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Additions of maize root mucilage to soil changed the structure of the bacterial community
ARTICLE IN PRESS
Soil Biology & Biochemistry 39 (2007) 1230–1233 www.elsevier.com/locate/soilbio
Short communication
Additions of maize root mucilage to soil changed the structure of the bacterial community Emile Benizria,, Christophe Nguyena, Se´verine Piuttia, Sophie Slezack-Deschaumesa, Laurent Philippotb a
UMR Agronomie et Environnement, INPL-INRA-ENSAIA, 2, avenue de la Foreˆt de Haye BP 172, 54505 Vandoeuvre-le`s-Nancy, France b UMR INRA 1220 Microbiologie et Ge´ochimie des sols, 17 avenue de Sully, BV 86510, 21065 Dijon Cedex, France Received 10 July 2006; received in revised form 7 December 2006; accepted 17 December 2006 Available online 30 January 2007
Abstract The organic compounds released from roots (rhizodeposits) stimulate the growth of the rhizosphere microbial community. They may be responsible for the differences in the structure of the microbial communities commonly observed between the rhizosphere and the bulk soil. Rhizodeposits consists of a broad range of compounds including root mucilage. The aim of this study was to investigate if additions of maize root mucilage, at a rate of 70 mg C g1 day1 for 15 days, to an agricultural soil could affect the structure of the bacterial community. Mucilage additions moderately increased microbial C (+23% increase relative to control), which suggests that the turnover rate of microorganisms consuming this substrate was high. Consistent with this, the number of cultivable bacteria was enhanced by +450%. Catabolic (Biologs GN2) and 16S–23S intergenic spacer fingerprints exhibited significant differences between control and mucilage treatments. These data indicate that mucilage can affect both the metabolic and genetic structure of the bacterial community as shown by a greater catabolic potential for carbohydrates. We concluded that mucilage is likely to significantly contribute to differences in the structure of the bacterial communities present in the rhizosphere compared to the bulk soil. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bacterial community structure; Maize; Rhizosphere; Mucilage; Biologs; RISA
In the rhizosphere, bacteria are not only more numerous (Newman, 1985) but also exhibit a different diversity than in the bulk soil (Bowen and Rovira, 1999; Lugtenberg et al., 1999; Gomes et al., 2001; Baudoin et al., 2002, 2003). The effect of plant roots on the microbial community are mainly attributed to the release of C compounds by plant roots via rhizodeposition (Griffiths et al., 1999). The major rhizodeposits are primarily diffusible soluble root exudates and secondly, root mucilage (Nguyen, 2003). Unlike soluble root exudates that are simple, readily available low molecular weight compounds, mucilage is composed of polymerized sugars and of up to 6% protein (review in Nguyen, 2003). Bacteria utilizing mucilage must have the capabilities of hydrolyzing these high molecular weight root polysaccharides to gain access to monomers that Corresponding author. Tel.: +33 383 59 58 48; fax: +33 383 59 57 99.
E-mail address: [email protected] (E. Benizri). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.12.026
support growth. Knee et al. (2001) demonstrated that strains of Rhizobium leguminosarum, Pseudomonas fluorescens and Burkholderia cepacia can use maize root mucilage as sole C source but with different growth rates. Numerous studies have investigated the effect of diffusible soluble root exudates on the structure of the soil microbial community (Benizri et al., 2002; Baudoin et al., 2003). In contrast, few studies were conducted on mucilage although it is quantitatively the second major rhizodeposit (Nguyen, 2003). Mounier et al. (2004) reported that addition of mucilage stimulated the activity of the denitrifying community but had a minor impact on its structure. In contrast, Lopez-Gutierrez et al. (2005) showed that in atrazine-treated soil mucilage decreased atrazine mineralization and caused a temporary increase in the size of the atrazine degrading community. Therefore, the objective of the present work was to investigate the significance of mucilage on the structure of
ARTICLE IN PRESS E. Benizri et al. / Soil Biology & Biochemistry 39 (2007) 1230–1233
