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The role of soil fungi and bacteria in plant litter decomposition and macroaggregate formation determined using phospholipid fatty acids
Applied Soil Ecology 96 (2015) 261–264
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Short communication
The role of soil fungi and bacteria in plant litter decomposition and macroaggregate formation determined using phospholipid fatty acids Mirjam Helfricha,* , Bernard Ludwigb , Carolin Thomsc , Gerd Gleixnerd , Heinz Flessaa a
Thünen-Institut für Agrarklimaschutz, Bundesallee 50, 38116 Braunschweig, Germany Fachgebiet Umweltchemie, Universität Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany c Nordwestdeutsche Forstliche Versuchsanstalt, Grätzelstr. 2, 37079 Göttingen, Germany d Max-Planck-Institut für Biogeochemie, Postbox 100164, 7701 Jena, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 March 2015 Received in revised form 20 July 2015 Accepted 28 August 2015 Available online xxx
Although microbial-derived carbon (C) inputs to soil are increasingly acknowledged as an important source of soil organic matter (SOM), the contribution of different microbial compounds to soil C transformation and their role in aggregation remain poorly understood. This study assessed the contribution of soil fungi and bacteria to the decomposition of maize residues by means of extracted phospholipid fatty acids (PLFAs) and 13C in specific PLFAs and investigated the importance of soil fungi in the formation of macroaggregates. Sieved soil (<250 mm) was incubated for 28 days with and without addition of maize residues and fungicide. Our results show a significant relation between the amount of fungal PLFA 18:2v6 and the amount of macroaggregates. Further, the amount of macroaggregates was higher in the treatment with the higher amount of maize-derived C in fungal PLFA, suggesting that fungal activity is important for macroaggregate formation. Based on an increased incorporation of maizederived C into actinomycetes in fungicide treatments, we suggest that actinomycetes may take over the role of soil fungi in the decomposition of SOM. Our study underpins the important role of soil fungi in the decomposition of organic matter and structure formation in the soil, and shows that during inhibition of soil fungi other soil microorganisms are promoted and adopt their function in the soil food web. ã 2015 Elsevier B.V. All rights reserved.
Keywords: C natural abundance Soil incubation Macroaggregates 13 C PLFA Plant residues Captan 13
1. Introduction Soil fungi have been recognized to positively affect soil aggregation (e.g. Bossuyt et al., 2001; Denef et al., 2001), which in turn is important for maintaining high soil quality, i.e. conditions favourable for plant and microbial growth like soil porosity, aeration, infiltration of water, and stability against erosion of soils. In a previous study, Helfrich et al. (2008) investigated the effect of maize residue decomposability and fungal biomass on the dynamics of macroaggregate (>250 mm) formation and the associated partitioning of litter-derived C and N in a three months’ incubation experiment. They found that the application of fungicide decreased the amount and catabolic activity of the microbial biomass (decreasing CO2-emissions and decreasing proportions of maize-derived C in CO2 C) and led to less macroaggregation. However, Ergosterol, which was used as a fungal biomarker was found inappropriate in periods of rapid decline of the microbial biomass, such as in the fungicide
treatments (Helfrich et al., 2008). Phospholipid fatty acids (PLFAs) are regarded a good measure of living microbial biomass. Because they are major constituents of the membranes of all living cells that are not found within storage products and are degraded quickly after cell death, they have the potential to mirror even rapid changes in the microbial community (Amelung et al., 2008). Combined with substance specific 13C contents, PLFA analyses allow C source elucidation in living microbial biomass (Amelung et al., 2008; Kramer and Gleixner, 2006; Kaur et al., 2005). The present study assessed (i) the contribution of soil fungi and bacteria to the decomposition of maize residues of different decomposability (leaves, roots) by means of specific PLFA contents and 13C in specific PLFAs and investigated (ii) their importance in the short-term formation of macroaggregates. The results from this study give the opportunity to get new insights into the initial C uptake upon degradation of plant litter by different microbial groups and its role for macroaggregation in an agricultural soil. 2. Materials and methods
* Corresponding author. Fax: +49 531 5962699. E-mail address: [email protected] (M. Helfrich). http://dx.doi.org/10.1016/j.apsoil.2015.08.023 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.
Soil samples for PLFA analysis were derived from the first 28 days of an incubation experiment (Helfrich et al., 2008; Table 1).
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M. Helfrich et al. / Applied Soil Ecology 96 (2015) 261–264
Table 1 Overview of the treatments. Quantification of PLFA was carried out on samples taken after 0, 14 and 28 days of incubation. Determination of specific 13C values was done on samples taken at the start (0 days) and after 28 days of incubation. Treatment
Description
Days incubated
S0 SC0 S14 SC14 SL14 SLC14 SR14 SRC14 S28 SC28 SL28 SLC28 SR28 SRC28
Control soil Control soil + Captan Control soil Control soil + Captan Soil + maize leaves Soil + maize leaves + Captan Soil + maize roots Soil + maize roots + Captan Control soil Control soil + Captan Soil + maize leaves Soil + maize leaves + Captan Soil + maize roots Soil + maize roots + Captan
0 0 14 14 14 14 14 14 28 28 28 28 28 28
In brief, soil of a silty loam (stagnic Luvisol derived from loess) under wheat monoculture (C3 vegetation; soil organic C 12.6 0.1 g kg 1; d13C –26.5 0.1m V-PDB; means standard deviation) was incubated after destruction of all naturally occurring macroaggregates (sieving <250 mm) in the dark at 15 C and 60% of the maximum water-holding capacity. Maize leaves (C/N 27.4; d13C 12.7 0.2m V-PDB; means standard deviation) or roots (C/N 86.4; d13C 11.9 0.2m V-PDB; means standard deviation) were oven-dried at 40 C, milled <500 mm and added to the soil at a rate of 2.1 mg C g 1 soil. Each treatment was carried out with and without addition of 0.3 g Captan per 100 g soil (50 W wettable powder, 89% active ingredient), which had been used as a fungicide in a number of studies (e.g. Bailey et al., 2002; Bossuyt et al., 2001; Denef et al., 2001) and was found to have little effect on soil bacteria (Ingham, 1985). Macroaggregates were obtained by wetsieving >250 mm (Helfrich et al., 2008). Phospholipid fatty acids were extracted following the method of Bligh and Dyer (1959) and Zelles and Bai (1993). Soil lipids were extracted using a mixture of chloroform, methanol and phosphate buffer (1:2:0.8 vol). Lipid extracts were separated into neutral, glycol- and phospholipids on silicic acid columns. Phospholipids
Fig. 1. Linear regression between the amount of lnPLFA 18:2v6 and water-stable macroaggregates.
