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Applied and Environmental Soil Science
Volume 2012, Article ID 307415, 4 pages
Research Article

Control of Cultivable IAA-Producing Bacteria by the Plant Arabidopsis thaliana and the Earthworm Aporrectodea caliginosa

UMR Biogéochimie et Ecologie des Milieux Continentaux, Université Paris-Est Créteil Val de Marne, 61 Avenue du Général De Gaulle, 94010 Créteil Cedex, France

Received 2 April 2012; Revised 9 May 2012; Accepted 16 May 2012

Academic Editor: Marco Trevisan

Copyright © 2012 Ruben Puga-Freitas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Some soil microorganisms are involved in the complex interactions with plants and earthworms, through the production of indole acetic acid (IAA) which modifies plant growth and development. In a factorial experiment testing the impact of the presence/absence of plants and earthworms on IAA production by cultivable bacteria, we observed that plants were decreasing IAA production of 43%, whereas earthworms were increasing it of 46%. In the presence of both plant and earthworms, IAA production was as low as in the presence of plant control, showing that plants influence on IAA production by microorganisms prevails on earthworm influence. We discuss functional reasons which could explain this result.

1. Introduction

Soil microorganisms can be closely associated with soil animals such as earthworms for the delivery of ecosystem functions such as organic matter mineralization [13]. The release of nutrients due to this dual interaction has consequences on plant growth [46]. In addition to nutrients, interdependence between microorganisms, plants, and earthworms has led to the selection of diversified signal molecules, from low-molecular weight compounds such as auxin (e.g., IAA), amino acids, and secondary metabolites, to high-molecular weight compounds such as proteins and polysaccharides (for review see [7]). Emission of signal molecules by microorganisms can be deleterious for plants in the case of pathogenic bacteria and invertebrate herbivores, or positive with mycorrhizas, nitrogen-fixing symbiotic bacteria, or free living bacteria such as plant growth promoting rhizobacteria (PGPR) [79]. Auxin-like compounds have also been detected in earthworm dejections [10, 11], and could be responsible for the positive effect of earthworms [12] and vermicompost [10] on plant growth. The same molecule can have positive or negative effects according to its concentration and plant sensitivity [13].

There are very few studies which consider simultaneously the impact of plants and earthworms on microbial activities. However, it is of prior importance to establish a hierarchy in biotic factors influencing soil microorganisms. The purpose of our study was to determine the hierarchy of biotic factors on IAA-producing bacteria: do microorganisms obey to plants or earthworms?

2. Materials and Methods

To answer this question, we set up an experiment with 4 treatments: soil without macroorganisms (S), with the plant Arabidopsis thaliana (P), and with the earthworm Aporrectodea caliginosa (E), with both plant and earthworms (PE). Nine cm diameter-10 cm high PVC experimental units ( per treatment) were filled with 1 kg of dry sandy soil from the CEREEP research station (Saint-Pierre-Lès-Nemours, France).

It was dried at 25°C for a week and sieved at 2 mm mesh size. This soil has the following properties: total organic carbon content, 14.7 g kg−1; total nitrogen content, 1.19 g kg−1; pH, 5.22; CEC, 4.08 cmol kg−1; texture: 6.9% clay, 19.0% silt, 74.1% sand. During the experiment, it was then maintained at 80% of the field capacity. Three earthworms ( ) were introduced one week after soil moistening in treatments E and PE. One seed of Arabidopsis thaliana cv. Columbia was sown per experimental unit two weeks after soil watering in treatment P and PE. Plants were grown for 42 days under controlled conditions (Conviron Growth Chamber, Canada) at 20 ± 1°C and 18 ± 1°C day and night temperatures, relative humidity, and 200 μmol m−2 s−1 photon flux for 10 h per day.

