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Evaluation of the “bait-lamina test” to assess soil microfauna feeding activity in mixed grassland
applied soil ecology 36 (2007) 199–204
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/apsoil
Evaluation of the ‘‘bait-lamina test’’ to assess soil microfauna feeding activity in mixed grassland Chantal Hamel a,b,*, Michael P. Schellenberg a, Keith Hanson a, Hong Wang a a b
Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK S9H 3X2 Canada Department of Soil Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8 Canada
article info
abstract
Article history:
The soil fauna plays an important role in regulating nutrient cycling through predation and
Accepted 27 February 2007
comminution of organic residues. The bait-lamina test was proposed as a practical mean to assess soil faunal feeding activity. The test consists of vertically inserting 16-hole-bearing
Keywords:
plastic strips filled with a plant material preparation into the soil. We assessed the value of the
Soil faunal feeding activity
bait-lamina test in the mixed grassland ecoregion of Canada, using 5-year-old seeded grass
assessment
field plots. There were five treatments of grass communities, i.e. monocultures of (1) Russian
Methodology
wild rye, (2) switchgrass, (3) green needlegrass, or (4) western wheatgrass, and (5) a grass
Soil biological activity
mixture of western wheatgrass + green needlegrass + switchgrass + little blue stem, which
Soil animals
were distributed in four complete blocks, and six bait mix treatments, i.e. bait made with
Field experiment
ground tissue of (1) Russian wild rye, (2) switchgrass, (3) green needlegrass, (4) western
Bait-lamina test
wheatgrass, (5) alfalfa, or with (6) wheat bran, which were randomized in each plot. Baitlamina strips were used in groups of five strips inserted in the soil; they were equally spaced across the inter-row. The analysis revealed a marginally significant effect (P = 0.065) of plant community on soil faunal feeding activity with more activity in mixed grass species compared to green needlegrass plots. Bait flavour had no significant effect (P = 0.22) on feeding. More (P < 0.0005) feeding activity was detected close to the soil surface (0–5 mm deep) than at 20 mm below the surface and at deeper depths. Feeding activity was relatively low over the 65-day period of the test (13 June to 17 August 2005), with 8.3% and 2.0% of the lamina (holes filled) showing signs of feeding in the 0–5 and 5–15 mm soil layer. We conclude that the bait-lamina test is appropriate to assess the activity of surface litter processing fauna in mixed grassland, but it is not a good indicator of the decomposition occurring in the soil matrix, where most litter is produced. We recommend the use of a large number of replicated bait-lamina strips and a whole growing season incubation period in soils of the semiarid prairie ecoregion where the surface litter layer is thin and faunal saprobes are relatively scarce. # 2007 Published by Elsevier B.V.
1.
Introduction
The soil is a complex environment where plants, microorganisms and small animals interact in the recycling of live and dead organic matter, which impacts soil chemistry. In terrestrial ecosystems, above- and belowground plant inputs
constitute the main sources of carbon and energy for a large and extraordinarily diverse community of living organisms (Ha¨ttenschwiler et al., 2005). Soil bacteria and fungi have the largest biomass and are responsible for most of the mineralization of nutrients from soil organic matter. Although bacteria and fungi can
* Corresponding author at: Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK S9H 3X2 Canada. Tel.: +1 306 778 7264; fax: +1 306 778 3188. E-mail address: [email protected] (C. Hamel). 0929-1393/$ – see front matter # 2007 Published by Elsevier B.V. doi:10.1016/j.apsoil.2007.02.004
200
applied soil ecology 36 (2007) 199–204
accomplish the complete degradation of organic matter, they rarely act alone (Whalen and Hamel, 2004). Some protozoa and nematodes are saprotrophic and directly contribute to decomposition, whereas others are predatory and regulate the activity of fungal and bacterial populations. The mesofauna mainly represented by springtails (Collembola) and mites (Acarina) are involved in the regulation of soil microorganisms but also consume and process considerable amounts of litter. Millipedes (Diplopoda), isopods (Isopoda), enchytraeids (Tubificida) and earthworms (Haplotaxida) displace and fragment litter producing large amounts of feces, which are easily utilized by soil microorganisms. They move surface litter into the soil where the environment is more favourable for microbial decomposers. Understanding soil processes requires the consideration of the soil fauna. Clearly, litter-feeding invertebrates play an important role in the decomposition and recycling of dead organic matter, and their contribution to nutrient cycling needs to be considered together with that of soil fungi and bacteria. Different methods have been developed to study soil animals’ contribution to organic matter decomposition. Among these, the bait-lamina test, proposed by Von To¨rne (1990), was found to best reflects the feeding activity of soil animals (Helling et al., 1998; Von Gestel et al., 2003). The test consists of vertically inserting 16-hole-bearing plastic strips filled with a plant material preparation into the soil. After a period of time, the bait-lamina strips are removed and examined for evidence of feeding. Helling et al. (1998) have shown high feeding activity of Collembola and Enchytraeidae using bait-lamina strips in microcosm experiments, and a reduced impact of microorganisms on the disappearance of the bait. They also demonstrated the preference of the organisms for bait-lamina made with nettle tissues over those made with wheat bran, as initially proposed by Von To¨rne (1990). Soil organisms preferentially feed on certain litter types (Ha¨ttenschwiler et al., 2005). Consequently, bait materials more closely resembling substrates found in different plant communities may be preferred by soil fauna occurring within. The bait-lamina test has been repeatedly used to assess litterfeeders’ activity (Paulus et al., 1999; Filzek et al., 2004), but it remains unclear how different plant