soil bacterial communities of an agricultural soil. Soil microcosms were amended daily with either maize mucilage or water over a 15-day period and microbial community structure was assessed by both Ribosomal Intergenic Spacer Analysis (RISA) and community level physiological profiling (CLPP). The mucilage was collected according to the protocol described by Morel et al. (1986). At flowering, maize plants grown in the field at the experimental station of the Ecole Nationale Supe´rieure d’Agronomie et des Industries Alimentaires in Champenoux (North-east of France) were cut at ground level, below the crown of nodal roots that grew above the soil surface. In the laboratory, the cut end and nodal roots were dip into water and the plants were exposed to daylight. After a few minutes, the mucilage covering the root tips swelled and it was removed by aspiration using a vacuum device. The bulk of mucilage was centrifuged for 15 min at 5000g to discard debris and then dialyzed (12,000–14,000 Da) against deionized water to remove solutes. The purified mucilage was stored at 20 1C. The dry/fresh weight ratio of mucilage was 0.0086 and its C and N contents were 38.3% and 0.7% dry matter, respectively. Petri dishes were individually filled with 25 g of a dry calcareous soil (Rendzic Leptosol) and were supplied daily either with 2 ml water (n ¼ 5) or mucilage solution (70 mg C kg1 dry soil, n ¼ 5) for 15 days. Bacterial density was estimated by counting the colony-forming unit (CFU) after 10 days of incubation. Microbial C was determined from two subsamples of 5 g dry soil by the fumigation–extraction method (Vance et al., 1987) using a KEC of 0.45 (Wu et al., 1990). The structure of the bacterial community was investigated by (i) CLPP using sole C source Biologs GN2 microplates after 48 h of incubation (Garland, 1996) and (ii) analysis of the rrl gene intergenic spacer 16S–23S (RISA) after DNA extraction from soil from each Petri dish according to the method described by Martin-Laurent et al. (2001). The total C deposited in soil by roots is commonly estimated around 100 mg C kg1 soil day1 (Trofymow et al., 1987; Iijima et al., 2000). The contribution of mucilage to total rhizodeposition was estimated to be 2–12% (Nguyen, 2003) from experiments on roots growing without mechanical impedance. However, in soil conditions, mechanical constraint experienced by roots is likely to stimulate the production of mucilage (Boeuf-Tremblay et al., 1995; Groleau-Renaud et al., 1998; Iijima et al., 2000). Hence, the rate of mucilage additions used in the present work was within the upper range of values that are relevant to rhizosphere conditions. The few reports on mucilage degradation in soil focused only on metabolic activity (Mary et al., 1992, 1993) and did not investigate microbial density or structure. Here, mucilage amendments increased microbial C by 23% (+29 mg C kg1 soil; Table 1). This increase in microbial C after mucilage additions was the difference between gross growth and microbial decay. The gross growth derived
1231
Table 1 Abundance of cultivable bacteria and microbial C in soils amended with 70 mg C kg1 soil for 15 days
Cultivable bacteria Log (CFU g1 dry soil) Microbial C (mg C kg1 soil)
Control soils
Mucilage amended soils
7.8b
8.5a
123b (12.6)
152a (17.4)
Standard deviation is given within brackets, n ¼ 5: Letters indicate mean grouping by the Tukey test at Po0:05.
from mucilage was estimated by using the following model: dM ¼ kM þ A, dt
(1)
d Cm ¼ kYM, (2) dt where M is the amount of mucilage in soil (mg C kg1 soil), A the exogenous supply of mucilage to the soil (70 mg C kg1 soil day1), k the rate of mucilage utilization by microorganisms (0.5 day1, unpublished results), Cm the microbial carbon derived from mucilage (mg C kg1 soil) and Y the yield of conversion of mucilage into microbial C (Y ¼ 0:6; Mary et al., 1993). After integration, Eqs. (1) and (2) give A ð1 ekt Þ, k 1 kt Cm ¼ AY t þ ðe 1Þ þ Cm0 , k M¼
(3) (4)
