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Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola
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Soil Biology & Biochemistry xxx (2015) 1e4
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Q6
Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola
Q5
kou F.M. Coulibaly, Matthieu Chauvat Estelle Forey*, Se Ecodiv URA/EA-1293, Normandie Universit e, Universit e de Rouen, IRSTEA, SFR Scale 4116, UFR Sciences et Techniques, 76821 Mont Saint Aignan Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2015 Received in revised form 23 July 2015 Accepted 27 July 2015 Available online xxx
The role of soil organisms as possible driver of flowering has never been investigated. We hypothesized that Collembola (microarthropods) will change plant allocation to reproductive modes by changing soil nutrient availability. Individual seedlings of Poa annua were planted in microcosms, in the presence or absence of Collembola. Collembola affected biotic (fungal biomass) and abiotic (N NO3 , P2O5) soil properties and some morphological (number of leaves, root biomass) and chemical (C:N, K, Mg, N) traits of P. annua. As a result, flowering of P. annua was promoted by the presence of Collembola. This provides experimental evidence that soil microarthropods can affect the reproduction strategy and phenology of a plant. © 2015 Published by Elsevier Ltd.
Keywords: Aboveebelow ground interactions Soil fauna Plant performances Soil properties Springtails
The belowground compartment is a fundamental driver of the aboveground system and therefore of ecosystem fate and properties (De Deyn et al., 2003; Wardle et al., 2004; Wagg et al., 2014). Collembola are among the most abundant microarthropods in the rhizosphere of plants (Coleman et al., 2004). Several studies have highlighted a strong influence of various Collembolan species on plant roots either indirectly by regulating soil nutrient availability (Bardgett and Chan, 1999; Filser, 2002; Ngosong et al., 2014) or directly by exerting rhizophagous pressure (Endlweber et al., 2009). Nevertheless, the consequences of such impacts on resource allocation for plant reproduction and on flowering phenology have apparently never been investigated. A small change in reproductive success or flowering time can affect plant fitness (e.g. Galloway and Burgess, 2012; Weis et al., 2015), and consequently might alter interactions with populations of pollinators, seed dispersers, or floral herbivores. The role of biotic interactions in shaping plant flowering phenology has been demonstrated for pollinators and herbivores (Elzinga et al., 2007). Therefore, in the present study, we tested in a microcosm experiment whether the presence of a natural assemblage of Collembola affected plant growth and sexual reproduction (flowering) of Poa annua L.
* Corresponding author. Tel.: þ33 2 32769455. E-mail address: [email protected] (E. Forey).
In a microcosm experiment, two treatments were established corresponding to the presence (“Coll.”) or absence (“None”) of a natural assemblage of Collembola. Each treatment was replicated 15 times in microcosms (plastic pots 10 9 9 cm) where individuals of P. annua were grown. The soil was a rendosol (Organic Matter content ¼ 6.17%; pH ¼ 7.79) collected from a chalk grassland (170 3000 E, 49 220 2200 N) in Normandy, France (further details are given in Supplemental information). A part of the soil was defaunated by repeated deep-freezing and thawing (see Supplemental information). The 30 microcosms were meshed (250 mm) to prevent any escape of microarthropods and were filled with a mixture of defaunated soil and sand (5:1). From the remaining part of the collected soil, microorganisms (fungi and bacteria) and Collembola were extracted and re-inoculated in order to set the two different treatments. Microorganisms were re-inoculated in all microcosms prior to Collembola by adding “soil extract” (see Supplemental information). Re-inoculation of microorganisms was followed, two weeks later, by Collembola addition into the 15 “Coll” microcosms. Collembola were extracted from collected soil using a BerleseeTullgren apparatus (see Supplemental information). The extraction of Collembola in microcosms at the end of the experiment, allowed us to confirm that (i) no contamination was observed in the treatment without Collembola, and that (ii) collembolan assemblages, in terms of abundance, were maintained until the end of the experiment, even if inevitably compositional
http://dx.doi.org/10.1016/j.soilbio.2015.07.024 0038-0717/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Forey, E., et al., Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.07.024
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E. Forey et al. / Soil Biology & Biochemistry xxx (2015) 1e4
Table 1 Effect of Collembolan inoculation on soil properties after 10 weeks of Poa annua growth. Data are means ± SE. Statistical significance was set at p < 0.05. Soil properties:
N
None
Coll.
