Plant and Soil 198: 109–116, 1998. 109c 1998 Kluwer Academic Publishers. Printed in the Netherlands.

An extraction method for nematodes in decomposition studies using theminicontainer-method

Ralf Lenz and Gerhard EisenbeisInstitut fur Zoologie, Johannes Gutenberg-Universitat, 55099 Mainz, Germany�

Received 18 June 1997. Accepted in revised form 10 November 1997

Key words: agro-ecosystem, biomonitoring, decomposition, extraction, minicontainer, Nematoda

Abstract

The minicontainer-method is a new method developed to study biological processes related to soil litter decomposi-tion. An adaptation of the classical Baermann-funnel technique is described which can be used, in association withthe minicontainer method, to investigate the role of Nematoda in litter decomposition. The use of the extractionmethod is illustrated in a study of the effects of different tillage systems on the decomposition of rye straw and onthe nematode density in minicontainers with different mesh sizes of 20 �m, 500 �m and 2 mm. Three tilled plots(conventional deep plough, cultivator and two-layer plough) and an untilled control were compared after periods of4 weeks and 38 weeks. On both sample dates there were significant main effects of treatment and mesh size on thenematode density, and additionally, after 38 weeks significant treatment x soil depth interactions. After 4 weeks,there were significant main effects of treatment and soil depth on the decomposition, but no mesh size effects,whereas after 38 weeks, all experimental factors had a significant effect on the decomposition of the straw. Dueto the small volume of litter substrate used in the minicontainer method, the efficiency of nematode extraction ishigh and the lack of oxygen in the minicontainers presents no serious problem during the extraction process. Themethod also allows the simultaneous extraction of a large number of samples within a short period of time. Ourresults indicate that the method is suitable to study the microdistribution of nematode activity within the soil profileand improves the possible applications of the minicontainer-method.

Introduction

The minicontainer-method (Eisenbeis, 1993; Eisen-beis et al., 1995) is a miniaturized litterbag-methodwith various applications in the study of decomposi-tion of organic matter on a microscale level. Hither-to it has been applied in several studies on the bio-logical activity of soils in agricultural and forest soilecosystems, and it offers, in contrast to larger-sizedlitterbags, an opportunity easily to measure the decom-position activity without serious disturbance of the soilecosystem (Eisenbeis et al., 1996 a,b). In general, soillitter decomposition is influenced by both biotic andabiotic factors and, while soil microorganisms play apredominant role in actual decomposition, larger ani-mals have a primarily regulating function influencing

� FAX No.: +49 6131393835.E-mail: lenzr000@mail.uni mainz.de

the rate of litter breakdown (Seastedt, 1984). Nema-todes are ubiquitous soil fauna that feed as herbivoreson living plants or as bacterivores on microflora, thusregulating decomposition and the release of nutrients.Since nematodes occur in most soils at high density andspecies number, they are very suitable as bioindicatorsof soil conditions. Recent interest in nematodes andtheir interactions with other soil organisms has addedto information regarding their role in soil ecosystems(Bongers, 1990; Yeates et al., 1993; Yeates, 1994).

It is especially important in agricultural soils thatplant residues are quickly processed and nutrients aremade available to the following crop. The vertical dis-tribution of plant residues is influenced by the tillagesystem. It is more homogeneous in ploughed soils,but residues are concentrated near the surface in soilsunder no tillage or disc tillage (Staricka et al., 1991).Agricultural practices also influence soil physical and

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chemical properties and lead to changes in microbialbiomass and enzyme activities (Franzluebbers et al.,1994; Deng and Tabatabai, 1996 a,b). The decompo-sition rate of plant litter reflects the total biologicalactivity in soils, thus integrating the activities of soilorganisms.

The objective of this study was to develop a sim-ple method to study the role of nematodes in litterdecomposition in minicontainers by developing a suit-able methodology for their efficient extraction from testsubstrates. The examination of nematodes enlarges thepossible applications of the minicontainer-method inmonitoring the biological activity of soils.