At the end of the experiment, plant shoots and roots were retrieved, dried at 45°C, and weighted. As extraction and purification of IAA from natural soils is very difficult due to soil adsorbing complex (organic matter, humus, clays etc.), we extracted bacterial communities from the soil of the different treatments and quantified the production of IAA due to cultivable bacteria present in these extracts. Soil samples (100 g) were collected from experimental units and dried at 40°C. For all treatments (S, P, E, and PE), microbial populations were extracted from these dried soil samples in aqueous solution (5 g L−1 NaCl) at 1 : 10 soil : water ratio with 40 rpm agitation for 1 h. After centrifugation (3 min, 1000 rpm), 1 mL of bacterial suspension was inoculated on 6.5 mL of liquid Czapek media (with only 5 g L−1 sucrose). Two hundred μg L−1 of L-Tryptophan (Sigma, USA) was added as precursor for IAA production. Bacteria culture was performed in the dark at 27°C with shaking at 180 rpm. At 24 h, 48 h, and 72 h, 500 μL of the bacterial culture supernatant were retrieved. Bacterial cells were removed by centrifugation at 5000 rpm during 1 min. Part of the supernatant (250 μL) free of bacterial cells was mixed with 750 μL of Salkowski’s reagent (50 mL HClO4 at 35% and 1 mL of 0.5 M FeCl3). IAA concentration was determined by absorbance at 530 nm with a Novaspec III spectrophotometer (Amersham Biosciences, USA), with the help of a standard curve obtained from serial dilutions of IAA stock solution (Sigma, USA).

3. Results

IAA production by cultivable soil microorganisms extracted from soil without macro-organisms (S) was about  mol L−1, whereas the presence of earthworms in soil (E) was responsible for a significant 46% (  mol L−1) increase in IAA production by cultivable microorganisms (Figure 1). In the presence of plants (P) or in the presence of plants and earthworms (PE), IAA production was at the same level, respectively, and  mol L−1 (Figure 1), suggesting that plants halved IAA production by soil microorganisms. A two-ways ANOVA confirmed the significance of plant negative effect (P value < 0.001, F = 58.33), and earthworm positive effect (P value = 0.02, F = 6.8) on IAA production by microorganisms (Table 1). The reason for this inhibition of IAA production by plants could be linked to the fact that too high IAA exogenous concentration can inhibit root growth and prevent plant development [14] (see Section 4). This was supported by the significant negative correlation ( = 0.71, P value = 0.005) between plant biomass and IAA production (Figure 2). As far as the interaction between plants and earthworms is concerned, we observed that the positive effect of earthworms on IAA production observed in treatment E disappeared in treatment PE (Figure 1). A two-ways ANOVA showed that the statistical interaction between the factors of plants and earthworms was significant (P value = 0.01, F = 8.25), which means that earthworm effect on IAA production was dependant on the presence of plants.

Table 1: Impact of the factors “plant,” “earthworm,” and their interaction on IAA production by cultivable microorganisms.
Figure 1: Average IAA concentration at 24, 48, and 72 h by cultivable soil bacteria from soil without macroorganisms (S), with the plant Arabidopsis thaliana (P), with the earthworm Aporrectodea caliginosa (E) and with both plant and earthworms (PE). Means ± s.e., n = 17, different letters indicate a significant difference, Tukey HSD, P < 0.05.
Figure 2: Relationship between IAA concentration and plant above-ground biomass (y = −24.35x + 54.53, = 0.75, P = 0.005).

Note that the discussed IAA concentration was due to the production of IAA by cultivable microorganisms, which represent only a small fraction of microorganisms living in a soil, despite the large spectrum of microorganisms growing on the Czapek medium. An alternative approach would be to extract all the IAA from the soil, but a given extraction protocol would not have the same extraction efficiency for soils deeply modified by plants or earthworms. Moreover, protocols developed to detect IAA in complex soils [10, 11, 15] do not provide reliable methods to quantify absolute amount of IAA in soil samples.