where Cm0 is the microbial carbon at the start of the experiment. Then, the gross increase in microbial C derived from mucilage is (Cm–Cm0), which gives 546 mg C kg1 soil after 15 days of mucilage addition. This calculated gross increase was much higher than the net increase in microbial C determined at the end of the experiment (+29 mg C kg1 soil). This suggests a possible high microbial turnover rate and consequently that mucilage may have been assimilated by copiotrophic microbes (Zelenev et al., 2005). This is supported by the increase in cultivable bacteria in the soil amended with mucilage (+450%; Table 1). The high turnover of microorganisms was also suggested by Mary et al. (1993) to explain the rapid remineralization of immobilized N during mucilage incubation in soil. In the rhizosphere, rapid turnover of microorganisms that grow on mucilage might accelerate nutrient cycling, which is beneficial to plant nutrition. Two approaches were used to investigate changes in bacterial community structure (CLPP and RISA) and a significant impact of mucilage addition was observed on both the catabolic profiles of the bacterial community (Fig. 1) and its genetic structure (Fig. 2). This shift in the genetic structure of the bacterial community observed by RISA suggests that some bacterial populations in the studied soil are able to utilize mucilage and are therefore
ARTICLE IN PRESS E. Benizri et al. / Soil Biology & Biochemistry 39 (2007) 1230–1233
1232
selected by the repeated mucilage additions. This hypothesis is strengthened by the CLPP results indicating that, on average, mucilage-treated soils exhibited greater catabolic
potential for carbohydrates and reduced catabolic potential for carboxylic acids compared to the control soils (Fig. 3). Mucilage is composed of polymerized sugars (galactose4fucose4arabinose4xylose4glucose; Bacic et al., 1987; Knee et al, 2001). In this study, although microbial galactose and arabinose oxidation by the mucilage-treated soils increased fucose, glucose, and mannose oxidation did not. Hence, results from the Biolog profiles did not match the expected catabolic ranges of the microbial community in soil amended with mucilage. In contrast, in a previous study, Grayston et al. (1998) observed that addition of sucrose to soil resulted in higher oxidation rates of sucrose and of its hydrolysis product, i.e. glucose, in Biolog plates. The literature suggests that addition of artificial root exudates to soil could change the structure of the bacterial community (Griffiths et al., 1999; Baudoin et al., 2003). We expected mucilage to have an effect on the bacterial community structure because the ability to utilize this substrate was shown to be variable among rhizosphere bacteria (Knee et al., 2001). Hence, the bacteria that extensively utilize mucilage were expected to grow more importantly and consequently to be relatively more abundant. Our results indicated that a rate of 70 mg Cmucilage g1 soil day1 was sufficient to affect both the catabolic and the genetic structure of the bacterial community. In contrast, the work of Mounier et al. (2004) and that of Lopez-Gutierrez et al. (2005) showed that the same rate of mucilage addition to the same soil had minor effect on the structure of the denitrifier community or on the size of the atrazine-degrading community, though their activities were either stimulated or decreased. Therefore, the impact of repeated additions of mucilage was more important at the total bacterial community level than at the level of the functional communities studied previously. In conclusion, our work showed that addition of mucilage to soil can stimulate microbial growth, presumably those having a high turnover rate such as bacteria. In addition, both physiological (Biolog) and genetic (RISA)
15 Mucilage 10 Control 5 PC1 (39%) -10
0 -5
0
5
10
PC2 (15%)
-5 -10 -15
Fig. 1. Ordination plot of bacterial communities generated by principal component analysis of Biologs GN2 signatures.
0.8 Mucilage
0.6
Control
0.4 0.2
-1.5
-1.0
-0.5
0.0 0.0 -0.2 -0.4 -0.6
0.5
1.0
1.5
Axis 2 (20.1%)
Axis 1 (29.4%)
-0.8 Fig. 2. Ordination plot of bacterial communities generated by factorial cluster analysis of RISA matrices.
2.5
-1.5 -2
*
L-leucine
*
D.L-carnitine
*
L-histidine
*
inositol
**
α-D-lactose
adonitol
D-arabitol
L-arabinose
D-sorbitol
D-galactose
uridine
inosine
L-rhamnose
-1
itaconic acid
0 -0.5
D-glucosaminic acid
0.5
*** **
α-keto-butyricacid
***
formic acid
**
succinic acid
**
quinic acid
**
D-saccharidic acid
*** ** *
L-ornithine
*** ** *
xylitol
***
L-phenylalanine
** *
1
p-hydroxy-phenylacetic acid
OD mucilage – OD control
1.5
**
Mono-methyl-succinate
Mucilage
2
**
** * *
*
* Control **
-2.5 Fig. 3. Differences in substrate utilization in Biolog plates between soils amended with 70 mg C kg1 soil for 15 days and control soils (water addition). Optical densities (OD) corresponded to the oxidization intensity of the substrate. Only substrates for which the difference was significant are presented (Tukey test at Po0:05, n ¼ 5).