were methylated and the obtained fatty acid methyl ester (FA-ME) were further separated into saturated, polyunsaturated and monounsaturated fatty acids and quantified by gas chromatography (GC: HP 6890 Series, AED: G 2350 A, Agilent Technologies, United States) using a HP ultra 2 column (50 m 0.32 mm I.D., 0.25 mm film thickness) in the split mode. Compound-specific determination of d13C values of individual PLFA-ME was performed with GC/MS-C-IRMS in triplicate (GCQ, Thermoquest, Germany); (Delta + XL, Finnigan MAT, Germany). To obtain d13C values of the PLFAs, the d13C values of PLFA-ME were corrected for the methyl-C which was added during methylation. The amount of maizederived C used as microbial C source in percent was calculated by dividing the isotopic shift between PLFAs from soil with and without addition of maize litter through the isotopic shift between the soil used for the incubation and the maize plant material (Balesdent and Mariotti, 1996). The obtained PLFAs were grouped into grampositive bacteria (Gram+) excluding actinomycetes
Table 2 Amounts of major and of biomarker PLFA for the investigated treatments. Amount of selected PLFAs (nmol g
i14:0 i15:0 a15:0 br16:0 i16:0 br17:0 i17:0 a17:0 br18:0r br19:0 18:2v6c n14:0 n15:0 n16:0 n17:0 n18:0 10Me17:0 10Me18:0 10Me19:0 cy19:0 16:1v7c 18:1v7c 18:1v9c % of total PLFA
+
Gram Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Fungal marker Universal Universal Universal Universal Universal Actinomycetes Actinomycetes Actinomycetes Gram Gram Gram Gram
1
soil)
S0
SC0
S14
SC14
S28
SC28
SL14
SLC14
SL28
SLC28
SR14
SRC14
SR28
SRC28
0.60 4.87 4.13 0.40 1.87 0.65 1.18 1.21 0.91 0.33 0.87 1.03 0.51 6.40 0.34 1.24 2.84 0.58 1.57 3.40 5.02 7.83 5.61 77.6
0.65 5.27 4.80 0.45 1.86 0.79 1.19 1.30 0.99 0.38 0.76 1.04 0.81 6.78 0.32 1.39 3.17 0.62 1.72 4.07 4.27 7.29 5.00 78.6
0.79 6.92 5.60 0.56 2.38 0.87 1.52 1.63 1.25 0.55 1.14 1.19 0.55 8.75 0.34 1.85 3.66 0.81 2.08 4.14 6.08 9.84 8.52 79.3
0.79 6.23 5.89 0.53 2.32 0.86 1.37 1.57 1.25 0.46 0.90 1.22 0.54 8.17 0.35 1.43 3.61 0.74 2.06 3.99 6.11 9.42 6.64 78.1
0.51 5.33 4.35 0.44 1.81 0.78 1.20 1.15 1.03 0.38 0.82 0.95 0.47 6.35 0.29 1.24 3.41 0.62 1.70 3.35 4.70 7.54 4.66 79.7
0.66 5.79 5.65 0.49 2.19 0.87 1.27 1.49 1.34 0.43 0.79 1.06 0.52 7.87 0.34 1.35 3.32 0.65 1.90 3.95 4.87 8.02 5.46 80.7
0.88 7.49 7.31 0.51 3.13 0.87 1.58 1.94 1.08 0.41 1.87 1.43 0.63 12.38 0.46 2.00 3.43 0.79 2.13 3.73 8.90 11.76 7.30 80.2
1.05 6.50 9.41 0.52 2.82 0.89 1.29 1.80 1.17 0.43 1.70 1.59 0.68 14.71 0.48 2.32 3.44 0.76 2.06 4.22 6.24 9.03 6.25 81.1
1.39 11.39 10.68 0.79 4.34 1.35 2.45 2.80 1.69 0.66 2.63 2.27 0.97 18.69 0.69 2.91 5.08 1.16 3.21 5.62 16.09 21.90 12.95 77.9
1.10 7.82 10.79 0.60 3.19 0.94 1.51 2.13 1.31 0.54 2.23 1.86 0.80 16.46 0.53 2.56 3.95 0.87 2.46 4.67 8.69 12.53 8.58 78.8
0.89 9.27 8.46 0.66 3.75 1.04 1.97 2.29 1.53 0.61 2.42 1.43 0.73 12.79 0.52 2.23 4.43 1.04 2.79 4.78 11.05 15.19 10.35 78.3
0.95 7.05 9.38 0.60 3.02 0.94 1.50 2.00 1.33 0.48 1.59 1.35 0.70 11.20 0.47 2.06 3.92 0.87 2.32 4.54 6.61 9.69 6.85 79.7
1.18 10.57 9.23 0.72 3.86 1.02 2.17 2.38 1.49 0.58 2.85 1.61 0.82 14.09 0.57 2.33 5.63 1.08 2.95 5.16 11.38 17.71 10.26 79.6
0.95 6.97 9.10 0.57 2.82 0.83 1.41 1.85 1.13 0.46 1.41 1.38 0.68 10.99 0.42 1.88 3.83 0.81 2.23 4.67 6.57 10.23 6.92 81.2
M. Helfrich et al. / Applied Soil Ecology 96 (2015) 261–264
(Lechevalier, 1977), actinomycetes (Kroppenstedt, 1985) and gramnegative bacteria (Gram ) (Kramer and Gleixner, 2006). Fatty acid 18:2v6c was used as fungal marker (Joergensen and Wichern, 2008). However, 18:2v6c is not only an indicator for fungi, but generally appears in eukaryotes (Kaur et al., 2005). Oneway analyses of variance (ANOVAs) were carried out to study treatment effects on the contents of maize-derived C in PLFAs for individual biomarkers followed by a Tukey test. In cases of nonhomogeneity of variances Welch ANOVAs were used after testing each group for normality followed by pairwise t-tests with Bonferroni corrections without pooled standard deviations and without the assumption of equal variances. For markers 18:1v9c, br18:0, 10Me18:0, and 10Me19:0 we observed non-homogeneity of variances and not all groups were normally distributed. Thus, a Kruskal–Wallis test was carried out for these markers followed by multiple comparisons in case of a significant group effect. A linear
263
regression for the content of fungal PLFA with water-stable macroaggregates as predictor was calculated. Transformation of the content of fungal PLFA to its natural logarithm was carried out to obtain normality of residuals in the linear regression. All statistical analyses were performed using the software R (version 3.1.1, R Core Team, 2014). 3. Results and discussion A total of 53 PLFAs with C chain lengths between 14 and 24 were detected in the soil extracts. Twenty three PLFAs comprised 77.6– 81.2% of total PLFAs (Table 2), and were each present in sufficient quantity to obtain accurate d13C values. The fungal marker 18:2v6 contributed 1.0–2.1% to total PLFAs. The amount of fungal PLFA was lower in all treatments with Captan compared to their non-Captan treated counterparts, with
