Soil Biology & Biochemistry 57 (2013) 950e952
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Utilization of microbial abundance and diversity as indicators of the origin of soil aggregates produced by earthworms Pascal Jouquet a, *, Pierre-Alain Maron b, Virginie Nowak b, Toan Tran Duc c a
Institute of Research for Development (IRD), UMR 211 Bioemco, Centre IRD Ile de France, 32 Av. Henry Varagnat, 93143 Bondy, France National Institute of Research in Agronomy (INRA), UMR MSE, Université de Bourgogne, 17 rue de Sully, 21065 Dijon, France c Soils and Fertilizers Research Institute (SFRI), Dong Ngac, Tu Liem, Hanoi, Viet Nam b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 May 2012 Received in revised form 1 August 2012 Accepted 27 August 2012 Available online 12 September 2012
This study aimed at testing the capability of microbial community structure and abundance to be used as bioindicators of the origin of earthworm cast aggregates. Compact surface casts produced by Amynthas khami and surrounding aggregates lacking visible signs of biological activity (control) were left to disaggregate by natural rainfall and separated into four size classes (5e2, 2e0.5, 0.5e0.25 and <0.25 mm). The genetic structure and the abundance of the bacterial and fungal communities were characterized using B- and F-ARISA ﬁngerprinting approach and quantitative PCR directly from DNA extracted from soil. Bacteria and to a lesser extent fungi were more abundant in casts than in control aggregates for all the size fractions. In addition, PCA carried out from B- and F-ARISA conﬁrmed the different microbial properties between cast and control aggregates for all the aggregate size fractions. In conclusion, this study conﬁrms the cryptic properties of earthworm casts when fragmented by the rain and the relevance of bacterial and fungal abundance and diversity as biological indicators of the origin of soil aggregates. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Earthworm Biogenic aggregates Physicogenic aggregates Microbial diversity Amynthas khami
Earthworms, as ecosystem engineers (Lavelle et al., 1997; Jouquet et al., 2006), have large effects on biotic and abiotic properties of the soil system. They create soil biogenic aggregates (i.e. earthworm casts) with speciﬁc physical, chemical and microbiological properties. Although easily identiﬁed from the surrounding soil aggregates when recently emitted, earthworm casts lose their initial speciﬁc spherical and smooth surface shapes when fragmented by the rain or other biological or physical variables (Jouquet et al., 2009). Earthworm casts then acquire cryptic properties with a similar aspect to the surrounding aggregates but with speciﬁc physical and chemical properties (Jouquet et al., 2009; Nguyen Hong et al., 2011; Bottinelli et al., in press). In addition to the above-mentioned studies, it has been shown that the different porosity and chemical properties in earthworm casts lead to the creation of speciﬁc microbial habitats, and as a consequence speciﬁc microbial properties (Mummey et al., 2006). The deﬁnition of microbial indicators of the origin of soil aggregates seems all the more relevant as microorganisms are on the basis of the biological functioning of soils. In this context, a microbial indicator could not * Corresponding author. Institute of Research for Development (IRD), UMR 211 Bioemco, Centre IRD Ile de France, 32 Av. Henry Varagnat, 93143 Bondy, France. E-mail address: [email protected]
(P. Jouquet). 0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.08.026
only constitute an indicator of the soil origin but also an indicator of soil functioning (Bastida et al., 2008). The objective of this study was therefore to test the capability of microbial community structure and abundance to discriminate altered earthworm casts from surrounding soil aggregates. Direct testing of this hypothesis was accomplished by using soil aggregates from the study carried out by Jouquet et al. (2009). Brieﬂy, soil aggregates were collected in a fallow ﬁeld located in the north-eastern Vietnam. Earthworm casts produced by Amynthas khami were collected on the soil surface, and aggregates of the same size without visible biological activity (control) were sampled from the top 10 cm of ploughed soil. Aggregates were sampled in ﬁve randomly chosen areas and then air-dried for three days. Approximately 80 g of soil aggregates (casts or control aggregates) were laid out on 0.5-cm-mesh grids and submitted to natural rainfalls during 2 months. Finally, aggregates remaining on the grid were gently sieved in deionised water and separated into four classes (5e2, 2e0.5, 0.5e0.25 and <0.25 mm). The chemical and physical properties of these different soil aggregates can be found in Jouquet et al. (2008, 2009, 2011), Nguyen Hong et al. (2011) and Bottinelli et al. (in press). The genetic structure and the abundance of the bacterial and fungal communities were characterized by using respectively the
P. Jouquet et al. / Soil Biology & Biochemistry 57 (2013) 950e952
Control <0.2 mm 0.2e0.5 mm 0.5e2 mm 2e5 mm
9.57 13.42 5.36 4.35
(1.86)c CV: 0.19 (3.4)b CV: 0.25 (1.97)d CV: 0.37 (1.56)d CV: 0.36