ARTICLE IN PRESS E. Benizri et al. / Soil Biology & Biochemistry 39 (2007) 1230–1233
profiles of the bacterial community were affected by mucilage addition. These results suggest that even though mucilage consists of high molecular weight polysaccharides, which are not easily broken down by soil microorganisms, it could contribute to the changes in the structure of the total microbial community in the rhizosphere. It would now be interesting to characterize the dynamics of microbial guilds able to utilize mucilage. This would enable us to better describe succession of populations within the bacterial community but also within the fungi community from the release of rhizodeposits. This work was funded by the ‘‘ACI Ecologie Quantitative’’ of the INSU-CNRS. The authors thank H.P. Guimont, manager of the Experimental Farm in Champenoux, for providing maize plots, P. Marchal for analyses and C. Robin for his valuable help for mucilage collection. References Bacic, A., Moody, S.F., McComb, J.A., Hinch, J.M., 1987. Extracellular polysaccharides from shaken liquid cultures of Zea mays. Australian Journal of Plant Physiology 14, 633–641. Baudoin, E., Benizri, E., Guckert, A., 2002. Impact of growth stage on the bacterial community structure along maize roots, as determined by metabolic and genetic fingerprinting. Applied Soil Ecology 19, 135–145. Baudoin, E., Benizri, E., Guckert, A., 2003. Impact of artificial root exudates on the bacterial community structure in bulk soil and maize rhizosphere. Soil Biology & Biochemistry 35, 1183–1192. Benizri, E., Dedourge, O., Di Battista-Leboeuf, C., Nguyen, C., Piutti, S., Guckert, A., 2002. Effect of maize rhizodeposits on soil microbial community structure. Applied Soil Ecology 21, 261–265. Boeuf-Tremblay, V., Plantureux, S., Guckert, A., 1995. Influence of mechanical impedance on root exudation of maize seedlings at two development stages. Plant and Soil 172, 279–287. Bowen, G.D., Rovira, A.D., 1999. The rhizosphere and its management to improve plant growth. In: Sparks, D.L. (Ed.), Advances in Agronomy (66). Academic Press, New York, pp. 1–102. Garland, J.L., 1996. Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization. Soil Biology & Biochemistry 28, 213–221. Gomes, N.C.M., Heuer, H., Scho¨nfeld, J., Costa, R., Mendoc- a-Hagler, L., Smalla, K., 2001. Bacterial diversity of the rhizosphere of maize (Zea mays) grown in tropical soil studied by temperature gradient gel electrophoresis. Plant and Soil 232, 167–180. Grayston, S.J., Campbell, C.D., Lutze, J.L., Gifford, R.M., 1998. Impact of elevated CO2 on the metabolic diversity of microbial communities in N-limited grass swards. Plant and Soil 203, 289–300. Griffiths, B.S., Ritz, K., Ebblwhite, N., Dobson, G., 1999. Soil microbial community structure: effects of substrate loading rates. Soil Biology & Biochemistry 31, 145–153.
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Groleau-Renaud, V., Plantureux, S., Guckert, A., 1998. Influence of plant morphology on root exudation of maize subjected to mechanical impedance in hydroponic conditions. Plant and Soil 201, 231–239. Iijima, M., Griffiths, B., Bengough, A.G., 2000. Sloughing of cap cells and carbon exudation from maize seedling roots in compacted sand. New Phytologist 145, 477–482. Knee, E.M., Gong, F.C., Gao, M., Teplitski, M., Jones, A.R., Foxworthy, A., 2001. Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source. Molecular Plant–Microbe Interactions 14, 775–784. Lopez-Gutierrez, J.C., Philippot, L., Martin-Laurent, F., 2005. Impact of maize mucilage on atrazine mineralization and atzC abundance. Pest Management Science 61, 838–844. Lugtenberg, B.J.J., Kravchenko, L.V., Simons, M., 1999. Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization. Environmental Microbiology 1, 439–446. Martin-Laurent, F., Philippot, L., Hallet, S., Chaussod, R., Germon, J.C., Soulas, G., Catroux, G., 2001. DNA extraction from soils: old bias for new microbial diversity analysis methods. Applied and Environmental Microbiology 67, 2354–2359. Mary, B., Mariotti, A., Morel, J.L., 1992. Use of 13C variations at natural abundance for studying the biodegradation of root mucilage, roots and glucose in soil. Soil Biology & Biochemistry 24, 1065–1072. Mary, B., Fresneau, C., Morel, J.L., Mariotti, A., 1993. C and N cycling during decomposition of root mucilage, roots and glucose in soil. Soil Biology & Biochemistry 25, 1005–1014. Morel, J.L., Mench, M., Guckert, A., 