Fig. 2. Amount of maize-derived C from added plant litter of (A) fungi and actinomycetes, (B) grampositive bacteria (Gram+) and (C) universal markers and gramnegative bacteria (Gram ) in the treatments after 28 days of incubation (means and standard deviation of measurement, n = 3). Different letters indicate significant differences between treatments for individual biomarkers (p < 0.05).
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M. Helfrich et al. / Applied Soil Ecology 96 (2015) 261–264
the soil and root treatments being most affected (Table 2). At day 28, fungal biomass was reduced by 15.0% and 50.4% in the SLC28 and SRC28 treatment compared to the respective nonfungicide treated variants, while the effect had levelled off in the control soil. However, as can be seen from Table 2, several bacterial groups including actinomycetes were even more inhibited by Captan than fungal PLFA (reduction upon the use of Captan by up to 46%). Similar to our study, Rousk et al. (2009) reported non-target effects of Captan on other microbial groups. The induced general toxity effects that are not specific to fungi which complicates interpretation of the obtained results and question the use of Captan as a fungicide although it has been used frequently as such in previous incubation (e.g. Bailey et al., 2002; Bossuyt et al., 2001; Denef et al., 2001) and field studies (e.g. Beare et al., 1997; Hu et al., 1995). The amount of fungal PLFA was related to the amount of waterstable macroaggregates (Fig. 1; R2 = 0.58). Analysis of 13C in specific PLFA analysis revealed that the incorporation of maize-derived C into the fungal biomass was considerably higher in the treatments without fungicide compared to fungicide-treated variants (Fig. 2A), pointing to decreased anabolic activity in fungicidetreated variants. This trend was observed for both, leaf-amended and root-amended soils. As seen for soil fungi, decreasing anabolic activity in fungicide-treated variants was observed for Gram bacteria (Fig. 2A). An increasing incorporation of maize-derived C upon use of fungicide was observed for actinomycetes 10Me19:0 as well as for the leaf treatments in 10Me18:0 and 10Me17:0—the latter two, however, were not significant (Fig. 2C). Gram+ bacteria and universal markers showed no general trend (Fig. 2B and C). The positive influence of soil fungi on the anabolic activity of Gram bacteria, but not on Gram+ bacteria may result from their dissimilar abilities to use different C sources. While Gram bacteria feed only on plant-derived material (which might be more readily degraded and therefore more easily available in the presence of an intact fungal community), Gram+ bacteria may use plant-derived C as well as more complex C sources such as soil organic C (Kramer and Gleixner, 2006; Thoms et al., 2010; Malik et al., 2015). The higher incorporation of maize-derived C into actinomycetes in the absence of fungi might be an indication that actinomycetes may take over the role of soil fungi in decomposition of SOM. However, Gram+ bacteria including actinomycetes were also found to feed on other microorganisms (Malik et al., 2015). Therefore, feeding on microbes that died upon the use of fungicide might be another possible reason for the increased 13C label in actinomycetes upon the use of fungicide. Furthermore, Gram+ bacterial cell envelopes are thicker and more complex than those of other microbial groups and could be turned over slower than those of other microbial groups. Yet, we do not think that the latter reason is of vital importance for the finding of a higher 13C label upon the use of fungicide in actinomycetes in our study, because (i) this was not a general trend found in Gram+ bacteria and (ii) we determined the label 28 days after the use of the fungicide which we think should be sufficient time for complete turnover of the microbial biomass that died upon the use of the fungicide. Overall, the use of the natural 13C label of maize plant litter and specific PLFA contents and 13C in specific PLFAs was a valuable tool to get new insights into the C transformation into different microbial groups upon plant litter degradation. Our results showed that the quality of plant residues is a key factor determining the composition as well as the activity of the soil microbial biomass, which is in accordance with findings from previous studies (Thoms and Gleixner, 2013). They further suggest that not only the contribution of fungi to the microbial biomass, but also fungal activity is of importance in the formation of macroaggregates. Due