Statistical test T or Z
1
Cmic (mgC g dw) Ergosterol (mg g1 dw) Fungal:bacterial ratio N NO4 þ (mg g1 dw) N NO3 (mg g1 dw) P2O5 (mg g1 dw) pH a b
10 10 10 10 10 10 10
300.1 1.84 0.63 0.097 0.063 34.7 7.78
± ± ± ± ± ± ±
20.2 0.08 0.03 0.007 0.002 1.6 0.03
315.6 2.45 0.79 0.109 0.100 53.0 7.76
± ± ± ± ± ± ±
18.3 0.12 0.03 0.013 0.011 2.5 0.02
P value a
0.570 4.166a 3.228a 0.189b 3.175b 6.153a 0.600a
0.575 <0.001 0.004 0.853 <0.001 <0.001 0.555
Student's t test. ManneWhitney non-parametric test.
changes appeared (see Supplemental information and supplemental Table 1). Basically we observed a shift from a community dominated by Folsomia quadrioculata (Tullberg 1871) at the €ffer 1896). start and finally dominated by Parisotoma notabilis (Scha However both species belong to the same ecological group (i.e. hemiedaphic species). A single seedling of P. annua was transplanted into each microcosm. All of them were incubated in a climate chamber (temperature: 20 C; daily light/dark 16 h/8 h) with soil moisture kept to 60% of the soil water holding capacity during the experiment. The experiment was stopped after 10 weeks, when more than 80% of P. annua were flowering in the Collembola treatment. Several plant traits were measured. Root parameters (root length, total root projected area and number of root tips) were assessed using an image analysis software. Shoot and root were oven dried at 65 C for 48 h to obtain root biomass (R), shoot biomass (S) and S:R ratio. The Chlorophyll Content Index (CCI) was measured on fresh leaves. Finally carbon, nitrogen, potassium and magnesium content of shoots were measured (see Supplemental information). Soil properties were also investigated at the end of the experimentation and included fungal biomass (i.e. ergosterol concentration), Microbial carbon biomass (Cmic), mineral N (N NO3 and N NO4 þ ), phosphorus content (P2O5), pH H2O (see Supplemental information for methods). Univariate analyses were performed to detect significant effects of Collembola on soil and plant. Student's t tests were used for parametric data, whereas ManneWhitney U tests were chosen for non-parametric data. All statistical analyses were conducted with Statistica software (Statsoft, Inc., V10).
The presence of Collembolan assemblages led to a significant increase of soil N NO3 (þ59%) and P2O5 (þ53%) after 3 months compared to the control treatment, but did not affect N NO4 þ content (Table 1). In parallel, the fungal biomass (ergosterol) also significantly increased in the presence of Collembola (þ39%, Table 1). Since there were no changes in microbial biomass between treatments (Table 1), the fungal:bacterial biomass ratio was also higher in the presence of Collembola (þ25%, Table 1). Past experiments have shown that Collembola stimulate soil fungi either directly by browsing or indirectly by dispersing propagules and excreting nutrient-rich excreta (Bardgett et al., 1993; Crowther et al., 2012). Additionally, several studies suggest that these Collembola e microorganisms interactions resulted in increased rates of soil nutrient mineralization through the grazing activities of the Collembola (Ineson et al., 1982; Cole et al., 2004; Crowther et al., 2012). The increase in soil N was associated with an increase in shoot N content (Table 2) and with a parallel decrease in the C:N ratio (T ¼ 2.181, p ¼ 0.038, Fig. 1C). Shoots of P. annua had significantly more leaves and more ramets when growing with Collembola (Table 2) but shoot height and biomass were not different between our treatments. There was no difference in root morphology between treatments beside root biomass which was 2.4 fold higher in the presence of Collembola (Table 2) conversely to the study of Scheu et al. (1999) that used two collembolan species. As a consequence, the shoot:root ratio was lower with Collembola (Z ¼ 2.67 and p ¼ 0.006, Fig. 1B). Plants generally invest less in their roots when soil nutrient is rich (Reynolds and D'Antonio, 1996; but see Hodge, 2000). This unexpected result might also reflect a Q1 feeding pressure of Collembola on P. annua roots, leading to compensatory growth of the roots (Endlweber et al., 2009). The effect of Collembola was evident in plant reproduction (Fig. 1A). After 10 weeks of monitoring, flower emergence was 6.5fold higher in the presence of Collembola (Z ¼ 3.54, P ¼ 0.0002). Specifically, 86.7% of P. annua were flowering when growing with Collembola, versus 13.33% in their absence. The onset of flowering was promoted by 2 weeks (14 days) by Collembola. Although flowering phenology strongly responds to climatic factors, other biotic and abiotic soil properties mediated by Collembola can act as a stimuli. Recently, Wagner et al. (2014) demonstrated that the flowering phenology of two herb species was slightly shifted (about 1.5e3 days) according to microbial communities. In our study, the regulation of microorganisms by Collembola might have promoted
Table 2 Effect of Collembolan inoculation on plant (Poa annua) parameters after 10 weeks of experimentation. Data are means ± SE. Statistical significance was set at p < 0.05. N
Shoot morphology Shoot height (cm) No. of leaves No. of ramets Shoot biomass (mg) Root morphology Root biomass (mg) Root length (cm) Root projected area (cm2) Number of root tips Plant chemistry (aerial part) N content (%) C content (%) Mg content (mg g1) Plant physiology Chlorophyll content index (CCI) a b
None
Coll.