4. Discussion

Interactions between organisms in soil are mainly based on trophic relationships and exchange of signals. Among signal molecules, auxins have been shown to play a major role in plant-microorganism interactions; for example, rhizobacteria producing high amounts of IAA are generally responsible for a negative effect on plant growth and have been called “Deleterious Rhizobacteria” [1618]. Rhizobacteria producing low amounts of IAA are generally responsible for a positive effect on plant growth and have been called “Plant Growth Promoting Rhizobacteria” [9, 19, 20]. This important effect of IAA can be related to its role in cell division and elongation: it is demonstrated that the auxin dose-response curve is bell shaped, with a maximal elongation rate reached for medium exogenous auxin concentration [14].

Many soil bacteria use signals to direct plant carbon for the build-up of additional root structures [2123], with a subsequent positive effect on root exudates and bacteria populations. As earthworms are supposed to consume soil microorganisms, the increase in IAA production observed in the presence of earthworms could be explained as a way to increase root exudates, bacterial biomass, and thus earthworm resources. This is supported by previous observations of higher IAA content in earthworms-worked soils as compared with soil without earthworms [10, 11] as well as the hormone-like effects observed in compost made by earthworms [2426].

However, initiation of additional root structures or increased exudation is probably not always beneficial to plants. Plants have indeed developed mechanisms to regulate IAA production by microorganisms. There are very few evidences of a direct control of IAA producing bacterial communities by plants. Nevertheless, it has been shown that transcription of ipdC, an Erwinia IAA biosynthetic gene, is induced in response to bean and tobacco compounds [27]. This kind of mechanism could be at the origin of the repression of IAA producing bacteria in the presence of plants observed in our study.

5. Conclusion

In this study, we confirmed that earthworms can be responsible for an increase in IAA production by microorganisms. We also observed a repression of IAA production in the presence of plants. In the presence of both earthworms and plants, we found that plants took the advantage on earthworms in the control of cultivable IAA-producing bacteria, by maintaining a weak level of IAA production by cultivable microorganisms.

Plants are responsible for energy inputs in terrestrial ecosystems (about 90% of soil organic matter). They are major providers of substrates to microorganisms through root exudates and dead roots, which could represent 20 to 50% of the total assimilated carbon [28, 29], or to earthworms through dead leaves and roots [30]. This could explain the fact that they control many soil processes such as the production of IAA, but also free-living bacteria nitrogenase activity [31], amoebae community structure [32], and hydrostructural stability of soil [33].