Material and methods

Study site and treatments

The development of the extraction method was carriedout within a study of the effects of different tillage sys-tems on the soil ecosystem. The study site is locatedat Worrstadt (Rhineland-Palatinate, Germany; latitude49�500 N, longitude 8�70 E), 230 m above sea level.Mean annual temperature is 10 �C and mean annualprecipitation is 530 mm. The bedrock is loess with aPararendzina (FAO: Calcaric Regosol) soil type. Theexperimental plots (each 12 m � 100 m) were sepa-rated from each other by 2 m untilled grassland. Eachtreatment was replicated twice. The crop was winter-wheat after green fallow in the previous year. Thetillage systems under comparison were:1. untilled control;2. conventional deep plough, inversion of the top30 cm;3. two-layer plough, inversion of the top 15 cm andripping tines from 15–30 cm;4. cultivator, ripping tines to 30 cm.

Experimental design and extraction method

The minicontainer-system consists of small polyeth-ylene-tubes (length 16 mm, diameter 11 mm) filledwith test substrate which are placed into PVC-bars(Figure 1a). The bars are then inserted into the soil. Theminicontainers were filled with 200-210 mg rye strawand closed on both sides with gauze discs of 20 �m,500 �m, or 2 mm mesh size to exclude soil fauna ofdifferent body size. The minicontainers of differentmesh size were placed in the bars in a randomizeddesign. In order to study the nematodes colonizing the

litter in the minicontainers, the bars were placed inthe soil vertically for periods of 4 and 38 weeks. Thedistance between the bars was 1 m. At each sampledate, in July 1995 and March 1996, 30 minicontainers(5 replicates per mesh size and soil depth 0–10 cm or10–20 cm) were taken from each treatment, a total of120 minicontainers per sample date. The fields weretilled and planted with the winter-wheat in October1995.

After removal of the minicontainers from the studysite, any external adhering soil was removed and thefine mesh ends were replaced by coarse 2 mm-gauze.To avoid contamination of extracts by soil, the lowerends of the containers were covered with tissue paperduring extraction. The minicontainers were then insert-ed into tightly fitting plastic funnels (Figure 1b) andsubmerged in plastic beakers filled with 20 ml tapwa-ter (Figure 1c). The samples were then placed in thelaboratory at 20 �C. Following an extraction time of24 h the funnels are removed and the nematodes insuspension in the beakers were counted. After extrac-tion, the litter was removed from a randomized chosensubset of 12 containers and flushed into petri dishes toassess the total nematode number in the minicontainersby counting any remaining nematodes trapped in thelitter. The extraction efficiency was calculated as thepercentage of nematodes counted in the suspension.The number of unextracted nematodes killed duringthe extraction process was negligible (approx. 5% oftotal nematode number).

After extraction of the nematodes, the litter wasremoved from the containers and mass loss and watercontent of the litter was determined gravimetrically.The litter was routinely contaminated with soil. There-fore the samples were dry ashed in a muffle furnace at600 �C for 2 h.

Statistical testing was performed using the STATIS-TICA package (StatSoft, Inc.). Differences betweenthe extraction efficiencies for the containers after expo-sure times of 4 weeks and 38 weeks were testedusing the Mann-Whitney-U-Test, correlations betweenextraction efficiency, nematode number and litter masswere calculated using the nonparametric Spearman-correlation. The significance of effects of cultivationtreatment, mesh size and soil depth on nematode densi-ty and decomposition rate was tested by means of a 3-way analysis of variance (ANOVA). Tests for normal-ity, for homogeneity of variances and for correlationof means and variances revealed that a natural log (x)transformation of the data was necessary. Differencesbetween the means were tested using the Tukey-Test.

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Figure 1. Apparatus for extraction of nematodes from minicontainers: a, bar with minicontainers (MC); b, minicontainer (MC) inserted intoplastic funnel (FU); c, funnel (FU) submerged into plastic beaker (PB) filled with tapwater.