Statistical test T or Z value
P Value
15 15 15 15
27.15 20.33 2.93 100.3
± ± ± ±
1.0 1.9 0.3 11.5
27.93 25.73 4.47 117.4
± ± ± ±
0.6 1.8 0.2 11.1
0.648a 2.083a 3.027b 1.071a
0.522 0.046 0.002 0.293
15 15 10 10
1399 110.3 1.2 867.6
± ± ± ±
304.6 27.2 0.3 248.3
3310 157.4 2.1 1181
± ± ± ±
660.9 37 0.6 217.7
5.641b 1.209b 1.361b 1.361b
0.008 0.247 0.190 0.190
1.679a 1.099b 2.175b
0.097 0.285 0.045
2.342a
0.027
15 15 9
2.6 ± 0.3 38.2 ± 0.1 1.8 ± 0.1
15
1.37 ± 0.04
3.2 ± 0.9 37.74 ± 0.2 1.5 ± 0.1 1.27 ± 0.03
Student's t test. ManneWhitney non-parametric test.
Please cite this article in press as: Forey, E., et al., Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.07.024
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E. Forey et al. / Soil Biology & Biochemistry xxx (2015) 1e4
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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 Fig. 1. Effect of Collembolan inoculation on Poa annua A/cumulative flowering emergence for the 15 individuals, B/Shoot:Root ratio (N ¼ 15), C/Shoot C:N ratio (N ¼ 15) and D/Shoot 95 K content (N ¼ 9) after 10 weeks of experiment. For A, the percentage of flower emergence was significantly higher with Collembola than without Collembolan inoculation 96 (Z ¼ 3.54, P ¼ 0.0002) at the end of the experiment (67 days, i.e. 10 weeks). For B, C and D, Asterisks indicate significant differences between the two treatments using Man97 neWhitney U non-parametric Test (p < 0.05). Asterisks indicate significant differences *p < 0.05 and ***p < 0.001. 98 99 100 insightful suggestions for the improvement of our manuscript. This this advanced flowering. Soil chemistry is also known to influence 101 study was financially supported by GRR TERA and SFR SCALE flowering time (e.g. Zandt and Mopper, 2002; Brun et al., 2003). In (Haute-Normandie, France). horticulture, K fertilization is sometime used to induce flowering Q2,3 102 103 (e.g. Dufault et al., 1990). This is consistent with the higher content 104 of K (þ37%) found in plants growing with Collembola (T ¼ 8.19, Appendix A. Supplementary information 105 p < 0.0001; Fig. 1D), suggesting that soil fauna might have increased 106 K availability. Other physiological parameters were also changed in Supplementary information related to this article can be found 107 the presence of Collembola. Both Mg and chlorophyll content at http://dx.doi.org/10.1016/j.soilbio.2015.07.024. 108 decreased in leaves (Table 2) suggesting a lower chlorophyll pro109 duction (Cakmak and Yazici, 2010). On the other hand, plants' 110 developmental transition from vegetative to reproductive form References 111 might also have induced changes in plant metabolism such as leaf 112 senescence. Manifold mechanisms may have caused the observed Bardgett, R.D., Chan, K.F., 1999. Experimental evidence that soil fauna enhance 113 effect by the Collembola community, e.g. fostering mycorrhizal nutrient mineralization and plant nutrient uptake in montane grassland eco114 inoculation, reducing pathogens or improving nutrient availability systems. Soil Biology and Biochemistry 31, 1007e1014. Bardgett, R.D., Whittaker, J.B., Frankland, J.C., 1993. The effect of collembolan grazing 115 by feeding on microorganisms. For a more detailed understanding on fungal activity in differently managed upland pastures: a microcosm study. 