1986. Measurement of Pb2+, Cu2+ and Cd2+ binding with mucilage exudates from maize (Zea mays L.) roots. Biology and Fertility of Soils 2, 29–34. Mounier, E., Hallet, S., Cheneby, D., Benizri, E., Gruet, Y., Nguyen, C., Piutti, S., Robin, C., Slezack-Deschaumes, S., Martin-Laurent, F., Germon, J.C., Philippot, L., 2004. Influence of maize mucilage on the diversity and activity of the denitrifying community. Environmental Microbiology 6, 301–312. Newman, E.I., 1985. The rhizosphere: carbon sources and microbial populations. In: Fitter, A.D. (Ed.), Ecological Interactions in Soil. Blackwell Scientific Publications, London, pp. 107–122. Nguyen, C., 2003. Rhizodeposition of organic C by plant: mechanisms and controls. Agronomie 23, 375–396. Trofymow, J.A., Coleman, D.C., Cambardella, C., 1987. Rates of rhizodeposition and ammonium depletion in the rhizosphere of axenic oat roots. Plant and Soil 97, 333–344. Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology & Biochemistry 19, 703–707. Wu, J., Joergensen, R.G., Pommerening, B., Chaussod, R., Brookes, P.C., 1990. Measurement of soil microbial biomass C by fumigation–extraction—an automated procedure. Soil Biology & Biochemistry 22, 1167–1169. Zelenev, V.V., van Bruggen, A.H.C., Semenov, A.M., 2005. Modeling wave-like dynamics of oligotrophic and copiotrophic bacteria along wheat roots in response to nutrient input from a growing root tip. Ecological Modelling 188, 404–417.
Soil Biology & Biochemistry 39 (2007) 1230–1233 www.elsevier.com/locate/soilbio
Short communication
Additions of maize root mucilage to soil changed the structure of the bacterial community Emile Benizria,, Christophe Nguyena, Se´verine Piuttia, Sophie Slezack-Deschaumesa, Laurent Philippotb a
UMR Agronomie et Environnement, INPL-INRA-ENSAIA, 2, avenue de la Foreˆt de Haye BP 172, 54505 Vandoeuvre-le`s-Nancy, France b UMR INRA 1220 Microbiologie et Ge´ochimie des sols, 17 avenue de Sully, BV 86510, 21065 Dijon Cedex, France Received 10 July 2006; received in revised form 7 December 2006; accepted 17 December 2006 Available online 30 January 2007
Abstract The organic compounds released from roots (rhizodeposits) stimulate the growth of the rhizosphere microbial community. They may be responsible for the differences in the structure of the microbial communities commonly observed between the rhizosphere and the bulk soil. Rhizodeposits consists of a broad range of compounds including root mucilage. The aim of this study was to investigate if additions of maize root mucilage, at a rate of 70 mg C g1 day1 for 15 days, to an agricultural soil could affect the structure of the bacterial community. Mucilage additions moderately increased microbial C (+23% increase relative to control), which suggests that the turnover rate of microorganisms consuming this substrate was high. Consistent with this, the number of cultivable bacteria was enhanced by +450%. Catabolic (Biologs GN2) and 16S–23S intergenic spacer fingerprints exhibited significant differences between control and mucilage treatments. These data indicate that mucilage can affect both the metabolic and genetic structure of the bacterial community as shown by a greater catabolic potential for carbohydrates. We concluded that mucilage is likely to significantly contribute to differences in the structure of the bacterial communities present in the rhizosphere compared to the bulk soil. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bacterial community structure; Maize; Rhizosphere; Mucilage; Biologs; RISA
In the rhizosphere, bacteria are not only more numerous (Newman, 1985) but also exhibit a different diversity than in the bulk soil (Bowen and Rovira, 1999; Lugtenberg et al., 1999; Gomes et al., 2001; Baudoin et al., 2002, 2003). The effect of plant roots on the microbial community are mainly attributed to the release of C compounds by plant roots via rhizodeposition (Griffiths et al., 1999). The major rhizodeposits are primarily diffusible soluble root exudates and secondly, root mucilage (Nguyen, 2003). Unlike soluble root exudates that are simple, readily available low molecular weight compounds, mucilage is composed of polymerized sugars and of up to 6% protein (review in Nguyen, 2003). Bacteria utilizing mucilage must have the capabilities of hydrolyzing these high molecular weight root polysaccharides to gain access to monomers that Corresponding author. Tel.: +33 383 59 58 48; fax: +33 383 59 57 99.