to an increasing incorporation of maize-derived C into actinomycetes upon the use of fungicide, we assume that actinomycetes may take over the role of soil fungi in the decomposition of soil organic matter. Yet, this finding needs to be verified and a more detailed quantitative understanding of soil microbial trophic interactions is vitally needed. Further, the generalizability of the results to other soil types and climates remains unclear. We strongly recommend future studies which aim at a better mechanistic understanding of microbial processes contributing to the persistence and transformation of C in soil using a combination of isotope tracers combined with biomarker analyses. Acknowledgements This study was supported by a grant from the Deutsche Forschungsgemeinschaft. We thank Steffen Rühlow for excellent technical assistance and compound-specific 13C analysis as well as Anne-Gret Seifert and Andrea Scheibe for helpful discussions and support in the lab. References Amelung, W., Brodowski, S., Sandhage-Hofmann, A., Bol, R., 2008. Combining biomarker with stable isotope analyses for assessing the transformation and turnover of soil organic matter. Adv. Agron. 100, 155–250. Bailey, V.L., Smith, J.L., Bolton, H., 2002. Fungal-to-bacterial ratios in soils investigated for enhanced C sequestration. Soil Biol. Biochem. 34, 997–1007. Balesdent, J., Mariotti, A., 1996. Measurement of soil organic matter turnover using 13 C natural abundance. In: Boutton, T.W., Yamasaki, S. (Eds.), Mass Spectrometry of Soils. Marcel Dekker, New York, pp. 83–111. Beare, M.H., Hus, S., Coleman, D.C., Hendrix, P.F., 1997. Influences of mycelial fungi on soil aggregation and organic matter storage in conventional and no-tillage soils. Appl. Soil Ecol. 5, 211–219. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Bossuyt, H., Denef, K., Six, J., Frey, S.D., Merckx, R., Paustian, K., 2001. Influence of microbial populations and residue quality on aggregate stability. Appl. Soil Ecol. 16, 195–208. Denef, K., Six, J., Bossuyt, H., Frey, S.D., Elliott, E.T., Merckx, R., Paustian, K., 2001. Influence of dry–wet cycles on the interrelationship between aggregate, particulate organic matter, and microbial community dynamics. Soil Biol. Biochem. 33, 1599–1611. Helfrich, M., Ludwig, B., Potthoff, M., Flessa, H., 2008. Effect of litter quality and soil fungi on macroaggregate dynamics and associated partitioning of litter carbon and nitrogen. Soil Biol. Biochem. 40, 1823–1835. Hu, S., Coleman, D.C., Beare, M.H., Hendrix, P.F., 1995. Soil carbohydrates in aggrading and degrading agroecosystems—influences of fungi and aggregates. Agric. Ecosyst. Environ. 54, 77–88. Ingham, E.R., 1985. Review of the effects of 12 selected biocides on target and nontarget soil organisms. Crop Prot. 4, 3–32. Joergensen, R.G., Wichern, F., 2008. Quantitative assessment of the fungal contribution to microbial tissue in soil. Soil Biol. Biochem. 40, 2977–2991. Kaur, A., Chaudhary, A., Choudhary, R., Kaushik, R., 2005. Phospholipid fatty acid—a bioindicator of environment monitoring and assessment in soil ecosystem. Curr. Sci. 89, 1103–1112. Kramer, C., Gleixner, G., 2006. Variable use of plant- and soil-derived carbon by microorganisms in agricultural soils. Soil Biol. Biochem. 38, 3267–3278. Kroppenstedt, R.M., 1985. Fatty acid and menaquinone analysis of actinomycetes and related organisms. In: Goodfellow, M., Minnikin, D.E. (Eds.), Chemical Methods in Bacterial Systematics. Academic Press, London, pp. 173–199. Lechevalier, M.P., 1977. Lipids in bacterial taxonomy—a taxonomists view. Crit. Rev. Microbiol. 5, 109–210. Malik, A.A., Dannert, H., Griffiths, R.I., Thomson, B.C., Gleixner, G., 2015. Rhizosphere bacterial carbon turnover is higher in nucleic acids than membrane lipids: implications for understanding soil carbon cycling. Front. Microbiol. 6, 268. Rousk, J., Demoling, L.A., Bååth, E., 2009. Contrasting short-term antibiotic effects on respiration and bacterial growth compromises the validity of the selective respiratory inhibition technique to distinguish fungi and bacteria. Microb. Ecol. 58, 75–85. Thoms, C., Gleixner, G., 2013. Seasonal differences in tree species’ influence on soil microbial communities. Soil Biol. Biochem. 66, 239–248. Thoms, C., Gattinger, A., Jacob, M., Thomas, F.M., Gleixner, G., 2010. Direct and indirect effects of tree diversity drive soil microbial diversity in temperate deciduous forest. Soil Biol. Biochem. 42, 1558–1565. Zelles, L., Bai, Q.Y., 1993. Fractionation of fatty acids derived from soil lipids by solid phase extraction and their quantitative analysis by GC–MS. Soil Biol. Biochem. 25, 495–507.