  1. G. G. Brown, I. Barois, and P. Lavelle, “Regulation of soil organic matter dynamics and microbial activity in the drilosphere and the role of interactions with other edaphic functional domains,” European Journal of Soil Biology, vol. 36, no. 3-4, pp. 177–198, 2000. View at Publisher · View at Google Scholar · View at Scopus
  2. P. Lavelle, “Functional domains in soils,” Ecological Research, vol. 17, no. 4, pp. 441–450, 2002. View at Publisher · View at Google Scholar · View at Scopus
  3. P. Lavelle and A. V. Spain, Soil Ecology, Kluwer Scientific Publications, Amsterdam, The Netherlands, 2001.
  4. R. E. Ingham, J. A. Trofymow, E. R. Ingham, and D. C. Coleman, “Interactions of bacteria, fungi, and their nematode grazers: effects on nutrient cycling and plant growth,” Ecological Monographs, vol. 55, no. 1, pp. 119–140, 1985. View at Google Scholar
  5. M. Blouin, S. Barot, and P. Lavelle, “Earthworms (Millsonia anomala, Megascolecidae) do not increase rice growth through enhanced nitrogen mineralization,” Soil Biology and Biochemistry, vol. 38, no. 8, pp. 2063–2068, 2006. View at Publisher · View at Google Scholar · View at Scopus
  6. U. Jana, S. Barot, M. Blouin, P. Lavelle, D. Laffray, and A. Repellin, “Earthworms influence the production of above- and belowground biomass and the expression of genes involved in cell proliferation and stress responses in Arabidopsis thaliana,” Soil Biology and Biochemistry, vol. 42, no. 2, pp. 244–252, 2010. View at Publisher · View at Google Scholar · View at Scopus
  7. H. P. Bais, T. L. Weir, L. G. Perry, S. Gilroy, and J. M. Vivanco, “The role of root exudates in rhizosphere interactions with plants and other organisms,” Annual Review of Plant Biology, vol. 57, pp. 233–266, 2006. View at Publisher · View at Google Scholar · View at Scopus
  8. D. V. Badri and J. M. Vivanco, “Regulation and function of root exudates,” Plant, Cell & Environment, vol. 32, no. 6, pp. 666–681, 2009. View at Publisher · View at Google Scholar · View at Scopus
  9. F. Persello-Cartieaux, L. Nussaume, and C. Robaglia, “Tales from the underground: molecular plant-rhizobacteria interactions,” Plant, Cell & Environment, vol. 26, no. 2, pp. 189–199, 2003. View at Publisher · View at Google Scholar · View at Scopus
  10. L. P. Canellas, F. L. Olivares, A. L. Okorokova-Façanha, and A. R. Façanha, “Humic acids isolated from earthworm compost enhance root elongation, lateral root emergence, and plasma membrane H+-ATPase activity in maize roots,” Plant Physiology, vol. 130, no. 4, pp. 1951–1957, 2002. View at Publisher · View at Google Scholar · View at Scopus
  11. A. Muscolo, S. Cutrupi, and S. Nardi, “IAA detection in humic substances,” Soil Biology and Biochemistry, vol. 30, no. 8-9, pp. 1199–1201, 1998. View at Publisher · View at Google Scholar · View at Scopus
  12. G. G. Brown, C. A. Edwards, and L. Brussaard, “How earthworms affect plant growth: burrowing into the mechanisms,” in Earthworm Ecology, pp. 13–49, CRC Press, Boca Raton, Fla, USA, 2004. View at Google Scholar
  13. O. Barazani and J. Friedman, “Is IAA the major root growth factor secreted from plant-growth-mediating bacteria?” Journal of Chemical Ecology, vol. 25, no. 10, pp. 2397–2406, 1999. View at Google Scholar · View at Scopus
  14. L. Taiz and E. Zeiger, Plant Physiology, Sinauer Associates, Sunderland, Mass, USA, 3rd edition, 2010.
  15. S. Quaggiotti, B. Ruperti, D. Pizzeghello, O. Francioso, V. Tugnoli, and S. Nardi, “Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.),” Journal of Experimental Botany, vol. 55, no. 398, pp. 803–813, 2004. View at Publisher · View at Google Scholar · View at Scopus
  16. D. B. Nehl, S. J. Allen, and J. F. Brown, “Deleterious rhizosphere bacteria: an integrating perspective,” Applied Soil Ecology, vol. 5, no. 1, pp. 1–20, 1997. View at Google Scholar · View at Scopus
  17. B. Schippers, A. W. Bakker, and P. Bakker, “Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practices,” Annual Review of Phytopathology, vol. 25, pp. 339–358, 1987. View at Google Scholar