116 further studies specifically addressing such points are required. Biology and Fertility of Soils 16, 255e262. 117 In conclusion, this study demonstrated that Collembola might Brun, L.A., Le Corff, J., Maillet, J., 2003. Effects of elevated soil copper on phenology, growth and reproduction of five ruderal plant species. Environmental Pollution 118 have short term-effects at the individual plant scale, and can 122, 361e368. 119 regulate flowering phenology. Underlying mechanisms are not yet Cakmak, I., Yazici, A.M., 2010. Magnesium: a forgotten element in crop production. 120 elucidated could be both direct and indirect. This study stresses the Better Crops 94, 23e25. Cole, L., Dromph, K.M., Boaglio, V., Bardgett, R.D., 2004. Effect of density and species 121 functional importance of belowground communities regarding richness of soil mesofauna on nutrient mineralisation and plant growth. Biology 122 plant reproduction that is still to be fully elucidated. and Fertility of Soils 39, 337e343. 123 Coleman, D.C., Crossley Jr., D.A., Hendrix, P.F., 2004. Fundamentals of Soil Ecology, second ed. Elsevier Academic Press, San Diego. 385 pp. 124 Acknowledgements Crowther, T.W., Boddy, L., Jones, T.H., 2012. Functional and ecological consequences 125 of saprotrophic fungus-grazer interactions. ISME Journal 6, 1992e2001. 126 The authors would like to thank the ECODIV lab and Valentin De Deyn, G.B., Raaijmakers, C.E., Zoomer, H.R., Berg, M.P., de Ruiter, P.C., Verhoef, H.A., Bezemer, T.M., van der Putten, W.H., 2003. Soil invertebrate fauna 127 Cheron for technical assistance, the Botanical Garden of Caen for enhances grassland succession and diversity. Nature 422, 711e713. 128 providing seeds, David Houben from Institut polytechnique LasalleDufault, R.J., Phillips, T.L., Kelly, J.W., 1990. Nitrogen and potassium fertility and 129 Beauvais, and Matt Robson for helpful comments on this manuplant populations influence field production of gerbera. Hortscience 25, 130 script. Lastly, we thank two anonymous reviewers for their very 1599e1602. Please cite this article in press as: Forey, E., et al., Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.07.024
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Elzinga, J.A., Atlan, A., Biere, A., Gigord, L., Weis, A.E., Bernasconi, G., 2007. Time after time: flowering phenology and biotic interactions. Trends in Ecology and Evolution 22, 432e439. Endlweber, K., Ruess, L., Scheu, S., 2009. Collembola switch diet in presence of plant roots thereby functioning as herbivores. Soil Biology and Biochemistry 41, 1151e1154. Filser, J., 2002. The role of Collembola in carbon and nitrogen cycling in soil. Pedobiologia 46, 234e245. Galloway, L.F., Burgess, K.S., 2012. Artificial selection on flowering time: influence on reproductive phenology across natural light environments. Journal of Ecology 100, 852e861. Hodge, A., 2000. Root decisions. Plant Cell and Environment 32, 628e640. Ineson, P., Leonard, M.A., Anderson, J.M., 1982. Effect of collembolan grazing upon nitrogen and cation leaching from decomposing leaf litter. Soil Biology and Biochemistry 14, 601e605. Ngosong, C., Gabriel, E., Ruess, L., 2014. Collembola grazing on arbuscular mycorrhiza fungi modulates nutrient allocation in plants. Pedobiologia 57, 171e179. Reynolds, H.L., D'Antonio, C., 1996. The ecological significance of plasticity in root weight ratio in response to nitrogen: opinion. Plant and Soil 185, 75e97.