stands can influence the results of the test. The bait-lamina test was successfully used in mesic environments, in micro- (Helling et al., 1998) and mesocosms (Von Gestel et al., 2003), in forest ecosystems (Von To¨rne, 1990; Paulus et al., 1999), and in grassland sites (Filzek et al., 2004), to assess the activity of the soil fauna. Moisture deficit characterizes the mixed grassland ecoregion, which offer soil environments less conducive to biological activity than more mesic soils, but soil invertebrates are present. A 5year survey conducted in the Northern part of the North American mixed grassland region at Matador Saskatchewan, approximately 60 km north of our site, found averages of 490 m2 Elatridae; 74,823 m2 Collembola; 31,191 m2 Acarina; 2.7 106 Nematoda; 34,083 m2 Enchytraeidae in the top 0– 30 cm soil layer (Willard, 1974). These numbers are similar to those reported by others from prairie ecosystems (Stanton, 1988), but generally lower than those reported for European grasslands (Paul and Clark, 1996). The bait-lamina test has never been used to assess the feeding activity of soil fauna in the mixed grassland ecoregion,
and the usefulness of this test in a semiarid environment is only speculative. We therefore evaluated the bait-lamina test in mixed grassland soils. The trial was conducted in an agricultural soil growing 5-year-old stands of different forage grasses. We wanted to assess the value of the bait-lamina test for mixed grassland soil environments and, in particular, we wanted to determine if the plant material used to prepare the bait strips would be differentially utilized by the soil faunal population present under differing plant species.
2.
Materials and methods
2.1.
Site description
The experiment was conducted on a Brown Chernozem (Haverhill clay loam) located at the South Farm of the Semiarid Prairie Agricultural Research Centre in Swift Current Saskatchewan, Canada (latitude: 508170 N; longitude: 1078410 W; elevation 825 m). The region receives 361 mm of annual precipitation and has a yearly mean temperature of 3.6 8C with mean monthly temperatures ranging from 13.2 8C in January to 18.6 8C in June (54-year averages). Soil temperature at 5 cm depth under a sod and rainfall received during the period of the study were measured at a weather station located approximately 300 m from the study sites.
2.2.
Experimental design
The bait-lamina method was evaluated in 5-year-old field plots planted with monocultures of Russian wild rye (Elymus junceus Fisch.), switchgrass (Panicum virgatum L.), green needlegrass (Stipa viridula Trin.), or western wheatgrass (Agropyron smithii Rydb.), or with a mixture of western wheatgrass, green needlegrass, little bluestem (Schizachyrium scoparium Michx.) and switchgrass. Each plant species treatment was grown in 4 m 8 m plots arranged in four randomized complete blocks. Six bait flavour treatments were factorially arranged and randomized within each plot. Baitlamina strips are 1-mm thick 6 mm 150 mm plastic strips bearing a series of 16 holes (2 mm diameter) pierced at 5 mm interval in their lower end, which is inserted into the soil. Holes are filled with a ground plant-based mix and, thus, called ‘‘bait-lamina’’. Five bait-lamina strip replicates constituted one experimental unit. Thus, 600 bait-lamina strips were used in this experiment. These strips were inserted in the soil on 13 June 2005. A wooden template containing holes was laid down between two plant rows and the tool used to prepare holes in the soil. The strips were then placed into the holes in the soil using a machined steel insertion tool, which supported and protected the strips during insertion. Bait-lamina strips were placed just deep enough in the soil for the edge of the uppermost bait-lamina to be 3 mm below soil surface (Fig. 1). Four supplementary bait-lamina strips of each flavour were also inserted in the soil of each plot. They were harvested at different time intervals to monitor feeding and to determine the incubation time most appropriate for the test, under mixed grassland field conditions. Experimental bait-lamina strips were removed from the soil on 17 August 2005, prior to forage harvest. The number of lamina showing feeding activity were
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applied soil ecology 36 (2007) 199–204
Quebec), cellulose powder and bentonite (Sigma–Aldrich Inc., St. Louis, MO) in a 1/6.5/1.5/1 proportion calculated on a weight basis. Several applications of the bait preparation were required to load the lamina, because of shrinkage. The recipe was modified by excluding bentonite in later applications. Plant tissue from the 2004 harvest and commercial wheat bran was used to prepare the bait. This material was analysed for: N by the Kjeldahl method (McGill and Figueiredo, 1993), P according to Murphy and Riley (1962), and crude protein by multiplying Kjeldahl N by 6.25. The percentages of N, P and crude protein in the plant materials are presented in Table 1.
2.4. Fig. 1 – Arrangement of one experimental subplot, each of which received one bait flavour treatment.
The significance of treatment effects could not be assessed by ANOVA due to lack of normality, according to the Shapiro Wilks’ test calculated using JMP1 v. 3.2.6 (SAS Institute, Cary, USA). The nonparametric Kruskal–Wallis test (Mcbean and Rovers, 1998) was conducted by PROC NPAR1WAY and treatments were compared by PROC MULTTEST using Bonferroni–Holms correction (Westfall and Wolfinger, 1997). The data were averaged over the 16 depths of each bait-lamina strip to assess plant species treatment effect. The bait flavour effects on soil faunal feeding activity within each plant community treatments as well as the overall bait flavour effect were also computed on data averaged over the 16 bait-lamina of each strip. Percentage of bait-lamina position with evidence of feeding was calculated over each plot, and these data were used to assess the impact of soil depth on feeding across all
counted and their location on the strips, i.e. their depth in soil, recorded. Some bait-lamina had dislodged during manipulation, and some had cracked. They were recorded as such but were not considered as indicators of feeding. Feeding activity was expressed as percentage of individual bait-lamina showing evidence of feeding.