Table 1. Efficiency (percentage of extracted nematodes, mean � standard error, n = 12)of the extraction of nematodes from minicontainers sampled after 4 and 38 weeks

Sampling after 4 weeks Sampling after 38 weeks

Suspension Litter Efficiency % Suspension Litter Efficiency %

232.83 88.83 72.7 176.92 43.92 79.1

� 42.70 � 17.23 � 25.53 � 6.03

Results

Extraction efficiency

The average extraction efficiency for living nema-todes (Table 1) was 72.7% for the containers thatwere sampled after 4 weeks and 79.1% for that after38 weeks. The difference is statistically significant(Mann-Whitney U-Test, p = 0.049). No correlation wasfound between extraction efficiency and total number

of nematodes, and between extraction efficiency andextracted sample mass.

Water content

After 4 weeks, the mean water content of the litter inthe minicontainers was 284.9% (20 �m gauze), 208.3(500 �m), and 263.1 (2 mm). After 38 weeks, it was336.4% (20 �m), 230.8 (500 �m), and 329.7 (2 mm).On both sampling dates, the water content was signif-icantly lower in the 500 �m containers than in the fine

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Figure 2. Water contents [% dry mass] in minicontainers with dif-ferent mesh sizes exposed for 4 and 38 weeks.

and coarse mesh-containers. Differences between themesh sizes in the tillage plots are shown in Figure 2.After 4 weeks, the mean water content in all contain-ers did not differ between the plots, whereas after 38weeks, the water content was significantly higher inthe untilled control.

Nematode density

Table 2a shows the results of a 3-way ANOVA onthe effects of cultivation treatment, mesh size and soildepth on nematode density in the minicontainers.

After a period of 4 weeks, there were significantmain effects of treatment and mesh size. Nematodedensity was lowest in the minicontainers in the untilledcontrol, while the largest number of nematodes werefound in containers from the deep ploughed soil. Onlythe differences between control and plough were sig-nificant (Table 3). Similar differences were seen after38 weeks when, additionally, there was a significant‘treatment�soil depth’ interaction and significant dif-ferences between the means of control on the one handand the two ploughed plots on the other hand. On thedeep ploughed site, nematode density was significantlyhigher in the lower 10 cm than in the upper, whereasthe other treatments and the control showed the reversedistribution (Figure 3).

Figure 3. Effect of different tillage systems on the vertical distri-bution of nematode density (mean � standard error, n = 15) in theminicontainers exposed for 4 and 38 weeks).

Figure 4. Effect of mesh size on the nematode density (mean �standard error, n = 10) in the minicontainers exposed for 4 and 38weeks).

The fine-mesh containers in all treatments and sam-ple times contained significantly more nematodes thanthe medium and coarse-mesh containers (Figure 4).After 4 weeks, the strongest main effect on nema-

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Table 2. Analysis of variance on the effects of treatment, mesh size and soil depth on nematodedensity and remaining litter mass in minicontainers. Only significant effects are shown (� = p<0.05,�� = p<0.01, ��� = p<0.001)

a) Nematode density

Time of exposure Source of variation df effect df error F p-level

4 weeks Treatment 3 96 2.56 �

Mesh-size 2 96 4.47 ��

Soil depth 1 96 4.47 �

38 weeks Treatment 3 96 15.21 ���

Mesh-size 2 96 12.78 ���

Treatment�soil depth 3 96 6.21 ���

Treatment�mesh size 6 96 2.51 �

b) Remaining litter mass

Time of exposure Source of variation df effect df error F p-level

4 weeks Treatment 3 96 4.51 ��

Soil depth 1 96 25.04 ���

38 weeks Treatment 3 96 5.67 ��

Mesh-size 2 96 3.39 �

Soil depth 6 96 4.43 �

Tillage � mesh size 6 96 5.15 �

Table 3. Results of the Tukey HSD-Test on the differences between the means of nematode density in the minicontainersexposed for 4 and 38 weeks. Only the main effects of tillage, mesh size and soil depth were tested (ns = not significant,� = p<0.05, �� = p<0.01)

Sampling after 4 weeks Sampling after 38 weeks

Tillage Control Plough Cultivator Two-layer Control Plough Cultivator Two-layer

plough plough

Mean 721 1235 1039 1082 752 1484 1172 1559

Control � ns ns � ns �

Plough ns ns ns ns

Cultivator ns ns

Mesh size 20 �m 500 �m 2 mm 20 �m 500 �m 2 mm

Mean 1405 797 839 1552 961 1212

20 �m �� �� � ns

500 �m ns ns

Soil depth 0–10 cm 10–20 cm 0–10 cm 10–20 cm

Mean 1124 906 1295 1188

0–10 cm ns ns

tode density was found for mesh size, whereas after38 weeks, cultivation treatment showed the strongestmain effect. After 4 weeks, the mean nematode den-sity in all containers was significantly higher in theploughed soil than in the control, after 38 weeks alltilled plots, except the fine-mesh containers in the cul-

tivator plot, showed higher nematode densities thanthe control.