  18. T. V. Suslow and M. N. Schroth, “Role of deleterious rhizobacteria as minor pathogens in reducing crop growth,” Phytopathology, vol. 72, no. 1, pp. 111–115, 1982. View at Google Scholar
  19. Y. Bashan and G. Holguin, “Proposal for the division of plant growth-promoting rhizobacteria into two classifications: biocontrol-PGPB (plant growth-promoting bacteria) and PGPB,” Soil Biology and Biochemistry, vol. 30, no. 8-9, pp. 1225–1228, 1998. View at Publisher · View at Google Scholar · View at Scopus
  20. J. W. Kloepper and M. N. Schroth, “Plant growth-promoting rhizobacteria on radishes,” in Proceedings of the 4th International Conference on Plant Pathogenic Bacteria, pp. 879–882, Station de Pathologie Vegetale et Phytobacteriologie, 1978.
  21. A. M. Hirsch, Y. Fang, S. Asad, and Y. Kapulnik, “The role of phytohormones in plant-microbe symbioses,” Plant and Soil, vol. 194, no. 1-2, pp. 171–184, 1997. View at Google Scholar · View at Scopus
  22. P. E. Jameson, “Cytokinins and auxins in plant-pathogen interactions—an overview,” Plant Growth Regulation, vol. 32, no. 2-3, pp. 369–380, 2000. View at Publisher · View at Google Scholar · View at Scopus
  23. M. Bonkowski, “Protozoa and plant growth: the microbial loop in soil revisited,” New Phytologist, vol. 162, no. 3, pp. 617–631, 2004. View at Publisher · View at Google Scholar · View at Scopus
  24. N. Q. Arancon, C. A. Edwards, S. Lee, and R. Byrne, “Effects ofhumic acids from vermicomposts onplant growth,” European Journal of Soil Biology, vol. 42, no. 1, pp. S65–S69, 2006. View at Publisher · View at Google Scholar · View at Scopus
  25. R. M. Atiyeh, N. Arancon, C. A. Edwards, and J. D. Metzger, “Influence of earthworm-processed pig manure on the growth and yield of greenhouse tomatoes,” Bioresource Technology, vol. 75, no. 3, pp. 175–180, 2000. View at Publisher · View at Google Scholar · View at Scopus
  26. H. Chaoui, C. A. Edwards, M. Brickner, S. Lee, and N. Q. Arancon, “Suppression of the plant deseases Pythium, Rhizoctonia, and Verticillium by vermicompost,” Proceedings of the Brighton Crop Protection Conference on Pests Diseases, vol. 2, no. 8B-3, pp. 711–715, 2002. View at Google Scholar
  27. M. T. Brandl and S. E. Lindow, “Environmental signals modulate the expression of an indole-3-acetic acid biosynthetic gene in Erwinia herbicola,” Molecular Plant-Microbe Interactions, vol. 10, no. 4, pp. 499–505, 1997. View at Google Scholar · View at Scopus
  28. Y. Kuzyakov and G. Domanski, “Carbon input by plants into the soil. Review,” Journal of Plant Nutrition and Soil Science, vol. 163, no. 4, pp. 421–431, 2000. View at Google Scholar · View at Scopus
  29. J. M. Lynch and J. M. Whipps, “Substrate flow in the rhizosphere,” Plant and Soil, vol. 129, no. 1, pp. 1–10, 1990. View at Publisher · View at Google Scholar · View at Scopus
  30. L. Brussaard, “On the mechanisms of interactions between earthworms and plants,” Pedobiologia, vol. 43, no. 6, pp. 880–885, 1999. View at Google Scholar · View at Scopus
  31. M. J. Zarea, A. Ghalavand, E. M. Goltapeh, F. Rejali, and M. Zamaniyan, “Effects of mixed cropping, earthworms (Pheretima sp.), and arbuscular mycorrhizal fungi (Glomus mosseae) on plant yield, mycorrhizal colonization rate, soil microbial biomass, and nitrogenase activity of free-living rhizosphere bacteria,” Pedobiologia, vol. 52, no. 4, pp. 223–235, 2009. View at Publisher · View at Google Scholar · View at Scopus
  32. J. Alphei, M. Bonkowski, and S. Scheu, “Protozoa, nematoda and lumbricidae in the rhizosphere of Hordelymus europaeus (Poaceae): faunal interactions, response of microorganisms and effects on plant growth,” Oecologia, vol. 106, no. 1, pp. 111–126, 1996. View at Google Scholar · View at Scopus
  33. R. Milleret, R. C. Le Bayon, F. Lamy, J. M. Gobat, and P. Boivin, “Impact of roots, mycorrhizas and earthworms on soil physical properties as assessed by shrinkage analysis,” Journal of Hydrology, vol. 373, no. 3-4, pp. 499–507, 2009. View at Publisher · View at Google Scholar · View at Scopus