2.3.
Statistical analysis
Preparation of the bait-lamina
Bait materials were prepared by mixing ground dry plant tissues with agar-agar (Anachemia Canada Inc., Montreal,
Table 1 – Analysis of the plant materials used to make the bait-lamina filling Analyses (mg g1 dry weight basis)
Plant tissues used as bait-lamina material
Russian wild rye Western wheatgrass Green needlegrass Switchgrass Alfalfa Wheat bran
Nitrogen
Phosphorus
9.5 8.8 7.2 4.7 22.2 11.5
0.78 0.88 0.95 0.90 4.20 7.90
Crude protein 59 55 45 29 144 75
All the material was harvested from the field experiment the previous year except alfalfa, which was used as a nitrogen rich control, and wheat bran, which was initially proposed as the bait ingredient.
Table 2 – Feeding activity, revealed by the bait-lamina test, in plots with different grass species and pair-wise comparisons of treatment meansa Grass community
Holes fed upon
Comparison
x2
P
Russian wild rye (RWR) Western wheatgrass (WWG) Green needlegrass (GNG) Switchgrass (SG) Mix community (WGLS)
3.2 2.6 1.9 2.4 3.2
GNG vs. RWR GNG vs. SG GNG vs. WGLS GNG vs. WWG RWR vs. SG
5.62 0.29 7.39 1.11 2.99
0.017 0.592 0.007 0.291 0.083
Average
2.7
RWR vs. WGLS RWR vs. WWG SG vs. WGLS SG vs. WWG WGLS vs. WWG
0.21 1.63 4.11 0.21 2.28
0.648 0.202 0.043 0.649 0.131
a
Significant ns ns b
ns ns ns ns ns ns ns
Whole model P value = 0.065. Differences between treatment means are considered significant only after applying Bonferroni–Holms correction for multiple comparisons. Each comparison represent a Kruskal–Wallis chi-square approximation with 1 d.f.
b
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applied soil ecology 36 (2007) 199–204
treatments. Analyses were conducted using the SAS/STAT1 software (SAS Institute Inc., Cary, NC, USA).
3.
Results
Overall soil faunal feeding activity was low over the 65-day period of the trial, with an average rate of 2.7% of the baitlamina positions showing evidence of feeding (Table 2). Under the conditions of the test, bait-lamina strips prepared with different plant materials were not differentially consumed (P = 0.22; mean range: 2.0–3.6% of lamina with evidence of feeding). Furthermore, the plant species did not influence soil faunal feeding preference, as indicated by a lack of significant bait-lamina flavour effect when tested within all five plant species treatments used in this trial (data not shown). Marginally larger feeding activity (P = 0.065) was found in the grass mix than in green needlegrass plots, and the difference between treatment means was small with feeding activity values spanning from 1.9% for green needlegrass, to 3.2% for the grass species mix (Table 2). Feeding activity decreased (P < 0.0005) from 8.3% of lamina showing evidence of feeding at 5 mm below soil surface to 2.0%, on average, in the 20–80 mm soil layer (Fig. 2). Air temperature and precipitation during the study period were close to normal (120-year average). June was wetter (123.2 mm, normal: 73.2 mm) and July drier (21.4 mm, normal: 52.0 mm) than normal, resulting in a slightly drier 1 June to 14 August period (134.2 mm, normal: 146.7 mm). Air temperature during the same period averaged 16.8 8C, i.e. 0.5 8C below
Fig. 2 – Variation in soil animal feeding activity with depth across all treatments, as revealed by the bait-lamina test (P = 0.0005; N = 20). Multiple mean comparisons are considered significant (P = 0.05) only after applying Bonferroni–Holms correction for multiple comparisons to the test. Each pair-wise comparison represent a Kruskal– Wallis chi-square approximation with 1 d.f.
Fig. 3 – Daily precipitation, and minimum (~) and maximum (^) soil temperature at 5 cm below soil surface from 1 June to 31 August 2005.
normal. The soil temperature and the distribution of the precipitation received during the study period are presented in Fig. 3.
4.