Cast 8.67 8.48 14.31 18.30
(0.79)c CV: 0.09 (0.72)c CV: 0.09 (1.05)b CV: 0.07 (3.39)a CV: 0.19
Control Bacteria (B) (109) <0.2 mm 0.2e0.5 mm 0.5e2 mm 2e5 mm Fungi (F) (108) <0.2 mm 0.2e0.5 mm 0.5e2 mm 2e5 mm
2.51 3.24 0.77 0.62
(0.97)de CV: 0.39 (1.37)cd CV: 0.42 (0.44)ef CV: 0.57 (0.31)f CV: 0.50
4.64 5.33 7.16 8.54
(0.83)bc CV: 0.18 (1.33)b CV: 0.25 (2.56)a CV: 0.36 (1.82)a CV: 0.21
0.38 0.43 0.15 0.10
(0.13)de CV: 0.35 (0.18)de CV: 0.41 (0.07)e CV: 0.42 (0.03)e CV: 0.25
0.80 1.10 1.57 2.10
(0.09)c CV: 0.11 (0.30)c CV: 0.27 (0.56)b CV: 0.36 (0.74)a CV: 0.35
SOM content and quality) than control surrounding soil aggregates (Jouquet et al., 2009, 2011; Nguyen Hong et al., 2011). In agreement with this lesser variability of the physical and chemical properties of soil aggregates, this study therefore pinpoints a higher
C B A
Axis 1: 24% A B
CD B A
Axis 1: 49% A C D
Axis 2: 25%
Table 1 Molecular biomass (mg DNA g1 soil) in control and cast samples according to their sizes (<0.2, 0.2e0.5, 0.5e2, 2e5 mm). Standard errors are in parenthesis. The coefﬁcient of variation (CV) is also indicated. Values with the same letters are not signiﬁcantly different (LSD test, n ¼ 5).
Table 2 Abundance of bacteria and fungi (16/18S rDNA g soil1) in control and cast samples according to their sizes (<0.2, 0.2e0.5, 0.5e2, 2e5 mm). Standard errors are in parenthesis. The coefﬁcient of variation (CV) is also indicated. Values with the same letters are not signiﬁcantly different (LSD test, n ¼ 5).
Axis 2: 16%
B- and F-ARISA (Bacterial- and Fungal-Automated Ribosomal Intergenic Spacer Analysis) ﬁngerprinting approach (Pascault et al., 2010) and quantitative PCR bacteria (341F/515R; López-Gutiérrez et al., 2004) and fungi (FF390/FR1; Vainio and Hantula, 2000) directly from DNA extracted from soil. Soil DNA was extracted following the procedure of Dequiedt et al. (2011) and the amount of soil DNA recovered was expressed in terms of soil molecular biomass. Before analyses, data were tested for homogeneity of variance using Levene’s test and log-transformed when necessary. The molecular biomass (i.e. amount of soil DNA extracted, Dequiedt et al., 2011) and the abundance of bacteria and fungi were analysed by two-way analysis of variance (ANOVA) with the origin of the soil aggregates and the soil aggregate size as the independent variables. Comparisons between means were tested with LSD test. A Principal Component Analysis (PCA) was carried out to differentiate the samples based on bacterial and fungal genetic diversities (Pascault et al., 2010). In our study, microbial molecular biomass and bacteria and fungi abundances were dependent on the origin and size of the soil aggregates (P < 0.05 in all the cases). Despite similar quantity of molecular biomass in aggregates <0.2 mm between cast and control aggregates, signiﬁcant differences were observed for larger aggregates. Values measured were higher in 0.2e0.5 mm control aggregates and lower in >0.5 mm control aggregates compared to the corresponding size classes of earthworm casts (Table 1). Bacteria and to a lesser extent fungi were always more abundant in casts than in control aggregates (P < 0.05 in all cases, Table 2). The increase of bacterial density is usually explained by the enrichment in earthworm casts of readily assimilable organic matter (mucus) and the possible presence of fresh organic residues in the ingested soil, which are the cases with the casts of A. khami (Nguyen Hong et al., 2011). The higher abundance of fungi in earthworm casts is somewhat surprising since earthworms are suspected to decrease fungal biomass by grazing and disrupting their hyphae during the gut passage (Butenschoen et al., 2007; Bernard et al., 2012) but might be explained by the ecological category of earthworms. A. khami is an anecic earthworm, thereby collecting part of its diet on the soil surface (litter) which is enriched in fungi (Jouquet et al., 2008). The evolution of microbial properties in earthworm casts over time has also been previously evidenced (Scullion et al., 2003; Aira et al., 2005), but the spatial distribution of bacteria and fungi in earthworm casts remains poorly known (Winding et al., 1997; Gorres et al., 2001; Mummey et al., 2006; Marhan et al., 2007). This study also showed that the abundance of bacteria and fungi in casts increased with the aggregate size and that the opposite trend was observed for the control. These results can be explained by the different organization of earthworm casts compared to the control surrounding aggregates (Jouquet et al., 2011). Interestingly, the coefﬁcient of variation (CV) was higher in control than in cast aggregates for both bacteria and fungi, except for fungi and aggregates >0.5 mm (Tables 1 and 2). As suggested in previous studies, earthworm casts produced by A. khami are suspected to have a more homogeneous organization (with respect to porosity,
Fig. 1. Principal component (PC1 PC2) plots generated from ARISA proﬁles of bacteria (a) and fungal (b) communities in the different size classes (<0.2 mm: ‘A’, 0.2e0.5 mm: ‘B’, 0.5e2 mm: ‘C’, >2 mm: ‘D’) of control (in white) and cast aggregates (in grey).