Scheu, S., Theenhaus, A., Jones, T.H., 1999. Links between the detritivore and the herbivore system: effects of earthworms and Collembola on plant growth and aphid development. Oecologia 119, 541e551. Wagner, M.R., Lundberg, D.S., Coleman-Derr, D., Tringe, S.G., Dangl, J.L., MitchellOlds, T., 2014. Natural soil microbes alter flowering phenology and the intensity of selection on flowering time in a wild Arabidopsis relative. Ecology Letters 17, 717e726. Wagg, C., Bender, S.F., Widmer, F., van der Heijden, M.G.A., 2014. Soil biodiversity and soil community composition determine ecosystem multifunctionality. Proceedings of the National Academy of Sciences of the United States of the America 111, 5266e5270. Wardle, D.A., Bardgett, R.D., Klironomos, J.N., Setala, H., van der Putten, W.H., Wall, D.H., 2004. Ecological linkages between aboveground and belowground biota. Science 304, 1629e1633. Weis, A.E., Turner, K.M., Petro, B., Austen, E.J., Wadgymar, S.M., 2015. Hard and soft selection on phenology through seasonal shifts in the general and social environments: a study on plant emergence time. Evolution 69, 1361e1374. Zandt, P.A.V., Mopper, S., 2002. Delayed and carryover effects of salinity on flowering in Iris hexagona (Iridaceae). American Journal of Botany 89, 1847e1851.
Please cite this article in press as: Forey, E., et al., Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.07.024
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Soil Biology & Biochemistry xxx (2015) 1e4
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Q6
Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola
Q5
kou F.M. Coulibaly, Matthieu Chauvat Estelle Forey*, Se Ecodiv URA/EA-1293, Normandie Universit e, Universit e de Rouen, IRSTEA, SFR Scale 4116, UFR Sciences et Techniques, 76821 Mont Saint Aignan Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2015 Received in revised form 23 July 2015 Accepted 27 July 2015 Available online xxx
The role of soil organisms as possible driver of flowering has never been investigated. We hypothesized that Collembola (microarthropods) will change plant allocation to reproductive modes by changing soil nutrient availability. Individual seedlings of Poa annua were planted in microcosms, in the presence or absence of Collembola. Collembola affected biotic (fungal biomass) and abiotic (N NO3 , P2O5) soil properties and some morphological (number of leaves, root biomass) and chemical (C:N, K, Mg, N) traits of P. annua. As a result, flowering of P. annua was promoted by the presence of Collembola. This provides experimental evidence that soil microarthropods can affect the reproduction strategy and phenology of a plant. © 2015 Published by Elsevier Ltd.
Keywords: Aboveebelow ground interactions Soil fauna Plant performances Soil properties Springtails
The belowground compartment is a fundamental driver of the aboveground system and therefore of ecosystem fate and properties (De Deyn et al., 2003; Wardle et al., 2004; Wagg et al., 2014). Collembola are among the most abundant microarthropods in the rhizosphere of plants (Coleman et al., 2004). Several studies have highlighted a strong influence of various Collembolan species on plant roots either indirectly by regulating soil nutrient availability (Bardgett and Chan, 1999; Filser, 2002; Ngosong et al., 2014) or directly by exerting rhizophagous pressure (Endlweber et al., 2009). Nevertheless, the consequences of such impacts on resource allocation for plant reproduction and on flowering phenology have apparently never been investigated. A small change in reproductive success or flowering time can affect plant fitness (e.g. Galloway and Burgess, 2012; Weis et al., 2015), and consequently might alter interactions with populations of pollinators, seed dispersers, or floral herbivores. The role of biotic interactions in shaping plant flowering phenology has been demonstrated for pollinators and herbivores (Elzinga et al., 2007). Therefore, in the present study, we tested in a microcosm experiment whether the presence of a natural assemblage of Collembola affected plant growth and sexual reproduction (flowering) of Poa annua L.
* Corresponding author. Tel.: þ33 2 32769455. E-mail address: [email protected] (E. Forey).