E-mail address: [email protected] (E. Benizri). 0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.12.026
support growth. Knee et al. (2001) demonstrated that strains of Rhizobium leguminosarum, Pseudomonas fluorescens and Burkholderia cepacia can use maize root mucilage as sole C source but with different growth rates. Numerous studies have investigated the effect of diffusible soluble root exudates on the structure of the soil microbial community (Benizri et al., 2002; Baudoin et al., 2003). In contrast, few studies were conducted on mucilage although it is quantitatively the second major rhizodeposit (Nguyen, 2003). Mounier et al. (2004) reported that addition of mucilage stimulated the activity of the denitrifying community but had a minor impact on its structure. In contrast, Lopez-Gutierrez et al. (2005) showed that in atrazine-treated soil mucilage decreased atrazine mineralization and caused a temporary increase in the size of the atrazine degrading community. Therefore, the objective of the present work was to investigate the significance of mucilage on the structure of
ARTICLE IN PRESS E. Benizri et al. / Soil Biology & Biochemistry 39 (2007) 1230–1233
soil bacterial communities of an agricultural soil. Soil microcosms were amended daily with either maize mucilage or water over a 15-day period and microbial community structure was assessed by both Ribosomal Intergenic Spacer Analysis (RISA) and community level physiological profiling (CLPP). The mucilage was collected according to the protocol described by Morel et al. (1986). At flowering, maize plants grown in the field at the experimental station of the Ecole Nationale Supe´rieure d’Agronomie et des Industries Alimentaires in Champenoux (North-east of France) were cut at ground level, below the crown of nodal roots that grew above the soil surface. In the laboratory, the cut end and nodal roots were dip into water and the plants were exposed to daylight. After a few minutes, the mucilage covering the root tips swelled and it was removed by aspiration using a vacuum device. The bulk of mucilage was centrifuged for 15 min at 5000g to discard debris and then dialyzed (12,000–14,000 Da) against deionized water to remove solutes. The purified mucilage was stored at 20 1C. The dry/fresh weight ratio of mucilage was 0.0086 and its C and N contents were 38.3% and 0.7% dry matter, respectively. Petri dishes were individually filled with 25 g of a dry calcareous soil (Rendzic Leptosol) and were supplied daily either with 2 ml water (n ¼ 5) or mucilage solution (70 mg C kg1 dry soil, n ¼ 5) for 15 days. Bacterial density was estimated by counting the colony-forming unit (CFU) after 10 days of incubation. Microbial C was determined from two subsamples of 5 g dry soil by the fumigation–extraction method (Vance et al., 1987) using a KEC of 0.45 (Wu et al., 1990). The structure of the bacterial community was investigated by (i) CLPP using sole C source Biologs GN2 microplates after 48 h of incubation (Garland, 1996) and (ii) analysis of the rrl gene intergenic spacer 16S–23S (RISA) after DNA extraction from soil from each Petri dish according to the method described by Martin-Laurent et al. (2001). The total C deposited in soil by roots is commonly estimated around 100 mg C kg1 soil day1 (Trofymow et al., 1987; Iijima et al., 2000). The contribution of mucilage to total rhizodeposition was estimated to be 2–12% (Nguyen, 2003) from experiments on roots growing without mechanical impedance. However, in soil conditions, mechanical constraint experienced by roots is likely to stimulate the production of mucilage (Boeuf-Tremblay et al., 1995; Groleau-Renaud et al., 1998; Iijima et al., 2000). Hence, the rate of mucilage additions used in the present work was within the upper range of values that are relevant to rhizosphere conditions. The few reports on mucilage degradation in soil focused only on metabolic activity (Mary et al., 1992, 1993) and did not investigate microbial density or structure. Here, mucilage amendments increased microbial C by 23% (+29 mg C kg1 soil; Table 1). This increase in microbial C after mucilage additions was the difference between gross growth and microbial decay. The gross growth derived
1231
Table 1 Abundance of cultivable bacteria and microbial C in soils amended with 70 mg C kg1 soil for 15 days
Cultivable bacteria Log (CFU g1 dry soil) Microbial C (mg C kg1 soil)
Control soils
Mucilage amended soils
7.8b
8.5a
123b (12.6)
152a (17.4)
Standard deviation is given within brackets, n ¼ 5: Letters indicate mean grouping by the Tukey test at Po0:05.