P. Jouquet et al. / Soil Biology & Biochemistry 57 (2013) 950e952
reproducibility of bacterial and fungal abundances in earthworm casts, compared to surrounding aggregates, and therefore more similar ecological functions with regards to nutrient cycling within these aggregates (i.e., emission of CO2, Jouquet et al., 2011). Fingerprinting of bacterial (B-ARISA) and fungal (F-ARISA) community structure provided complex proﬁles. The PCA carried out from these data are shown in Fig. 1 and conﬁrm the different microbial properties between cast and control aggregates. Casts were clearly differentiated from control aggregates for all the size classes mainly along the second axis. These ﬁndings are in agreement with others who showed signiﬁcant impact of earthworms on soil microbial communities (Egert et al., 2004; Mummey et al., 2006; Butenschoen et al., 2007; Marhan et al., 2007; ChapuisLardy et al., 2010; Bernard et al., 2012). The PCA did not allow us to discriminate the bacterial communities in aggregates lower than 2 mm, for both types of aggregates, therefore suggesting that bacteria were homogeneously distributed in soil aggregates <2 mm and that larger size aggregates were described by speciﬁc bacterial communities. Fungal communities were less homogeneous and the high variability did not allow us to pinpoint an inﬂuence of the aggregate size. Strongest variations of the fungal community structure compared to bacteria could be explained by the highly heterogeneous distribution of fungal species in soil, which are known to be spatially clustered leading to hot spot distribution of their biomass and diversity (Horton and Bruns, 2001; Ranjard et al., 2003). In conclusion, soil undergoes many transformations in the gut after being ingested by earthworms. Aggregates are broken down and all the constituting functional units observed in the control aggregates are mixed into a more homogenous new matrix than the surrounding control aggregates. When fragmented by the rain, earthworm casts acquire cryptic properties and the speciﬁc microbial abundance and diversity produce biological indicators of the origin of soil aggregates. Acknowledgements This project was supported ﬁnancially by CNRS/INSU (VERAGREGAT project, EC2CO program) and IRD French institutes. References Aira, M., Monroy, F., Dominguez, J., 2005. Ageing effects on nitrogen dynamics and enzyme activities in casts of Aporrectodea caliginosa (lumbricidae). Pedobiologia 49, 467e473. Bastida, F., Zsolnay, A., Hernandez, T., Garcia, C., 2008. Past, present and future of soil quality indices: a biological perspective. Geoderma 147, 159e171. Bernard, L., Chapuis-Lardy, L., Razaﬁmbelo, T., Razaﬁndrakoto, M., Pablo, A.-L., Legname, E., Poulain, J., Bruls, T., O’Donohue, M., Brauman, A., Chotte, J.-L., Blanchart, E., 2012. Endogeic earthworms shape bacterial functional communities and affect organic matter mineralization in a tropical soil. The ISME Journal 6, 213e222. Bottinelli, N., Jouquet, P., Tran, T., Hallaire, V. Morphological characterisation of weathered earthworm casts by 2D-image analysis. Biology and Fertility of Soils, in press.