Contents lists available at ScienceDirect
Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil
Short communication
The role of soil fungi and bacteria in plant litter decomposition and macroaggregate formation determined using phospholipid fatty acids Mirjam Helfricha,* , Bernard Ludwigb , Carolin Thomsc , Gerd Gleixnerd , Heinz Flessaa a
Thünen-Institut für Agrarklimaschutz, Bundesallee 50, 38116 Braunschweig, Germany Fachgebiet Umweltchemie, Universität Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany c Nordwestdeutsche Forstliche Versuchsanstalt, Grätzelstr. 2, 37079 Göttingen, Germany d Max-Planck-Institut für Biogeochemie, Postbox 100164, 7701 Jena, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 March 2015 Received in revised form 20 July 2015 Accepted 28 August 2015 Available online xxx
Although microbial-derived carbon (C) inputs to soil are increasingly acknowledged as an important source of soil organic matter (SOM), the contribution of different microbial compounds to soil C transformation and their role in aggregation remain poorly understood. This study assessed the contribution of soil fungi and bacteria to the decomposition of maize residues by means of extracted phospholipid fatty acids (PLFAs) and 13C in specific PLFAs and investigated the importance of soil fungi in the formation of macroaggregates. Sieved soil (<250 mm) was incubated for 28 days with and without addition of maize residues and fungicide. Our results show a significant relation between the amount of fungal PLFA 18:2v6 and the amount of macroaggregates. Further, the amount of macroaggregates was higher in the treatment with the higher amount of maize-derived C in fungal PLFA, suggesting that fungal activity is important for macroaggregate formation. Based on an increased incorporation of maizederived C into actinomycetes in fungicide treatments, we suggest that actinomycetes may take over the role of soil fungi in the decomposition of SOM. Our study underpins the important role of soil fungi in the decomposition of organic matter and structure formation in the soil, and shows that during inhibition of soil fungi other soil microorganisms are promoted and adopt their function in the soil food web. ã 2015 Elsevier B.V. All rights reserved.
Keywords: C natural abundance Soil incubation Macroaggregates 13 C PLFA Plant residues Captan 13
1. Introduction Soil fungi have been recognized to positively affect soil aggregation (e.g. Bossuyt et al., 2001; Denef et al., 2001), which in turn is important for maintaining high soil quality, i.e. conditions favourable for plant and microbial growth like soil porosity, aeration, infiltration of water, and stability against erosion of soils. In a previous study, Helfrich et al. (2008) investigated the effect of maize residue decomposability and fungal biomass on the dynamics of macroaggregate (>250 mm) formation and the associated partitioning of litter-derived C and N in a three months’ incubation experiment. They found that the application of fungicide decreased the amount and catabolic activity of the microbial biomass (decreasing CO2-emissions and decreasing proportions of maize-derived C in CO2 C) and led to less macroaggregation. However, Ergosterol, which was used as a fungal biomarker was found inappropriate in periods of rapid decline of the microbial biomass, such as in the fungicide
treatments (Helfrich et al., 2008). Phospholipid fatty acids (PLFAs) are regarded a good measure of living microbial biomass. Because they are major constituents of the membranes of all living cells that are not found within storage products and are degraded quickly after cell death, they have the potential to mirror even rapid changes in the microbial community (Amelung et al., 2008). Combined with substance specific 13C contents, PLFA analyses allow C source elucidation in living microbial biomass (Amelung et al., 2008; Kramer and Gleixner, 2006; Kaur et al., 2005). The present study assessed (i) the contribution of soil fungi and bacteria to the decomposition of maize residues of different decomposability (leaves, roots) by means of specific PLFA contents and 13C in specific PLFAs and investigated (ii) their importance in the short-term formation of macroaggregates. The results from this study give the opportunity to get new insights into the initial C uptake upon degradation of plant litter by different microbial groups and its role for macroaggregation in an agricultural soil. 2. Materials and methods
* Corresponding author. Fax: +49 531 5962699. E-mail address: [email protected] (M. Helfrich). http://dx.doi.org/10.1016/j.apsoil.2015.08.023 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.
Soil samples for PLFA analysis were derived from the first 28 days of an incubation experiment (Helfrich et al., 2008; Table 1).
262
M. Helfrich et al. / Applied Soil Ecology 96 (2015) 261–264
Table 1 Overview of the treatments. Quantification of PLFA was carried out on samples taken after 0, 14 and 28 days of incubation. Determination of specific 13C values was done on samples taken at the start (0 days) and after 28 days of incubation. Treatment
Description
Days incubated
S0 SC0 S14 SC14 SL14 SLC14 SR14 SRC14 S28 SC28 SL28 SLC28 SR28 SRC28
Control soil Control soil + Captan Control soil Control soil + Captan Soil + maize leaves Soil + maize leaves + Captan Soil + maize roots Soil + maize roots + Captan Control soil Control soil + Captan Soil + maize leaves Soil + maize leaves + Captan Soil + maize roots Soil + maize roots + Captan
0 0 14 14 14 14 14 14 28 28 28 28 28 28
In brief, soil of a silty loam (stagnic Luvisol derived from loess) under wheat monoculture (C3 vegetation; soil organic C 12.6 0.1 g kg 1; d13C –26.5 0.1m V-PDB; means standard deviation) was incubated after destruction of all naturally occurring macroaggregates (sieving <250 mm) in the dark at 15 C and 60% of the maximum water-holding capacity. Maize leaves (C/N 27.4; d13C 12.7 0.2m V-PDB; means standard deviation) or roots (C/N 86.4; d13C 11.9 0.2m V-PDB; means standard deviation) were oven-dried at 40 C, milled <500 mm and added to the soil at a rate of 2.1 mg C g 1 soil. Each treatment was carried out with and without addition of 0.3 g Captan per 100 g soil (50 W wettable powder, 89% active ingredient), which had been used as a fungicide in a number of studies (e.g. Bailey et al., 2002; Bossuyt et al., 2001; Denef et al., 2001) and was found to have little effect on soil bacteria (Ingham, 1985). Macroaggregates were obtained by wetsieving >250 mm (Helfrich et al., 2008). Phospholipid fatty acids were extracted following the method of Bligh and Dyer (1959) and Zelles and Bai (1993). Soil lipids were extracted using a mixture of chloroform, methanol and phosphate buffer (1:2:0.8 vol). Lipid extracts were separated into neutral, glycol- and phospholipids on silicic acid columns. Phospholipids
Fig. 1. Linear regression between the amount of lnPLFA 18:2v6 and water-stable macroaggregates.