In a microcosm experiment, two treatments were established corresponding to the presence (“Coll.”) or absence (“None”) of a natural assemblage of Collembola. Each treatment was replicated 15 times in microcosms (plastic pots 10 9 9 cm) where individuals of P. annua were grown. The soil was a rendosol (Organic Matter content ¼ 6.17%; pH ¼ 7.79) collected from a chalk grassland (170 3000 E, 49 220 2200 N) in Normandy, France (further details are given in Supplemental information). A part of the soil was defaunated by repeated deep-freezing and thawing (see Supplemental information). The 30 microcosms were meshed (250 mm) to prevent any escape of microarthropods and were filled with a mixture of defaunated soil and sand (5:1). From the remaining part of the collected soil, microorganisms (fungi and bacteria) and Collembola were extracted and re-inoculated in order to set the two different treatments. Microorganisms were re-inoculated in all microcosms prior to Collembola by adding “soil extract” (see Supplemental information). Re-inoculation of microorganisms was followed, two weeks later, by Collembola addition into the 15 “Coll” microcosms. Collembola were extracted from collected soil using a BerleseeTullgren apparatus (see Supplemental information). The extraction of Collembola in microcosms at the end of the experiment, allowed us to confirm that (i) no contamination was observed in the treatment without Collembola, and that (ii) collembolan assemblages, in terms of abundance, were maintained until the end of the experiment, even if inevitably compositional
http://dx.doi.org/10.1016/j.soilbio.2015.07.024 0038-0717/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Forey, E., et al., Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.07.024
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119
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E. Forey et al. / Soil Biology & Biochemistry xxx (2015) 1e4
Table 1 Effect of Collembolan inoculation on soil properties after 10 weeks of Poa annua growth. Data are means ± SE. Statistical significance was set at p < 0.05. Soil properties:
N
None
Coll.
Statistical test T or Z
1
Cmic (mgC g dw) Ergosterol (mg g1 dw) Fungal:bacterial ratio N NO4 þ (mg g1 dw) N NO3 (mg g1 dw) P2O5 (mg g1 dw) pH a b
10 10 10 10 10 10 10
300.1 1.84 0.63 0.097 0.063 34.7 7.78
± ± ± ± ± ± ±
20.2 0.08 0.03 0.007 0.002 1.6 0.03
315.6 2.45 0.79 0.109 0.100 53.0 7.76
± ± ± ± ± ± ±
18.3 0.12 0.03 0.013 0.011 2.5 0.02
P value a
0.570 4.166a 3.228a 0.189b 3.175b 6.153a 0.600a
0.575 <0.001 0.004 0.853 <0.001 <0.001 0.555
Student's t test. ManneWhitney non-parametric test.
changes appeared (see Supplemental information and supplemental Table 1). Basically we observed a shift from a community dominated by Folsomia quadrioculata (Tullberg 1871) at the €ffer 1896). start and finally dominated by Parisotoma notabilis (Scha However both species belong to the same ecological group (i.e. hemiedaphic species). A single seedling of P. annua was transplanted into each microcosm. All of them were incubated in a climate chamber (temperature: 20 C; daily light/dark 16 h/8 h) with soil moisture kept to 60% of the soil water holding capacity during the experiment. The experiment was stopped after 10 weeks, when more than 80% of P. annua were flowering in the Collembola treatment. Several plant traits were measured. Root parameters (root length, total root projected area and number of root tips) were assessed using an image analysis software. Shoot and root were oven dried at 65 C for 48 h to obtain root biomass (R), shoot biomass (S) and S:R ratio. The Chlorophyll Content Index (CCI) was measured on fresh leaves. Finally carbon, nitrogen, potassium and magnesium content of shoots were measured (see Supplemental information). Soil properties were also investigated at the end of the experimentation and included fungal biomass (i.e. ergosterol concentration), Microbial carbon biomass (Cmic), mineral N (N NO3 and N NO4 þ ), phosphorus content (P2O5), pH H2O (see Supplemental information for methods). Univariate analyses were performed to detect significant effects of Collembola on soil and plant. Student's t tests were used for parametric data, whereas ManneWhitney U tests were chosen for non-parametric data. All statistical analyses were conducted with Statistica software (Statsoft, Inc., V10).