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Table 4. Results of the Tukey HSD-Test on the differences between the means of the remaining litter mass in the minicontainersexposed for 4 and 38 weeks. Only the main effects of tillage, mesh size and soil depth were tested (ns = not significant,� = p<0.05, �� = p<0.01, ��� = p<0.001)

Sampling after 4 weeks Sampling after 38 weeks

Tillage Control Plough Cultivator Two-layer Control Plough Cultivator Two-layer

plough plough

Mean 77.55 77.24 74.23 78.78 51.71 43.57 44.20 42.60

Control ns � ns � � ��

Plough ns ns ns ns

Cultivator �� ns

Mesh size 20 �m 500 �m 2 mm 20 �m 500 �m 2 mm

Mean 77.50 76.84 76.44 43.99 48.65 43.92

20 �m ns ns � ns

500 �m ns ns

Soil depth 0–10 cm 10–20 cm 0–10 cm 10–20 cm

Mean 74.77 79.11 43.37 47.67

0–10 cm ��� �

Decomposition

After 4 weeks, the analysis of variance showed sig-nificant main effects of tillage and soil depth, but nosignificant effects of mesh size and no significant inter-actions between the factors on the decomposition of thestraw in the minicontainers (Table 2b). As shown bythe Tukey-Test (Table 4), the main effects of tillage aremainly based on a higher mass loss in the cultivatorplot (74.2% remaining litter mass), but only the differ-ences between cultivator and two layer-plough weresignificant. The significant main effects of soil depthwere due to higher mass loss in the upper soil layer(0–10 cm).

After 38 weeks, significant main effects of tillage,mesh size and soil depth were found. The averageremaining litter mass in the minicontainers in theuntilled control was significantly higher than in thetilled plots. Differences between the three tillage sys-tems were not significant. The significant main effectsof mesh size based on a lower mass loss in the contain-ers with 500 �m mesh size (48.7%), in contrast to thefine and coarse-mesh containers with both 43.9%. Theaverage remaining litter mass over all treatments wassignificantly higher in 10–20 cm depth than in 0–10 cmdepth.

After 4 weeks, no correlation was found betweenmass loss and nematode density in the minicontainers,whereas after 38 weeks the correlation was highly sig-nificant (Spearman R = �0.486, p = 0.001). On both

sample dates, the remaining litter mass was significantnegatively correlated with the water content (4 weeks:Spearman R = – 0.66, p = 0.001, 38 weeks: SpearmanR = – 0.52, p = 0.001).

Discussion

Previous studies on soil nematode densities, usingpopulation counts in soil samples, have shown a sig-nificant influence of different tillage systems (Bairdand Bernard, 1984; Freckman and Ettema, 1993;Yeates and Bird, 1994). The first results using theminicontainer-method also reveal significant effectson the nematode community. At both sampling times,the nematode density in exposed litter was higherin the tilled soils than in the control. Similar resultswere reported by Sohlenius and Sandor (1989), whoobserved an increase in total numbers of nematodes inploughed soil. A full evaluation of the results is impos-sible without detailed information about the speciescomposition of the nematode community extractedfrom the minicontainers. Greater incorporation of soilorganic matter as a result of cultivation, and a reductionin the incidence of macrofauna, primarily earthworms,resulting in lower competition may explain largernematode numbers in the cultivated plots. In gener-al, cultivation of soil reduces numbers and diversity ofsoil arthropods. Ploughing of soils, especially, has beenreported to reduce populations of Mesostigmatid mites,

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whereas tillage without inversion of the soil showedlower reduction (El Titi, 1984). Nematophagous mitescan play an important role as predators of nematodes(Karg, 1983). Lower nematode density in the coarse-mesh containers compared with fine mesh providessupporting evidence for the latter point. Additionally,laboratory experiments have shown that the presenceof earthworms can reduce the abundance of soil nema-todes (Hyvonen et al., 1994).