Discussion
The soil faunal feeding activity measured over a period of 65 days under summer mixed grassland conditions was relatively low (2.7% on average). As a comparison, mean feeding activities measured with the bait-lamina test ranged from about 50 to 1% over 6 days in grasslands sites in UK (Filzek et al., 2004). Values between 15.8 and 4.1% over 44 h were found in German forest soils (Von To¨rne, 1990), and Helling et al. (1998) reported mean feeding activities ranging from 13% to less than 4% with incubation times of 15–43 days, in microcosms containing various combinations of Enchytraeidae, Collembola and microorganisms. Differentiating evidence of feeding from cracked or lost bait is largely subjective. Our determinations were conservative and this may have also contributed to the low percentage of feeding we found in comparison with similar studies. In spite of the relatively low activity found in our study plots, our analysis was precise enough to detect a significant effect of soil depth. The difference in feeding activity in the grass mix and green needlegrass plots was marginally significant (P = 0.065) and small, with feeding activity values of 1.9% in green needlegrass and 3.2% for the grass species mix (Table 2). Greater feeding in mixed grasses stands may reflect lower invertebrate abundance under monoculture, as it was reported by others (St. John et al., 2006). We conclude from these observations that, technically, the bait-lamina test can be used to assess soil faunal feeding over a normal North American mixed grassland summer period and that using wheat bran in the bait is as appropriate as using other plant materials. Feeding activity revealed by the bait-lamina test was reduced below 15 mm of the soil surface. This feeding reduction may be surprising considering that plant residues
applied soil ecology 36 (2007) 199–204
are abundant in soil. Sixty to 80% of total net primary productivity (NPP) occurs belowground in mixed grassland (Stanton, 1988). Mean root:shoot ratio vary from 2:1 to 13:1 across North American prairie regions, with higher values associated with cooler regions like the Swift Current area. The bait-lamina test detected lower activity belowground where most NPP occurs suggesting that this test does not assess belowground decomposition processes, at least in our mixed grassland. While the general principle of the technique seems appropriate for the assessment of epigeic animal feeding associated with surface litter decomposition, its value to assess belowground nutrient cycling-related activity appears questionable. The bait-lamina test has yielded results different from that of other methods of biological activity assessment suggesting that different methods reflect the activity of different groups (Kula and Rombke, 1998; Paulus et al., 1999). The activity of Collembola and Enchytraeidae may be revealed by the test, but the activity of microorganisms is hard to detect (Helling et al., 1998) probably due to the low surface to volume ratio of the baitlamina. Von Gestel et al. (2003) found increasing bait-lamina consumption rate with increasing earthworm density, and no significant consumption in mesocosms containing only Collembola and Acarina, over a period of 60 days. Earthworms appear as very effective feeders on bait-lamina strips and the bait-lamina test seems to largely reflect their activity. Dry soils are not a favourable habitat for earthworms, which are rarely or never found in arid and semiarid grasslands (Stanton, 1988). No earthworms were ever found at the field research station where our study was located and no earthworms were reported in a 5-year survey conducted 60 km away from our study site (Willard, 1974). The absence of earthworms in our study site may explain the low feeding activity revealed by the bait-strip test, in particular at lower depths (15–80 mm). Standing microbial biomass is about equivalent to half that of net primary productivity (NPP) and in grassland, mineralization was explained by the activity of bacterivores and fungivores, mostly protozoa and nematodes (Stanton, 1988), which are quite evenly distributed throughout the top 300 mm of the soil in mixed grass prairie (Willard, 1974). Such activities are not revealed by the bait-lamina test. Our results support the conclusion that animal feeding on plant materials in mixed grassland soils is significant only in the surface litter layer, which is well colonized by epigeic saprobes such as the Acarina. Belowground mineralization processes should be better described through enumeration of bacterivorous and fungivorous invertebrates extracted from soil. Another limitation of the bait-lamina test comes from the fact that the data produced with this test has a non-normal distribution (Von To¨rne, 1990), which can rarely be corrected with data transformation. This prevents the use of ANOVA, the most powerful statistical analysis for experimental data (Scherrer, 1984) and restricts statistical treatment to one-way analyses.
5.
Conclusion
The bait-lamina test is appropriate to assess the activity of soil animals involved in the comminution of soil litter of
203
plant origin, a process that seems largely restricted to the few uppermost millimetres of the soil, in mixed grassland soils. We measured low feeding activity in our mixed grassland plots indicating that the number of bait-lamina strips per plot must be high for the test to achieve good precision. We used 30 bait-lamina strips per plot. This level of duplication should be maintained or increased in further work. We also recommend to incubate the bait-lamina strips from early spring to the first killing frost, in Canadian mixed grassland soils.
Acknowledgements Thanks to D. Wiebe for his perseverance in loading the baitlamina strips and in data acquisition and to D. Judiesch for providing the weather data.
references
Filzek, P.D.B., Spurgeon, D.J., Broll, G., Svendsen, C., Hankard, P.K., Parekh, N., Stubberud, H.E., Weeks, J.M., 2004. Metal effects on soil invertebrate feeding: measurements using the bait lamina method. Ecotoxicology 13, 807–816. Ha¨ttenschwiler, S., Tiunov, A.V., Scheu, S., 2005. Biodiversity and litter decomposition in terrestrial ecosystems. Ann. Rev. Ecol. Syst. 36, 191–218. Helling, B., Pfeiff, G., Larink, O., 1998. A comparison of feeding activity of collembolan and enchytraeid in laboratory studies using the bait-lamina test. Appl. Soil Ecol. 7, 207–212. Kula, C., Rombke, J., 1998. Evaluation of soil ecotoxicity tests with functional endpoints for the risk assessment of plant protection products—state-of-the-art. Environ. Sci. Pollut. Res. 5, 55–60. Mcbean, E.A., Rovers, F.A., 1998. Statistical Procedures for Analysis of Environmental Monitoring Data and Risk Assessment. Prentice Hall, Upper Saddle River, NJ, pp. 313. McGill, W.B., Figueiredo, C.T., 1993. Total nitrogen. In: Carter, M. (Ed.), Soil Sampling and Methods of Soil Analysis. Lewis Publisher, Boca Raton, FL, pp. 201–212. Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chem. Acta 27, 109–112. Paul, E.A., Clark, F.E., 1996. Soil Microbiology and Biochemistry. Academic Press, San Diego. Paulus, R., Rombke, J., Ruf, A., Beck, L., 1999. A comparison of the litterbag-, minicontainer- and bait-lamina-methods in an ecotoxicological field experiment with diflubenzuron and btk. Pedobiologia 43, 120–133. Scherrer, B., 1984. Biostatistique. Gae¨tan Morin E´diteur. Chicoutimi, Canada, 850 p. St. John, M.G., Wall, D.D., Behan-Pelletier, V.M., 2006. Does plant species co-occurence influence soil mite diversity? Ecology 87, 625–633. Stanton, N.L., 1988. The underground in grasslands. Ann. Rev. Ecol. Syst. 19, 573–589. Von Gestel, C.A.M., Kruidenier, M., Berg, M.P., 2003. Suitability of wheat straw decomposition, cotton strip degradation and bait-lamina feeding tests to determine soil invertebrate activity. Biol. Fertil. Soils 37, 115–123.