The presence of Collembolan assemblages led to a significant increase of soil N NO3 (þ59%) and P2O5 (þ53%) after 3 months compared to the control treatment, but did not affect N NO4 þ content (Table 1). In parallel, the fungal biomass (ergosterol) also significantly increased in the presence of Collembola (þ39%, Table 1). Since there were no changes in microbial biomass between treatments (Table 1), the fungal:bacterial biomass ratio was also higher in the presence of Collembola (þ25%, Table 1). Past experiments have shown that Collembola stimulate soil fungi either directly by browsing or indirectly by dispersing propagules and excreting nutrient-rich excreta (Bardgett et al., 1993; Crowther et al., 2012). Additionally, several studies suggest that these Collembola e microorganisms interactions resulted in increased rates of soil nutrient mineralization through the grazing activities of the Collembola (Ineson et al., 1982; Cole et al., 2004; Crowther et al., 2012). The increase in soil N was associated with an increase in shoot N content (Table 2) and with a parallel decrease in the C:N ratio (T ¼ 2.181, p ¼ 0.038, Fig. 1C). Shoots of P. annua had significantly more leaves and more ramets when growing with Collembola (Table 2) but shoot height and biomass were not different between our treatments. There was no difference in root morphology between treatments beside root biomass which was 2.4 fold higher in the presence of Collembola (Table 2) conversely to the study of Scheu et al. (1999) that used two collembolan species. As a consequence, the shoot:root ratio was lower with Collembola (Z ¼ 2.67 and p ¼ 0.006, Fig. 1B). Plants generally invest less in their roots when soil nutrient is rich (Reynolds and D'Antonio, 1996; but see Hodge, 2000). This unexpected result might also reflect a Q1 feeding pressure of Collembola on P. annua roots, leading to compensatory growth of the roots (Endlweber et al., 2009). The effect of Collembola was evident in plant reproduction (Fig. 1A). After 10 weeks of monitoring, flower emergence was 6.5fold higher in the presence of Collembola (Z ¼ 3.54, P ¼ 0.0002). Specifically, 86.7% of P. annua were flowering when growing with Collembola, versus 13.33% in their absence. The onset of flowering was promoted by 2 weeks (14 days) by Collembola. Although flowering phenology strongly responds to climatic factors, other biotic and abiotic soil properties mediated by Collembola can act as a stimuli. Recently, Wagner et al. (2014) demonstrated that the flowering phenology of two herb species was slightly shifted (about 1.5e3 days) according to microbial communities. In our study, the regulation of microorganisms by Collembola might have promoted
Table 2 Effect of Collembolan inoculation on plant (Poa annua) parameters after 10 weeks of experimentation. Data are means ± SE. Statistical significance was set at p < 0.05. N
Shoot morphology Shoot height (cm) No. of leaves No. of ramets Shoot biomass (mg) Root morphology Root biomass (mg) Root length (cm) Root projected area (cm2) Number of root tips Plant chemistry (aerial part) N content (%) C content (%) Mg content (mg g1) Plant physiology Chlorophyll content index (CCI) a b
None
Coll.
Statistical test T or Z value
P Value
15 15 15 15
27.15 20.33 2.93 100.3
± ± ± ±
1.0 1.9 0.3 11.5
27.93 25.73 4.47 117.4
± ± ± ±
0.6 1.8 0.2 11.1
0.648a 2.083a 3.027b 1.071a
0.522 0.046 0.002 0.293
15 15 10 10
1399 110.3 1.2 867.6
± ± ± ±
304.6 27.2 0.3 248.3
3310 157.4 2.1 1181
± ± ± ±
660.9 37 0.6 217.7
5.641b 1.209b 1.361b 1.361b
0.008 0.247 0.190 0.190
1.679a 1.099b 2.175b
0.097 0.285 0.045
2.342a
0.027
15 15 9
2.6 ± 0.3 38.2 ± 0.1 1.8 ± 0.1
15
1.37 ± 0.04
3.2 ± 0.9 37.74 ± 0.2 1.5 ± 0.1 1.27 ± 0.03
Student's t test. ManneWhitney non-parametric test.
