounts of ammonium in the present study could be a result of nitrate being readily used by the plants (Miller and Cramer, 2005). The soil from pots with legumes, in particular T. repens, also had lower pH than the other plant treatments. This acidification by legumes is in agreement with other studies (Yan et al., 1996). This difference in pH is probably not harmful to the nematodes (Nicholas, 1984; Khanna et al., 1997), and is within the pH range at the BIODEPTH site in Umea˚ where the soil was originally collected (C. Palmborg, personal communication). Forbs in the experimental grassland (Viketoft et al., 2005) had high abundances of fungal feeders but this was not found in the present study. Instead fungal feeders seemed to be promoted by grasses, but in general they had low and very varying abundances within the different plant treatments. However, the decrease in NCR with time could be an indication for an increased importance of fungal decomposition in most plant treatments. It is possible that the centrifugation during preparation of the inoculate for microflora disfavoured fungi, so that it was mostly bacteria that were indeed inoculated. The results from the present glasshouse study do not contradict the results found in monocultures of the same plant species in an experimental grassland (Viketoft et al., 2005), but there are some differences (Fig. 2). For example,
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100%
80%
60%
Omn Bf Ff Pa Pf
40%
20%
0% Pp
Fo
Th
Tr
Am
Rua
Fig. 2. Comparison between the nematode fauna (at final sampling) under six plant species in this glasshouse study (left bar) and in monocultures in an experimental grassland (means for the autumn sampling) (right bar) (Viketoft et al., 2005). Proportional contribution of nematode-feeding groups: Omn, omnivores (black); Bf, bacterial feeders (light grey); Ff, fungal feeders (white); Pa, plant-associated (striped); Pf, plant feeders (dark grey). No, no plants; Pp, Phleum pratense; Fo, Festuca ovina; Th, Trifolium hybridum; Tr, Trifolium repens; Am, Achillea millefolium; Rua, Rumex acetosa.
there were only occasional occurrences of omnivores in the glasshouse and plant-associated nematodes were far more common in the experimental grassland. There were also some differences for individual plant species, e.g., T. hybridum had a lower proportion of plant feeders in the glasshouse than in the field experiment, while F. ovina had a higher proportion of plant feeders in the glasshouse. Due to the defaunation, the pots in the glasshouse represent a more artificial environment with a different competitive situation than the soil in the field experiment. It is likely that some of the nematodes that were added to the pots were negatively affected by the handling during the preparation and application of the inoculate. Also, the inoculation procedure favours nematode species with short generation times and high population growth rates, and the few species that do reproduce have the possibility to reach unnaturally high abundances. The number of nematodes of Paratylenchus and Acrobeloides (per gram soil dry weight) in the glasshouse clearly exceed the numbers found in the experimental grassland for all the investigated plant species (Viketoft et al., 2005). A comparison of the number of taxa and the Shannon diversity index between the glasshouse and the field reveals that the fauna indeed is depauperate in the glasshouse (6–8 and 15–24 taxa, respectively, H0 0.38–0.83 and 1.10–2.17, respectively). Naturally, the experimental grassland is more heterogeneous than the pots in the glasshouse and this can partly explain the higher diversity in the field, but a major point is that it was a limited number of nematodes that were inoculated. Another important factor is time because the experimental grassland has been established for 7 years while the present study lasted 16 weeks. It takes several years for the nematode populations to come to dynamic equilibrium
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with the management regime (Yeates et al., 1999) and after cessation of agriculture to recover from the stress associated with cropping (Ha´neˇl, 2003). The maintenance of the experimental grassland for 7 years has lead to the development of a nematode fauna requiring more stabilised conditions (Viketoft et al., unpublished data). In conclusion, this and several other recent studies demonstrate that plants affect the soil nematode community and that the identity of plant species is of importance for the nematode abundance and community composition. This applies for both field experiments and under more controlled glasshouse conditions as in this study. Despite some differences in the nematode species pool, the effects of plant species appear quite consistent between field experiments and the present glasshouse study. This implies that future results found in the glasshouse can be relevant for field conditions. Acknowledgements The author thanks Cecilia Palmborg for collecting the soil, Karin O¨nneby for help with the set-up of the experiment and Birgitta Vegerfors-Persson for statistical advice. Author also thanks Jan Bengtsson, Bjo¨rn Sohlenius and Cecilia Palmborg for valuable comments on previous versions of the manuscript and the anonymous reviewers for constructive comments on the manuscript. This study was funded by the Oscar and Lili Lamm Foundation and the Swedish Research Council (grant to Jan Bengtsson). References Athanasiadou, S., Kyriazakis, I., 2004. Plant secondary metabolites: antiparasitic effects and their role in ruminant production systems. Proceedings of the Nutrition Society 63, 631–639. Badejo, M.A., Tian, G., 1999. Abundance of soil mites under four agroforestry tree species with contrasting litter quality. Biology and Fertility of Soils 30, 107–112. Bardgett, R.D., Mawdsley, J.L., Edwards, S., Hobbs, P.J., Rodwell, J.S., Davies, W.J., 1999. Plant species and nitrogen effects on soil biological properties of temperate upland grasslands. Functional Ecology 13, 650–660. Bell, N.L., Watson, R.N., 2001a. Population dynamics of Paratylenchus nanus in soil under pasture: 1. Aggregation and abiotic factors. Nematology 3, 187–197. Bell, N.L., Watson, R.N., 2001b. Identification and host range assessment of Paratylenchus nanus (Tylenchida: Tylenchulidae) and Paratrichodorus minor (Triplonchida: Trichodoridae). Nematology 3, 483–490. Bernard, E.C., Gwinn, K.D., Griffin, G.D., 1998. Forage grasses. In: Barker, K.R., Pederson, G.A., Windham, G.L. (Eds.), Plant and Nematode Interactions. Agronomy Monograph 36, Madison, pp. 427–454. Bongers, T., 1990. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14–19. Coulson, S.J., Hodkinson, I.D., Webb, N.R., 2003. Microscale distribution patterns in high Arctic soil microarthropod communities: the influence of plant species within the vegetation mosaic. Ecography 26, 801–809. De Deyn, G.B., Raaijmakers, C.E., Van Ruijven, J., Berendse, F., Van der Putten, W.H., 2004. Plant species identity and diversity effects on
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