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Von To¨rne, E., 1990. Assessing feeding activities of soil-living animals. Pedobiologia 34, 89–101. Westfall, P.H., Wolfinger, R.D., 1997. Multiple tests with discrete distributions. Am. Stat. 51, 3–8. Whalen, J.K., Hamel, C., 2004. Effects of key soil organisms on nutrient dynamics in temperate agroecosystems. In:
Clements, D., Shrestha, A. (Eds.), New Dimensions in Agroecology. Haworth Food Products Press, New York, pp. 175–208. Willard, J.R., 1974. Soil Invertegrates: VIII. A Summary of Populations and Biomass. University of Saskatchewan, Saskatoon, p. 110.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/apsoil
Evaluation of the ‘‘bait-lamina test’’ to assess soil microfauna feeding activity in mixed grassland Chantal Hamel a,b,*, Michael P. Schellenberg a, Keith Hanson a, Hong Wang a a b
Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK S9H 3X2 Canada Department of Soil Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8 Canada
article info
abstract
Article history:
The soil fauna plays an important role in regulating nutrient cycling through predation and
Accepted 27 February 2007
comminution of organic residues. The bait-lamina test was proposed as a practical mean to assess soil faunal feeding activity. The test consists of vertically inserting 16-hole-bearing
Keywords:
plastic strips filled with a plant material preparation into the soil. We assessed the value of the
Soil faunal feeding activity
bait-lamina test in the mixed grassland ecoregion of Canada, using 5-year-old seeded grass
assessment
field plots. There were five treatments of grass communities, i.e. monocultures of (1) Russian
Methodology
wild rye, (2) switchgrass, (3) green needlegrass, or (4) western wheatgrass, and (5) a grass
Soil biological activity
mixture of western wheatgrass + green needlegrass + switchgrass + little blue stem, which
Soil animals
were distributed in four complete blocks, and six bait mix treatments, i.e. bait made with
Field experiment
ground tissue of (1) Russian wild rye, (2) switchgrass, (3) green needlegrass, (4) western
Bait-lamina test
wheatgrass, (5) alfalfa, or with (6) wheat bran, which were randomized in each plot. Baitlamina strips were used in groups of five strips inserted in the soil; they were equally spaced across the inter-row. The analysis revealed a marginally significant effect (P = 0.065) of plant community on soil faunal feeding activity with more activity in mixed grass species compared to green needlegrass plots. Bait flavour had no significant effect (P = 0.22) on feeding. More (P < 0.0005) feeding activity was detected close to the soil surface (0–5 mm deep) than at 20 mm below the surface and at deeper depths. Feeding activity was relatively low over the 65-day period of the test (13 June to 17 August 2005), with 8.3% and 2.0% of the lamina (holes filled) showing signs of feeding in the 0–5 and 5–15 mm soil layer. We conclude that the bait-lamina test is appropriate to assess the activity of surface litter processing fauna in mixed grassland, but it is not a good indicator of the decomposition occurring in the soil matrix, where most litter is produced. We recommend the use of a large number of replicated bait-lamina strips and a whole growing season incubation period in soils of the semiarid prairie ecoregion where the surface litter layer is thin and faunal saprobes are relatively scarce. # 2007 Published by Elsevier B.V.
1.