were methylated and the obtained fatty acid methyl ester (FA-ME) were further separated into saturated, polyunsaturated and monounsaturated fatty acids and quantified by gas chromatography (GC: HP 6890 Series, AED: G 2350 A, Agilent Technologies, United States) using a HP ultra 2 column (50 m 0.32 mm I.D., 0.25 mm film thickness) in the split mode. Compound-specific determination of d13C values of individual PLFA-ME was performed with GC/MS-C-IRMS in triplicate (GCQ, Thermoquest, Germany); (Delta + XL, Finnigan MAT, Germany). To obtain d13C values of the PLFAs, the d13C values of PLFA-ME were corrected for the methyl-C which was added during methylation. The amount of maizederived C used as microbial C source in percent was calculated by dividing the isotopic shift between PLFAs from soil with and without addition of maize litter through the isotopic shift between the soil used for the incubation and the maize plant material (Balesdent and Mariotti, 1996). The obtained PLFAs were grouped into grampositive bacteria (Gram+) excluding actinomycetes
Table 2 Amounts of major and of biomarker PLFA for the investigated treatments. Amount of selected PLFAs (nmol g
i14:0 i15:0 a15:0 br16:0 i16:0 br17:0 i17:0 a17:0 br18:0r br19:0 18:2v6c n14:0 n15:0 n16:0 n17:0 n18:0 10Me17:0 10Me18:0 10Me19:0 cy19:0 16:1v7c 18:1v7c 18:1v9c % of total PLFA
+
Gram Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Gram+ Fungal marker Universal Universal Universal Universal Universal Actinomycetes Actinomycetes Actinomycetes Gram Gram Gram Gram
1
soil)
S0
SC0
S14
SC14
S28
SC28
SL14
SLC14
SL28
SLC28
SR14
SRC14
SR28
SRC28
0.60 4.87 4.13 0.40 1.87 0.65 1.18 1.21 0.91 0.33 0.87 1.03 0.51 6.40 0.34 1.24 2.84 0.58 1.57 3.40 5.02 7.83 5.61 77.6
0.65 5.27 4.80 0.45 1.86 0.79 1.19 1.30 0.99 0.38 0.76 1.04 0.81 6.78 0.32 1.39 3.17 0.62 1.72 4.07 4.27 7.29 5.00 78.6
0.79 6.92 5.60 0.56 2.38 0.87 1.52 1.63 1.25 0.55 1.14 1.19 0.55 8.75 0.34 1.85 3.66 0.81 2.08 4.14 6.08 9.84 8.52 79.3
0.79 6.23 5.89 0.53 2.32 0.86 1.37 1.57 1.25 0.46 0.90 1.22 0.54 8.17 0.35 1.43 3.61 0.74 2.06 3.99 6.11 9.42 6.64 78.1
0.51 5.33 4.35 0.44 1.81 0.78 1.20 1.15 1.03 0.38 0.82 0.95 0.47 6.35 0.29 1.24 3.41 0.62 1.70 3.35 4.70 7.54 4.66 79.7
0.66 5.79 5.65 0.49 2.19 0.87 1.27 1.49 1.34 0.43 0.79 1.06 0.52 7.87 0.34 1.35 3.32 0.65 1.90 3.95 4.87 8.02 5.46 80.7
0.88 7.49 7.31 0.51 3.13 0.87 1.58 1.94 1.08 0.41 1.87 1.43 0.63 12.38 0.46 2.00 3.43 0.79 2.13 3.73 8.90 11.76 7.30 80.2
1.05 6.50 9.41 0.52 2.82 0.89 1.29 1.80 1.17 0.43 1.70 1.59 0.68 14.71 0.48 2.32 3.44 0.76 2.06 4.22 6.24 9.03 6.25 81.1
1.39 11.39 10.68 0.79 4.34 1.35 2.45 2.80 1.69 0.66 2.63 2.27 0.97 18.69 0.69 2.91 5.08 1.16 3.21 5.62 16.09 21.90 12.95 77.9
1.10 7.82 10.79 0.60 3.19 0.94 1.51 2.13 1.31 0.54 2.23 1.86 0.80 16.46 0.53 2.56 3.95 0.87 2.46 4.67 8.69 12.53 8.58 78.8
0.89 9.27 8.46 0.66 3.75 1.04 1.97 2.29 1.53 0.61 2.42 1.43 0.73 12.79 0.52 2.23 4.43 1.04 2.79 4.78 11.05 15.19 10.35 78.3
0.95 7.05 9.38 0.60 3.02 0.94 1.50 2.00 1.33 0.48 1.59 1.35 0.70 11.20 0.47 2.06 3.92 0.87 2.32 4.54 6.61 9.69 6.85 79.7
1.18 10.57 9.23 0.72 3.86 1.02 2.17 2.38 1.49 0.58 2.85 1.61 0.82 14.09 0.57 2.33 5.63 1.08 2.95 5.16 11.38 17.71 10.26 79.6
0.95 6.97 9.10 0.57 2.82 0.83 1.41 1.85 1.13 0.46 1.41 1.38 0.68 10.99 0.42 1.88 3.83 0.81 2.23 4.67 6.57 10.23 6.92 81.2
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(Lechevalier, 1977), actinomycetes (Kroppenstedt, 1985) and gramnegative bacteria (Gram ) (Kramer and Gleixner, 2006). Fatty acid 18:2v6c was used as fungal marker (Joergensen and Wichern, 2008). However, 18:2v6c is not only an indicator for fungi, but generally appears in eukaryotes (Kaur et al., 2005). Oneway analyses of variance (ANOVAs) were carried out to study treatment effects on the contents of maize-derived C in PLFAs for individual biomarkers followed by a Tukey test. In cases of nonhomogeneity of variances Welch ANOVAs were used after testing each group for normality followed by pairwise t-tests with Bonferroni corrections without pooled standard deviations and without the assumption of equal variances. For markers 18:1v9c, br18:0, 10Me18:0, and 10Me19:0 we observed non-homogeneity of variances and not all groups were normally distributed. Thus, a Kruskal–Wallis test was carried out for these markers followed by multiple comparisons in case of a significant group effect. A linear
263
regression for the content of fungal PLFA with water-stable macroaggregates as predictor was calculated. Transformation of the content of fungal PLFA to its natural logarithm was carried out to obtain normality of residuals in the linear regression. All statistical analyses were performed using the software R (version 3.1.1, R Core Team, 2014). 3. Results and discussion A total of 53 PLFAs with C chain lengths between 14 and 24 were detected in the soil extracts. Twenty three PLFAs comprised 77.6– 81.2% of total PLFAs (Table 2), and were each present in sufficient quantity to obtain accurate d13C values. The fungal marker 18:2v6 contributed 1.0–2.1% to total PLFAs. The amount of fungal PLFA was lower in all treatments with Captan compared to their non-Captan treated counterparts, with