Please cite this article in press as: Forey, E., et al., Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.07.024
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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 Fig. 1. Effect of Collembolan inoculation on Poa annua A/cumulative flowering emergence for the 15 individuals, B/Shoot:Root ratio (N ¼ 15), C/Shoot C:N ratio (N ¼ 15) and D/Shoot 95 K content (N ¼ 9) after 10 weeks of experiment. For A, the percentage of flower emergence was significantly higher with Collembola than without Collembolan inoculation 96 (Z ¼ 3.54, P ¼ 0.0002) at the end of the experiment (67 days, i.e. 10 weeks). For B, C and D, Asterisks indicate significant differences between the two treatments using Man97 neWhitney U non-parametric Test (p < 0.05). Asterisks indicate significant differences *p < 0.05 and ***p < 0.001. 98 99 100 insightful suggestions for the improvement of our manuscript. This this advanced flowering. Soil chemistry is also known to influence 101 study was financially supported by GRR TERA and SFR SCALE flowering time (e.g. Zandt and Mopper, 2002; Brun et al., 2003). In (Haute-Normandie, France). horticulture, K fertilization is sometime used to induce flowering Q2,3 102 103 (e.g. Dufault et al., 1990). This is consistent with the higher content 104 of K (þ37%) found in plants growing with Collembola (T ¼ 8.19, Appendix A. Supplementary information 105 p < 0.0001; Fig. 1D), suggesting that soil fauna might have increased 106 K availability. Other physiological parameters were also changed in Supplementary information related to this article can be found 107 the presence of Collembola. Both Mg and chlorophyll content at http://dx.doi.org/10.1016/j.soilbio.2015.07.024. 108 decreased in leaves (Table 2) suggesting a lower chlorophyll pro109 duction (Cakmak and Yazici, 2010). On the other hand, plants' 110 developmental transition from vegetative to reproductive form References 111 might also have induced changes in plant metabolism such as leaf 112 senescence. Manifold mechanisms may have caused the observed Bardgett, R.D., Chan, K.F., 1999. Experimental evidence that soil fauna enhance 113 effect by the Collembola community, e.g. fostering mycorrhizal nutrient mineralization and plant nutrient uptake in montane grassland eco114 inoculation, reducing pathogens or improving nutrient availability systems. Soil Biology and Biochemistry 31, 1007e1014. Bardgett, R.D., Whittaker, J.B., Frankland, J.C., 1993. The effect of collembolan grazing 115 by feeding on microorganisms. For a more detailed understanding on fungal activity in differently managed upland pastures: a microcosm study. 116 further studies specifically addressing such points are required. Biology and Fertility of Soils 16, 255e262. 117 In conclusion, this study demonstrated that Collembola might Brun, L.A., Le Corff, J., Maillet, J., 2003. Effects of elevated soil copper on phenology, growth and reproduction of five ruderal plant species. Environmental Pollution 118 have short term-effects at the individual plant scale, and can 122, 361e368. 119 regulate flowering phenology. Underlying mechanisms are not yet Cakmak, I., Yazici, A.M., 2010. Magnesium: a forgotten element in crop production. 120 elucidated could be both direct and indirect. This study stresses the Better Crops 94, 23e25. Cole, L., Dromph, K.M., Boaglio, V., Bardgett, R.D., 2004. Effect of density and species 121 functional importance of belowground communities regarding richness of soil mesofauna on nutrient mineralisation and plant growth. Biology 122 plant reproduction that is still to be fully elucidated. and Fertility of Soils 39, 337e343. 123 Coleman, D.C., Crossley Jr., D.A., Hendrix, P.F., 2004. Fundamentals of Soil Ecology, second ed. Elsevier Academic Press, San Diego. 385 pp. 124 Acknowledgements Crowther, T.W., Boddy, L., Jones, T.H., 2012. Functional and ecological consequences 125 of saprotrophic fungus-grazer interactions. ISME Journal 6, 1992e2001. 126 The authors would like to thank the ECODIV lab and Valentin De Deyn, G.B., Raaijmakers, C.E., Zoomer, H.R., Berg, M.P., de Ruiter, P.C., Verhoef, H.A., Bezemer, T.M., van der Putten, W.H., 2003. Soil invertebrate fauna 127 Cheron for technical assistance, the Botanical Garden of Caen for enhances grassland succession and diversity. Nature 422, 711e713. 128 providing seeds, David Houben from Institut polytechnique LasalleDufault, R.J., Phillips, T.L., Kelly, J.W., 1990. Nitrogen and potassium fertility and 129 Beauvais, and Matt Robson for helpful comments on this manuplant populations influence field production of gerbera. Hortscience 25, 130 script. Lastly, we thank two anonymous reviewers for their very 1599e1602. Please cite this article in press as: Forey, E., et al., Flowering phenology of a herbaceous species (Poa annua) is regulated by soil Collembola, Soil Biology & Biochemistry (2015), http://dx.doi.org/10.1016/j.soilbio.2015.07.024
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