Introduction
The soil is a complex environment where plants, microorganisms and small animals interact in the recycling of live and dead organic matter, which impacts soil chemistry. In terrestrial ecosystems, above- and belowground plant inputs
constitute the main sources of carbon and energy for a large and extraordinarily diverse community of living organisms (Ha¨ttenschwiler et al., 2005). Soil bacteria and fungi have the largest biomass and are responsible for most of the mineralization of nutrients from soil organic matter. Although bacteria and fungi can
* Corresponding author at: Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada, Swift Current, SK S9H 3X2 Canada. Tel.: +1 306 778 7264; fax: +1 306 778 3188. E-mail address: [email protected] (C. Hamel). 0929-1393/$ – see front matter # 2007 Published by Elsevier B.V. doi:10.1016/j.apsoil.2007.02.004
200
applied soil ecology 36 (2007) 199–204
accomplish the complete degradation of organic matter, they rarely act alone (Whalen and Hamel, 2004). Some protozoa and nematodes are saprotrophic and directly contribute to decomposition, whereas others are predatory and regulate the activity of fungal and bacterial populations. The mesofauna mainly represented by springtails (Collembola) and mites (Acarina) are involved in the regulation of soil microorganisms but also consume and process considerable amounts of litter. Millipedes (Diplopoda), isopods (Isopoda), enchytraeids (Tubificida) and earthworms (Haplotaxida) displace and fragment litter producing large amounts of feces, which are easily utilized by soil microorganisms. They move surface litter into the soil where the environment is more favourable for microbial decomposers. Understanding soil processes requires the consideration of the soil fauna. Clearly, litter-feeding invertebrates play an important role in the decomposition and recycling of dead organic matter, and their contribution to nutrient cycling needs to be considered together with that of soil fungi and bacteria. Different methods have been developed to study soil animals’ contribution to organic matter decomposition. Among these, the bait-lamina test, proposed by Von To¨rne (1990), was found to best reflects the feeding activity of soil animals (Helling et al., 1998; Von Gestel et al., 2003). The test consists of vertically inserting 16-hole-bearing plastic strips filled with a plant material preparation into the soil. After a period of time, the bait-lamina strips are removed and examined for evidence of feeding. Helling et al. (1998) have shown high feeding activity of Collembola and Enchytraeidae using bait-lamina strips in microcosm experiments, and a reduced impact of microorganisms on the disappearance of the bait. They also demonstrated the preference of the organisms for bait-lamina made with nettle tissues over those made with wheat bran, as initially proposed by Von To¨rne (1990). Soil organisms preferentially feed on certain litter types (Ha¨ttenschwiler et al., 2005). Consequently, bait materials more closely resembling substrates found in different plant communities may be preferred by soil fauna occurring within. The bait-lamina test has been repeatedly used to assess litterfeeders’ activity (Paulus et al., 1999; Filzek et al., 2004), but it remains unclear how different plant stands can influence the results of the test. The bait-lamina test was successfully used in mesic environments, in micro- (Helling et al., 1998) and mesocosms (Von Gestel et al., 2003), in forest ecosystems (Von To¨rne, 1990; Paulus et al., 1999), and in grassland sites (Filzek et al., 2004), to assess the activity of the soil fauna. Moisture deficit characterizes the mixed grassland ecoregion, which offer soil environments less conducive to biological activity than more mesic soils, but soil invertebrates are present. A 5year survey conducted in the Northern part of the North American mixed grassland region at Matador Saskatchewan, approximately 60 km north of our site, found averages of 490 m2 Elatridae; 74,823 m2 Collembola; 31,191 m2 Acarina; 2.7 106 Nematoda; 34,083 m2 Enchytraeidae in the top 0– 30 cm soil layer (Willard, 1974). These numbers are similar to those reported by others from prairie ecosystems (Stanton, 1988), but generally lower than those reported for European grasslands (Paul and Clark, 1996). The bait-lamina test has never been used to assess the feeding activity of soil fauna in the mixed grassland ecoregion,
and the usefulness of this test in a semiarid environment is only speculative. We therefore evaluated the bait-lamina test in mixed grassland soils. The trial was conducted in an agricultural soil growing 5-year-old stands of different forage grasses. We wanted to assess the value of the bait-lamina test for mixed grassland soil environments and, in particular, we wanted to determine if the plant material used to prepare the bait strips would be differentially utilized by the soil faunal population present under differing plant species.
2.
Materials and methods
2.1.
Site description
The experiment was conducted on a Brown Chernozem (Haverhill clay loam) located at the South Farm of the Semiarid Prairie Agricultural Research Centre in Swift Current Saskatchewan, Canada (latitude: 508170 N; longitude: 1078410 W; elevation 825 m). The region receives 361 mm of annual precipitation and has a yearly mean temperature of 3.6 8C with mean monthly temperatures ranging from 13.2 8C in January to 18.6 8C in June (54-year averages). Soil temperature at 5 cm depth under a sod and rainfall received during the period of the study were measured at a weather station located approximately 300 m from the study sites.
2.2.
Experimental design
The bait-lamina method was evaluated in 5-year-old field plots planted with monocultures of Russian wild rye (Elymus junceus Fisch.), switchgrass (Panicum virgatum L.), green needlegrass (Stipa viridula Trin.), or western wheatgrass (Agropyron smithii Rydb.), or with a mixture of western wheatgrass, green needlegrass, little bluestem (Schizachyrium scoparium Michx.) and switchgrass. Each plant species treatment was grown in 4 m 8 m plots arranged in four randomized complete blocks. Six bait flavour treatments were factorially arranged and randomized within each plot. Baitlamina strips are 1-mm thick 6 mm 150 mm plastic strips bearing a series of 16 holes (2 mm diameter) pierced at 5 mm interval in their lower end, which is inserted into the soil. Holes are filled with a ground plant-based mix and, thus, called ‘‘bait-lamina’’. Five bait-lamina strip replicates constituted one experimental unit. Thus, 600 bait-lamina strips were used in this experiment. These strips were inserted in the soil on 13 June 2005. A wooden template containing holes was laid down between two plant rows and the tool used to prepare holes in the soil. The strips were then placed into the holes in the soil using a machined steel insertion tool, which supported and protected the strips during insertion. Bait-lamina strips were placed just deep enough in the soil for the edge of the uppermost bait-lamina to be 3 mm below soil surface (Fig. 1). Four supplementary bait-lamina strips of each flavour were also inserted in the soil of each plot. They were harvested at different time intervals to monitor feeding and to determine the incubation time most appropriate for the test, under mixed grassland field conditions. Experimental bait-lamina strips were removed from the soil on 17 August 2005, prior to forage harvest. The number of lamina showing feeding activity were
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Quebec), cellulose powder and bentonite (Sigma–Aldrich Inc., St. Louis, MO) in a 1/6.5/1.5/1 proportion calculated on a weight basis. Several applications of the bait preparation were required to load the lamina, because of shrinkage. The recipe was modified by excluding bentonite in later applications. Plant tissue from the 2004 harvest and commercial wheat bran was used to prepare the bait. This material was analysed for: N by the Kjeldahl method (McGill and Figueiredo, 1993), P according to Murphy and Riley (1962), and crude protein by multiplying Kjeldahl N by 6.25. The percentages of N, P and crude protein in the plant materials are presented in Table 1.