Fig. 2. Amount of maize-derived C from added plant litter of (A) fungi and actinomycetes, (B) grampositive bacteria (Gram+) and (C) universal markers and gramnegative bacteria (Gram ) in the treatments after 28 days of incubation (means and standard deviation of measurement, n = 3). Different letters indicate significant differences between treatments for individual biomarkers (p < 0.05).
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the soil and root treatments being most affected (Table 2). At day 28, fungal biomass was reduced by 15.0% and 50.4% in the SLC28 and SRC28 treatment compared to the respective nonfungicide treated variants, while the effect had levelled off in the control soil. However, as can be seen from Table 2, several bacterial groups including actinomycetes were even more inhibited by Captan than fungal PLFA (reduction upon the use of Captan by up to 46%). Similar to our study, Rousk et al. (2009) reported non-target effects of Captan on other microbial groups. The induced general toxity effects that are not specific to fungi which complicates interpretation of the obtained results and question the use of Captan as a fungicide although it has been used frequently as such in previous incubation (e.g. Bailey et al., 2002; Bossuyt et al., 2001; Denef et al., 2001) and field studies (e.g. Beare et al., 1997; Hu et al., 1995). The amount of fungal PLFA was related to the amount of waterstable macroaggregates (Fig. 1; R2 = 0.58). Analysis of 13C in specific PLFA analysis revealed that the incorporation of maize-derived C into the fungal biomass was considerably higher in the treatments without fungicide compared to fungicide-treated variants (Fig. 2A), pointing to decreased anabolic activity in fungicidetreated variants. This trend was observed for both, leaf-amended and root-amended soils. As seen for soil fungi, decreasing anabolic activity in fungicide-treated variants was observed for Gram bacteria (Fig. 2A). An increasing incorporation of maize-derived C upon use of fungicide was observed for actinomycetes 10Me19:0 as well as for the leaf treatments in 10Me18:0 and 10Me17:0—the latter two, however, were not significant (Fig. 2C). Gram+ bacteria and universal markers showed no general trend (Fig. 2B and C). The positive influence of soil fungi on the anabolic activity of Gram bacteria, but not on Gram+ bacteria may result from their dissimilar abilities to use different C sources. While Gram bacteria feed only on plant-derived material (which might be more readily degraded and therefore more easily available in the presence of an intact fungal community), Gram+ bacteria may use plant-derived C as well as more complex C sources such as soil organic C (Kramer and Gleixner, 2006; Thoms et al., 2010; Malik et al., 2015). The higher incorporation of maize-derived C into actinomycetes in the absence of fungi might be an indication that actinomycetes may take over the role of soil fungi in decomposition of SOM. However, Gram+ bacteria including actinomycetes were also found to feed on other microorganisms (Malik et al., 2015). Therefore, feeding on microbes that died upon the use of fungicide might be another possible reason for the increased 13C label in actinomycetes upon the use of fungicide. Furthermore, Gram+ bacterial cell envelopes are thicker and more complex than those of other microbial groups and could be turned over slower than those of other microbial groups. Yet, we do not think that the latter reason is of vital importance for the finding of a higher 13C label upon the use of fungicide in actinomycetes in our study, because (i) this was not a general trend found in Gram+ bacteria and (ii) we determined the label 28 days after the use of the fungicide which we think should be sufficient time for complete turnover of the microbial biomass that died upon the use of the fungicide. Overall, the use of the natural 13C label of maize plant litter and specific PLFA contents and 13C in specific PLFAs was a valuable tool to get new insights into the C transformation into different microbial groups upon plant litter degradation. Our results showed that the quality of plant residues is a key factor determining the composition as well as the activity of the soil microbial biomass, which is in accordance with findings from previous studies (Thoms and Gleixner, 2013). They further suggest that not only the contribution of fungi to the microbial biomass, but also fungal activity is of importance in the formation of macroaggregates. Due