Cultivation of soil causes a number of changes instructure and activity of soil organisms and leads to adecline of soil organic matter. As expected, the inves-tigations with the minicontainer-method showed sig-nificant effects of tillage on the decomposition of ryestraw with higher mass loss in the tilled plots than in theuntilled control. These results support the observationsof other authors that cultivation of soil leads to a lower-ing of soil organic matter content, while no-till systemsmay conserve soil organic matter (Douglas and Goss,1982; Dick, 1983; Dalal and Mayer, 1986). The corre-lations between water content and decomposition rateemphasize the importance of adequate moisture con-ditions in decomposition processes. In the first phaseof the decomposition, litter mass loss is mainly influ-enced by abiotic factors (leaching), while in the laterphase biotic influences become more important. In ourexperiment, the role of soil fauna is indicated by thecorrelations between nematode density and mass lossafter 38 weeks.

Studies of the role of nematodes in the decompo-sition of plant residues and the successional coloniza-tion of substrates by nematodes mainly involve the useof bigger-sized mesh-bags or modifications of them(Wasilewska et al., 1981; Wasilewska, 1992). The lit-terbag method is the most widely used technique tostudy decomposition processes, although its use some-times criticized. For example, there are drawbacks inhandling associated with sample size, problems withcontamination of the litter and sample size, and theanalysis of resulting data is quite variable among inves-tigators and at times inappropriate (Wieder and Lang,1982). The bigger-sized litterbags often lead to distur-bance of soil structure as a result of their incorporationinto the soil. This is a major problem in agriculturalsites where the placement of test substrate in deepersoil layers is desirable, but for which large litterbagsare unsuitable. Microbial and microfaunal activity insoil is highest at discrete and heterogenously distribut-ed sites in soil where organic matter accumulate. Theactivity of nematodes and protozoa is concentrated atthese so called hotspots (Griffiths, 1994). The small

dimensions of the minicontainers allow a better chanceof sampling these active microhabitats in soil. Further-more, the larger number of replicate units allows theanalysis of a larger amount of data within a compara-tively short period of time.

The very small number of unextracted dead nema-todes remaining in samples indicates that, in contrastto other Baermann-modifications (Southey, 1986), alack of oxygen in the extraction vessel leading to mor-tality during extraction is not a serious problem in oursystem. Although no significant correlation betweenextraction efficiency and litter mass was found, it maybe possible that some of the difference between litterexposure time is caused by changes in litter density inthe minicontainers. In a study by Kimpinski and Welch(1971) the nematode numbers extracted from compact-ed soils were less than from non-compacted soils. Forqualitative faunal comparisons absolute extraction effi-ciency is unimportant but in quantitative studies, it isimportant that extraction efficiency should be assessed.The small volume of the material in the minicontainermakes it relatively easy to assess the number of nema-todes remaining in the sample. It is, however, desir-able to obtain more information about the extractionof nematodes from other materials using this method-ology. Further improvement of the method’s efficien-cy by modification of extraction time, temperature orthe tissue paper used in sample preparation, is prob-able (Chawla et al., 1973; Viglierchio and Schmitt,1983) and, for that, further investigation of these influ-ences are needed. The minicontainer-method is capa-ble of handling a large numbers of samples within ashort time, and, simultaneously, many other parame-ters relating to microbiological and chemical soil prop-erties can be assessed. In combination with the newextraction method the minicontainer-system providesa new useful way to study the role of nematodes indecomposition processes.

Acknowledgements

We thank Ulrike Asal for the critical revision of theenglish text and two anonymous reviewers for helpfulcomments on an earlier form of the manuscript. Ourresearch is financially supported by the ‘Zentrum furUmweltforschung’, University of Mainz and the ‘Feld-bauschstiftung’, Department of Biology, University ofMainz.

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Section editor: J M Whipps

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