2.4. Fig. 1 – Arrangement of one experimental subplot, each of which received one bait flavour treatment.
The significance of treatment effects could not be assessed by ANOVA due to lack of normality, according to the Shapiro Wilks’ test calculated using JMP1 v. 3.2.6 (SAS Institute, Cary, USA). The nonparametric Kruskal–Wallis test (Mcbean and Rovers, 1998) was conducted by PROC NPAR1WAY and treatments were compared by PROC MULTTEST using Bonferroni–Holms correction (Westfall and Wolfinger, 1997). The data were averaged over the 16 depths of each bait-lamina strip to assess plant species treatment effect. The bait flavour effects on soil faunal feeding activity within each plant community treatments as well as the overall bait flavour effect were also computed on data averaged over the 16 bait-lamina of each strip. Percentage of bait-lamina position with evidence of feeding was calculated over each plot, and these data were used to assess the impact of soil depth on feeding across all
counted and their location on the strips, i.e. their depth in soil, recorded. Some bait-lamina had dislodged during manipulation, and some had cracked. They were recorded as such but were not considered as indicators of feeding. Feeding activity was expressed as percentage of individual bait-lamina showing evidence of feeding.
2.3.
Statistical analysis
Preparation of the bait-lamina
Bait materials were prepared by mixing ground dry plant tissues with agar-agar (Anachemia Canada Inc., Montreal,
Table 1 – Analysis of the plant materials used to make the bait-lamina filling Analyses (mg g1 dry weight basis)
Plant tissues used as bait-lamina material
Russian wild rye Western wheatgrass Green needlegrass Switchgrass Alfalfa Wheat bran
Nitrogen
Phosphorus
9.5 8.8 7.2 4.7 22.2 11.5
0.78 0.88 0.95 0.90 4.20 7.90
Crude protein 59 55 45 29 144 75
All the material was harvested from the field experiment the previous year except alfalfa, which was used as a nitrogen rich control, and wheat bran, which was initially proposed as the bait ingredient.
Table 2 – Feeding activity, revealed by the bait-lamina test, in plots with different grass species and pair-wise comparisons of treatment meansa Grass community
Holes fed upon
Comparison
x2
P
Russian wild rye (RWR) Western wheatgrass (WWG) Green needlegrass (GNG) Switchgrass (SG) Mix community (WGLS)
3.2 2.6 1.9 2.4 3.2
GNG vs. RWR GNG vs. SG GNG vs. WGLS GNG vs. WWG RWR vs. SG
5.62 0.29 7.39 1.11 2.99
0.017 0.592 0.007 0.291 0.083
Average
2.7
RWR vs. WGLS RWR vs. WWG SG vs. WGLS SG vs. WWG WGLS vs. WWG
0.21 1.63 4.11 0.21 2.28
0.648 0.202 0.043 0.649 0.131
a
Significant ns ns b
ns ns ns ns ns ns ns
Whole model P value = 0.065. Differences between treatment means are considered significant only after applying Bonferroni–Holms correction for multiple comparisons. Each comparison represent a Kruskal–Wallis chi-square approximation with 1 d.f.
b
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treatments. Analyses were conducted using the SAS/STAT1 software (SAS Institute Inc., Cary, NC, USA).
3.
Results
Overall soil faunal feeding activity was low over the 65-day period of the trial, with an average rate of 2.7% of the baitlamina positions showing evidence of feeding (Table 2). Under the conditions of the test, bait-lamina strips prepared with different plant materials were not differentially consumed (P = 0.22; mean range: 2.0–3.6% of lamina with evidence of feeding). Furthermore, the plant species did not influence soil faunal feeding preference, as indicated by a lack of significant bait-lamina flavour effect when tested within all five plant species treatments used in this trial (data not shown). Marginally larger feeding activity (P = 0.065) was found in the grass mix than in green needlegrass plots, and the difference between treatment means was small with feeding activity values spanning from 1.9% for green needlegrass, to 3.2% for the grass species mix (Table 2). Feeding activity decreased (P < 0.0005) from 8.3% of lamina showing evidence of feeding at 5 mm below soil surface to 2.0%, on average, in the 20–80 mm soil layer (Fig. 2). Air temperature and precipitation during the study period were close to normal (120-year average). June was wetter (123.2 mm, normal: 73.2 mm) and July drier (21.4 mm, normal: 52.0 mm) than normal, resulting in a slightly drier 1 June to 14 August period (134.2 mm, normal: 146.7 mm). Air temperature during the same period averaged 16.8 8C, i.e. 0.5 8C below
Fig. 2 – Variation in soil animal feeding activity with depth across all treatments, as revealed by the bait-lamina test (P = 0.0005; N = 20). Multiple mean comparisons are considered significant (P = 0.05) only after applying Bonferroni–Holms correction for multiple comparisons to the test. Each pair-wise comparison represent a Kruskal– Wallis chi-square approximation with 1 d.f.