from mucilage was estimated by using the following model: dM ¼ kM þ A, dt
(1)
d Cm ¼ kYM, (2) dt where M is the amount of mucilage in soil (mg C kg1 soil), A the exogenous supply of mucilage to the soil (70 mg C kg1 soil day1), k the rate of mucilage utilization by microorganisms (0.5 day1, unpublished results), Cm the microbial carbon derived from mucilage (mg C kg1 soil) and Y the yield of conversion of mucilage into microbial C (Y ¼ 0:6; Mary et al., 1993). After integration, Eqs. (1) and (2) give A ð1 ekt Þ, k 1 kt Cm ¼ AY t þ ðe 1Þ þ Cm0 , k M¼
(3) (4)
where Cm0 is the microbial carbon at the start of the experiment. Then, the gross increase in microbial C derived from mucilage is (Cm–Cm0), which gives 546 mg C kg1 soil after 15 days of mucilage addition. This calculated gross increase was much higher than the net increase in microbial C determined at the end of the experiment (+29 mg C kg1 soil). This suggests a possible high microbial turnover rate and consequently that mucilage may have been assimilated by copiotrophic microbes (Zelenev et al., 2005). This is supported by the increase in cultivable bacteria in the soil amended with mucilage (+450%; Table 1). The high turnover of microorganisms was also suggested by Mary et al. (1993) to explain the rapid remineralization of immobilized N during mucilage incubation in soil. In the rhizosphere, rapid turnover of microorganisms that grow on mucilage might accelerate nutrient cycling, which is beneficial to plant nutrition. Two approaches were used to investigate changes in bacterial community structure (CLPP and RISA) and a significant impact of mucilage addition was observed on both the catabolic profiles of the bacterial community (Fig. 1) and its genetic structure (Fig. 2). This shift in the genetic structure of the bacterial community observed by RISA suggests that some bacterial populations in the studied soil are able to utilize mucilage and are therefore
ARTICLE IN PRESS E. Benizri et al. / Soil Biology & Biochemistry 39 (2007) 1230–1233
1232
selected by the repeated mucilage additions. This hypothesis is strengthened by the CLPP results indicating that, on average, mucilage-treated soils exhibited greater catabolic
potential for carbohydrates and reduced catabolic potential for carboxylic acids compared to the control soils (Fig. 3). Mucilage is composed of polymerized sugars (galactose4fucose4arabinose4xylose4glucose; Bacic et al., 1987; Knee et al, 2001). In this study, although microbial galactose and arabinose oxidation by the mucilage-treated soils increased fucose, glucose, and mannose oxidation did not. Hence, results from the Biolog profiles did not match the expected catabolic ranges of the microbial community in soil amended with mucilage. In contrast, in a previous study, Grayston et al. (1998) observed that addition of sucrose to soil resulted in higher oxidation rates of sucrose and of its hydrolysis product, i.e. glucose, in Biolog plates. The literature suggests that addition of artificial root exudates to soil could change the structure of the bacterial community (Griffiths et al., 1999; Baudoin et al., 2003). We expected mucilage to have an effect on the bacterial community structure because the ability to utilize this substrate was shown to be variable among rhizosphere bacteria (Knee et al., 2001). Hence, the bacteria that extensively utilize mucilage were expected to grow more importantly and consequently to be relatively more abundant. Our results indicated that a rate of 70 mg Cmucilage g1 soil day1 was sufficient to affect both the catabolic and the genetic structure of the bacterial community. In contrast, the work of Mounier et al. (2004) and that of Lopez-Gutierrez et al. (2005) showed that the same rate of mucilage addition to the same soil had minor effect on the structure of the denitrifier community or on the size of the atrazine-degrading community, though their activities were either stimulated or decreased. Therefore, the impact of repeated additions of mucilage was more important at the total bacterial community level than at the level of the functional communities studied previously. In conclusion, our work showed that addition of mucilage to soil can stimulate microbial growth, presumably those having a high turnover rate such as bacteria. In addition, both physiological (Biolog) and genetic (RISA)
15 Mucilage 10 Control 5 PC1 (39%) -10
0 -5
0
5
10
PC2 (15%)
-5 -10 -15
Fig. 1. Ordination plot of bacterial communities generated by principal component analysis of Biologs GN2 signatures.