Butenschoen, O., Poll, C., Langel, R., Kandeler, E., Marhan, S., Scheu, S., 2007. Endogeic earthworms alter carbon translocation by fungi at the soil-litter interface. Soil Biology and Biochemistry 39, 2854e2864. Chapuis-Lardy, L., Brauman, A., Bernard, L., Pablo, A.L., Toucet, J., Mano, M.J., Weber, L., Brunet, D., Razaﬁmbelo, T., Chotte, J.L., Blanchart, E., 2010. Effect of the endogeic earthworm Pontoscolex corethrurus on the microbial structure and activity related to CO2 and N2O ﬂuxes from a tropical soil (Madagascar). Applied Soil Ecology 45, 201e208. Dequiedt, S., Saby, N.P.A., Lelievre, M., Jolivet, C., Thioulouse, J., Toutain, B., Arrouays, D., Bispo, A., Lemanceau, P., Ranjard, L., 2011. Biogeographical patterns of soil molecular microbial biomass as inﬂuenced by soil characteristics and management. Global Ecology and Biogeography 20, 641e652. Egert, M., Marhan, S., Wagner, B., Scheu, S., Friedrich, M.W., 2004. Molecular proﬁling of 16S rRNA genes reveals diet-related differences of microbial communities in soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta: Lumbricidae). FEMS Microbiology Ecology 48, 187e197. Gorres, J.H., Savin, M.C., Amador, J.A., 2001. Soil micropore structure and carbon mineralization in burrows and casts of an anecic earthworm (Lumbricus terrestris). Soil Biology and Biochemistry 33, 1881e1887. Horton, T.R., Bruns, T.D., 2001. The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Molecular Ecology 10, 1855e1871. Jouquet, P., Dauber, J., Lagerlof, J., Lavelle, P., Lepage, M., 2006. Soil invertebrates as ecosystem engineers: intended and accidental effects on soil and feedback loops. Applied Soil Ecology 32, 153e164. Jouquet, P., Bottinelli, N., Podwojewski, P., Hallaire, V., Tran Duc, T., 2008. Chemical and physical properties of earthworm casts as compared to bulk soil under a range of different land-use systems in Vietnam. Geoderma 146, 231e238. Jouquet, P., Zangerle, A., Rumpel, C., Brunet, D., Bottinelli, N., Tran Duc, T., 2009. Relevance and limitations of biogenic and physicogenic classiﬁcation: a comparison of approaches for differentiating the origin of soil aggregates. European Journal of Soil Science 60, 1117e1125. Jouquet, P., Phuong, N.T., Hanh, N.H., Henry-des-Tureaux, T., Chevallier, T., Tran Duc, T., 2011. Laboratory investigation of organic matter mineralization and nutrient leaching from earthworm casts produced by Amynthas khami. Applied Soil Ecology 47, 24e30. Lavelle, P., Bignell, D., Lepage, M., 1997. Soil function in a changing world: the role of invertebrate ecosystem engineers. European Journal of Soil Biology 33, 159e193. López-Gutiérrez, J.C., Henry, S., Hallet, S., Martin-Laurent, F., Catroux, G., Philippot, L., 2004. Quantiﬁcation of a novel group of nitrate-reducing bacteria in the environment by real time PCR. Journal of Microbiology Methods 57, 399e407. Marhan, S., Kandeler, E., Scheu, S., 2007. Phospholipid fatty acid proﬁles and xylanase activity in particle size fractions of forest soil and casts of Lumbriscus terrestris L. (Oligochaeta, Lumbricidae). Applied Soil Ecology 35, 414e422. Mummey, D.L., Rillig, M.C., Six, J., 2006. Endogeic earthworms differentially inﬂuence bacterial communities associated with different soil aggregate size fractions. Soil Biology and Biochemistry 32, 1608e1614. Nguyen Hong, H., Rumpel, C., Henry-des-Tureaux, T., Bardoux, G., Billou, D., Tran Duc, T., Jouquet, P., 2011. How do earthworms inﬂuence organic matter quantity and quality in tropical soils? Soil Biology and Biochemistry 43, 223e230. Pascault, N., Cécillon, L., Mathieu, O., Hénault, C., Sarr, A., 2010. In situ dynamics of microbial communities during decomposition of wheat, rape and alfalfa residues. Microbial Ecology 60, 816e828. Ranjard, L., Lejon, D.P.H., Mougel, C., Schehrer, L., Merdinoglu, D., Chaussod, R., 2003. Sampling strategy in molecular microbial ecology: inﬂuence of soil sample size on DNA ﬁngerprinting analysis of fungal and bacterial communities. Environmental Microbiology 5, 1111e1120. Scullion, J., Elliott, G.N., Huang, W.E., Goodacre, R., Worgan, H., Darby, R., Bailey, M.J., Gwynn-Jones, D., Grifﬁth, G.W., Winson, M.K., Williams, P.A., Clegg, C., Draper, J., 2003. Use of earthworm casts to validate FT-IR spectroscopy as a ‘sentinel’ technology for high-throughput monitoring of global changes in microbial ecology. Pedobiologia 47, 1e7. Vainio, E.J., Hantula, J., 2000. Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of ampliﬁed ribosomal DNA. Mycology Research 104, 927e936. Winding, A., Ronn, R., Hendriksen, N.B., 1997. Bacteria and protozoa in soil microhabitats as affected by earthworms. Biology and Fertility of Soils 24, 133e140.