Fig. 3 – Daily precipitation, and minimum (~) and maximum (^) soil temperature at 5 cm below soil surface from 1 June to 31 August 2005.
normal. The soil temperature and the distribution of the precipitation received during the study period are presented in Fig. 3.
4.
Discussion
The soil faunal feeding activity measured over a period of 65 days under summer mixed grassland conditions was relatively low (2.7% on average). As a comparison, mean feeding activities measured with the bait-lamina test ranged from about 50 to 1% over 6 days in grasslands sites in UK (Filzek et al., 2004). Values between 15.8 and 4.1% over 44 h were found in German forest soils (Von To¨rne, 1990), and Helling et al. (1998) reported mean feeding activities ranging from 13% to less than 4% with incubation times of 15–43 days, in microcosms containing various combinations of Enchytraeidae, Collembola and microorganisms. Differentiating evidence of feeding from cracked or lost bait is largely subjective. Our determinations were conservative and this may have also contributed to the low percentage of feeding we found in comparison with similar studies. In spite of the relatively low activity found in our study plots, our analysis was precise enough to detect a significant effect of soil depth. The difference in feeding activity in the grass mix and green needlegrass plots was marginally significant (P = 0.065) and small, with feeding activity values of 1.9% in green needlegrass and 3.2% for the grass species mix (Table 2). Greater feeding in mixed grasses stands may reflect lower invertebrate abundance under monoculture, as it was reported by others (St. John et al., 2006). We conclude from these observations that, technically, the bait-lamina test can be used to assess soil faunal feeding over a normal North American mixed grassland summer period and that using wheat bran in the bait is as appropriate as using other plant materials. Feeding activity revealed by the bait-lamina test was reduced below 15 mm of the soil surface. This feeding reduction may be surprising considering that plant residues
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are abundant in soil. Sixty to 80% of total net primary productivity (NPP) occurs belowground in mixed grassland (Stanton, 1988). Mean root:shoot ratio vary from 2:1 to 13:1 across North American prairie regions, with higher values associated with cooler regions like the Swift Current area. The bait-lamina test detected lower activity belowground where most NPP occurs suggesting that this test does not assess belowground decomposition processes, at least in our mixed grassland. While the general principle of the technique seems appropriate for the assessment of epigeic animal feeding associated with surface litter decomposition, its value to assess belowground nutrient cycling-related activity appears questionable. The bait-lamina test has yielded results different from that of other methods of biological activity assessment suggesting that different methods reflect the activity of different groups (Kula and Rombke, 1998; Paulus et al., 1999). The activity of Collembola and Enchytraeidae may be revealed by the test, but the activity of microorganisms is hard to detect (Helling et al., 1998) probably due to the low surface to volume ratio of the baitlamina. Von Gestel et al. (2003) found increasing bait-lamina consumption rate with increasing earthworm density, and no significant consumption in mesocosms containing only Collembola and Acarina, over a period of 60 days. Earthworms appear as very effective feeders on bait-lamina strips and the bait-lamina test seems to largely reflect their activity. Dry soils are not a favourable habitat for earthworms, which are rarely or never found in arid and semiarid grasslands (Stanton, 1988). No earthworms were ever found at the field research station where our study was located and no earthworms were reported in a 5-year survey conducted 60 km away from our study site (Willard, 1974). The absence of earthworms in our study site may explain the low feeding activity revealed by the bait-strip test, in particular at lower depths (15–80 mm). Standing microbial biomass is about equivalent to half that of net primary productivity (NPP) and in grassland, mineralization was explained by the activity of bacterivores and fungivores, mostly protozoa and nematodes (Stanton, 1988), which are quite evenly distributed throughout the top 300 mm of the soil in mixed grass prairie (Willard, 1974). Such activities are not revealed by the bait-lamina test. Our results support the conclusion that animal feeding on plant materials in mixed grassland soils is significant only in the surface litter layer, which is well colonized by epigeic saprobes such as the Acarina. Belowground mineralization processes should be better described through enumeration of bacterivorous and fungivorous invertebrates extracted from soil. Another limitation of the bait-lamina test comes from the fact that the data produced with this test has a non-normal distribution (Von To¨rne, 1990), which can rarely be corrected with data transformation. This prevents the use of ANOVA, the most powerful statistical analysis for experimental data (Scherrer, 1984) and restricts statistical treatment to one-way analyses.
5.
Conclusion
The bait-lamina test is appropriate to assess the activity of soil animals involved in the comminution of soil litter of
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plant origin, a process that seems largely restricted to the few uppermost millimetres of the soil, in mixed grassland soils. We measured low feeding activity in our mixed grassland plots indicating that the number of bait-lamina strips per plot must be high for the test to achieve good precision. We used 30 bait-lamina strips per plot. This level of duplication should be maintained or increased in further work. We also recommend to incubate the bait-lamina strips from early spring to the first killing frost, in Canadian mixed grassland soils.
Acknowledgements Thanks to D. Wiebe for his perseverance in loading the baitlamina strips and in data acquisition and to D. Judiesch for providing the weather data.
references
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