0.8 Mucilage
0.6
Control
0.4 0.2
-1.5
-1.0
-0.5
0.0 0.0 -0.2 -0.4 -0.6
0.5
1.0
1.5
Axis 2 (20.1%)
Axis 1 (29.4%)
-0.8 Fig. 2. Ordination plot of bacterial communities generated by factorial cluster analysis of RISA matrices.
2.5
-1.5 -2
*
L-leucine
*
D.L-carnitine
*
L-histidine
*
inositol
**
α-D-lactose
adonitol
D-arabitol
L-arabinose
D-sorbitol
D-galactose
uridine
inosine
L-rhamnose
-1
itaconic acid
0 -0.5
D-glucosaminic acid
0.5
*** **
α-keto-butyricacid
***
formic acid
**
succinic acid
**
quinic acid
**
D-saccharidic acid
*** ** *
L-ornithine
*** ** *
xylitol
***
L-phenylalanine
** *
1
p-hydroxy-phenylacetic acid
OD mucilage – OD control
1.5
**
Mono-methyl-succinate
Mucilage
2
**
** * *
*
* Control **
-2.5 Fig. 3. Differences in substrate utilization in Biolog plates between soils amended with 70 mg C kg1 soil for 15 days and control soils (water addition). Optical densities (OD) corresponded to the oxidization intensity of the substrate. Only substrates for which the difference was significant are presented (Tukey test at Po0:05, n ¼ 5).
ARTICLE IN PRESS E. Benizri et al. / Soil Biology & Biochemistry 39 (2007) 1230–1233
profiles of the bacterial community were affected by mucilage addition. These results suggest that even though mucilage consists of high molecular weight polysaccharides, which are not easily broken down by soil microorganisms, it could contribute to the changes in the structure of the total microbial community in the rhizosphere. It would now be interesting to characterize the dynamics of microbial guilds able to utilize mucilage. This would enable us to better describe succession of populations within the bacterial community but also within the fungi community from the release of rhizodeposits. This work was funded by the ‘‘ACI Ecologie Quantitative’’ of the INSU-CNRS. The authors thank H.P. Guimont, manager of the Experimental Farm in Champenoux, for providing maize plots, P. Marchal for analyses and C. Robin for his valuable help for mucilage collection. References Bacic, A., Moody, S.F., McComb, J.A., Hinch, J.M., 1987. Extracellular polysaccharides from shaken liquid cultures of Zea mays. Australian Journal of Plant Physiology 14, 633–641. Baudoin, E., Benizri, E., Guckert, A., 2002. Impact of growth stage on the bacterial community structure along maize roots, as determined by metabolic and genetic fingerprinting. Applied Soil Ecology 19, 135–145. Baudoin, E., Benizri, E., Guckert, A., 2003. Impact of artificial root exudates on the bacterial community structure in bulk soil and maize rhizosphere. Soil Biology & Biochemistry 35, 1183–1192. Benizri, E., Dedourge, O., Di Battista-Leboeuf, C., Nguyen, C., Piutti, S., Guckert, A., 2002. Effect of maize rhizodeposits on soil microbial community structure. Applied Soil Ecology 21, 261–265. Boeuf-Tremblay, V., Plantureux, S., Guckert, A., 1995. Influence of mechanical impedance on root exudation of maize seedlings at two development stages. Plant and Soil 172, 279–287. Bowen, G.D., Rovira, A.D., 1999. The rhizosphere and its management to improve plant growth. In: Sparks, D.L. (Ed.), Advances in Agronomy (66). Academic Press, New York, pp. 1–102. Garland, J.L., 1996. Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization. Soil Biology & Biochemistry 28, 213–221. Gomes, N.C.M., Heuer, H., Scho¨nfeld, J., Costa, R., Mendoc- a-Hagler, L., Smalla, K., 2001. Bacterial diversity of the rhizosphere of maize (Zea mays) grown in tropical soil studied by temperature gradient gel electrophoresis. Plant and Soil 232, 167–180. Grayston, S.J., Campbell, C.D., Lutze, J.L., Gifford, R.M., 1998. Impact of elevated CO2 on the metabolic diversity of microbial communities in N-limited grass swards. Plant and Soil 203, 289–300. Griffiths, B.S., Ritz, K., Ebblwhite, N., Dobson, G., 1999. Soil microbial community structure: effects of substrate loading rates. Soil Biology & Biochemistry 31, 145–153.
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