to an increasing incorporation of maize-derived C into actinomycetes upon the use of fungicide, we assume that actinomycetes may take over the role of soil fungi in the decomposition of soil organic matter. Yet, this finding needs to be verified and a more detailed quantitative understanding of soil microbial trophic interactions is vitally needed. Further, the generalizability of the results to other soil types and climates remains unclear. We strongly recommend future studies which aim at a better mechanistic understanding of microbial processes contributing to the persistence and transformation of C in soil using a combination of isotope tracers combined with biomarker analyses. Acknowledgements This study was supported by a grant from the Deutsche Forschungsgemeinschaft. We thank Steffen Rühlow for excellent technical assistance and compound-specific 13C analysis as well as Anne-Gret Seifert and Andrea Scheibe for helpful discussions and support in the lab. References Amelung, W., Brodowski, S., Sandhage-Hofmann, A., Bol, R., 2008. Combining biomarker with stable isotope analyses for assessing the transformation and turnover of soil organic matter. Adv. Agron. 100, 155–250. Bailey, V.L., Smith, J.L., Bolton, H., 2002. Fungal-to-bacterial ratios in soils investigated for enhanced C sequestration. Soil Biol. Biochem. 34, 997–1007. Balesdent, J., Mariotti, A., 1996. Measurement of soil organic matter turnover using 13 C natural abundance. In: Boutton, T.W., Yamasaki, S. (Eds.), Mass Spectrometry of Soils. Marcel Dekker, New York, pp. 83–111. Beare, M.H., Hus, S., Coleman, D.C., Hendrix, P.F., 1997. Influences of mycelial fungi on soil aggregation and organic matter storage in conventional and no-tillage soils. Appl. Soil Ecol. 5, 211–219. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Bossuyt, H., Denef, K., Six, J., Frey, S.D., Merckx, R., Paustian, K., 2001. Influence of microbial populations and residue quality on aggregate stability. Appl. Soil Ecol. 16, 195–208. Denef, K., Six, J., Bossuyt, H., Frey, S.D., Elliott, E.T., Merckx, R., Paustian, K., 2001. Influence of dry–wet cycles on the interrelationship between aggregate, particulate organic matter, and microbial community dynamics. Soil Biol. Biochem. 33, 1599–1611. Helfrich, M., Ludwig, B., Potthoff, M., Flessa, H., 2008. Effect of litter quality and soil fungi on macroaggregate dynamics and associated partitioning of litter carbon and nitrogen. Soil Biol. Biochem. 40, 1823–1835. Hu, S., Coleman, D.C., Beare, M.H., Hendrix, P.F., 1995. Soil carbohydrates in aggrading and degrading agroecosystems—influences of fungi and aggregates. Agric. Ecosyst. Environ. 54, 77–88. Ingham, E.R., 1985. Review of the effects of 12 selected biocides on target and nontarget soil organisms. Crop Prot. 4, 3–32. Joergensen, R.G., Wichern, F., 2008. Quantitative assessment of the fungal contribution to microbial tissue in soil. Soil Biol. Biochem. 40, 2977–2991. Kaur, A., Chaudhary, A., Choudhary, R., Kaushik, R., 2005. Phospholipid fatty acid—a bioindicator of environment monitoring and assessment in soil ecosystem. Curr. Sci. 89, 1103–1112. Kramer, C., Gleixner, G., 2006. Variable use of plant- and soil-derived carbon by microorganisms in agricultural soils. Soil Biol. Biochem. 38, 3267–3278. Kroppenstedt, R.M., 1985. Fatty acid and menaquinone analysis of actinomycetes and related organisms. In: Goodfellow, M., Minnikin, D.E. (Eds.), Chemical Methods in Bacterial Systematics. Academic Press, London, pp. 173–199. Lechevalier, M.P., 1977. Lipids in bacterial taxonomy—a taxonomists view. Crit. Rev. Microbiol. 5, 109–210. Malik, A.A., Dannert, H., Griffiths, R.I., Thomson, B.C., Gleixner, G., 2015. Rhizosphere bacterial carbon turnover is higher in nucleic acids than membrane lipids: implications for understanding soil carbon cycling. Front. Microbiol. 6, 268. Rousk, J., Demoling, L.A., Bååth, E., 2009. Contrasting short-term antibiotic effects on respiration and bacterial growth compromises the validity of the selective respiratory inhibition technique to distinguish fungi and bacteria. Microb. Ecol. 58, 75–85. Thoms, C., Gleixner, G., 2013. Seasonal differences in tree species’ influence on soil microbial communities. Soil Biol. Biochem. 66, 239–248. Thoms, C., Gattinger, A., Jacob, M., Thomas, F.M., Gleixner, G., 2010. Direct and indirect effects of tree diversity drive soil microbial diversity in temperate deciduous forest. Soil Biol. Biochem. 42, 1558–1565. Zelles, L., Bai, Q.Y., 1993. Fractionation of fatty acids derived from soil lipids by solid phase extraction and their quantitative analysis by GC–MS. Soil Biol. Biochem. 25, 495–507.