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Arbuscular Mycorrhiza Prevents Suppression of Actual Nitrification Rates in the (Myco-)Rhizosphere of Plantago lanceolata
Pedosphere 22(2): 225–229, 2012 ISSN 1002-0160/CN 32-1315/P c 2012 Soil Science Society of China Published by Elsevier B.V. and Science Press
Arbuscular Mycorrhiza Prevents Suppression of Actual Nitrification Rates in the (Myco-)Rhizosphere of Plantago lanceolata∗1 S. D. VERESOGLOU∗2 Faculty of Agriculture, Laboratory of Ecology and Environmental Protection, Aristotle University of Thessaloniki, 541 24 Thessaloniki (Greece) (Received June 9, 2011; revised January 5, 2012)
ABSTRACT The vast majority of herbaceous plants engage into arbuscular mycorrhizal (AM) symbioses and consideration of their mycorrhizal status should be embodied in studies of plant-microbe interactions. To establish reliable AM contrasts, however, a sterilized re-inoculation procedure is commonly adopted. It was questioned whether the specific approach is sufficient for the studies targeting the bacterial domain, specifically nitrifiers, a group of autotrophic, slow growing microbes. In a controlled experiment mycorrhizal and non-mycorrhizal Plantago lanceolata were grown up in compartmentalized pots to study the AM effect on nitrification rates in the plant rhizosphere. Nitrification rates were assayed following an extensive 3-week bacterial equilibration step of the re-inoculated soil and a 13-week plant growth period in a controlled environment. Under these specific conditions, the nitrification potential levels at harvest were exceptionally low, and actual nitrification rates of the root compartment of non-mycorrhizal P. lanceolata were significantly lower than those of any other compartment. It is then argued that the specific effects should be attributed to the alleged higher growth rates of non-mycorrhizal plants that are known to occur early in the AM experiment. It is concluded that the specific experimental approach is not suitable for the study of microbes with slow growth rates. Key Words:
ammonia oxidizers, nitrification potential, plant-microbe interaction, root compartment
Citation: Veresoglou, S. D. 2012. Arbuscular mycorrhiza prevents suppression of actual nitrification rates in the (myco-)rhizosphere of Plantago lanceolata. Pedosphere. 22(2): 225–229.
INTRODUCTION Nitrogen is the element most frequently limiting primary productivity in terrestrial ecosystems (Chapin III et al., 2004). Within the N cycle, nitrification, the − oxidation of ammonium (NH+ 4 ) to nitrate (NO3 ) via an intermediate step that generates nitrite (NO− 2 ), is of exceptional interest as it modifies the relative − availability of the two forms of N (NH+ 4 and NO3 ) that are most readily available for plant N nutrition (Falkengren-Grerup, 1995). NH+ 4 uptake for the plants is less expensive in terms of resources, but requires an extensive root system which results in lower maximum specific growth rates (Raven et al., 1992). As a result, relative availability of the two forms of inorganic N could determine the efficiency of a plant species in− vestment strategy towards either NH+ 4 or NO3 assimi∗1 ∗2
Supported by a PhD fellowship from the Chloros trust. Corresponding author. E-mail: [email protected].
− lation. Transformation of NO− 2 to NO3 , under usual − soil conditions, is faster than oxidation of NH+ 4 to NO2 − and, thus, generated NO2 is short-lived and of little ecological importance. Hence, literature on nitrification commonly addresses the transformation of NH+ 4 to , which is mediated by a group of predominantly NO− 2 autotrophic bacteria and archaea that are collectively known as ammonia oxidizers (AO) (Prosser, 2007). Generally, research on the N-cycle has overlooked the fact that the vast majority of plants engage in arbuscular mycorrhizal (AM) associations (Wang and Qui, 2006). AM fungi are believed to assist N-nutrition of host plants through assimilation of N, either exclusively (Tanaka and Yano, 2005) or preferentially (Govindarajulu et al., 2005), in the form of NH+ 4 . Thus there is a high likelihood that they can affect fitness of autotrophic ammonia oxidizers (AO) and influence ni-
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trification activity of soils. There have been three studies that addressed the effect of AM fungi on nitrification. Amora-Lazcano et al. (1998), in a 60 dayexperiment, detected that non-mycorrhizal maize supported lower numbers of autotrophic AO than AMinoculated maize. Cavagnaro et al. (2007) failed to detect the differences between mycorrhiza-defective mutant tomatos and their wild-type progenitors in either the composition or the density of AO bacteria. By contrast, Veresoglou et al. (2011a) in a series of experiments asserted that nitrification potential activity in the rhizosphere of mycotrophic plants consistently declined. Clearly further investigation is required to address why the results of Amora-Lazcano et al. (1998) have not been reproducible. A common source of bias, when the nitrifying community is addressed in the sterilized soils re-inoculated with bacteria, is the low growth rates of AO, which may require several days for a single doubling event even under laboratory conditions (Prosser, 2007). As a result, commonly the study of the AO community is performed under highly dynamic conditions. For example, the sequential harvesting approach in Amora-Lazcano et al. (1998) revealed that AO counts had been increasing rapidly with time till the end of the experiment. Most AM studies have proceeded with experimentation immediately after inoculating the soil with the bacterial filtrate (e.g., Amora-Lazcano et al., 1998). However, an optimization step that includes the equilibration of the soil with the bacterial community for approximately 2 weeks is optimally recommended before initiation of experiments targeting the microbial community (Shaw et al., 1999). The aim of the study was to assay the nitrification rates of the nutrient-limited sterilized soils in the rhizosphere of Plantago lanceolata inoculated with AM when an extensive 3-week equilibration step preceded initiation of the experiment. It was hypothesized that the slow growth rates of AO coupled to early encountering of competition for inorganic N with the plants and the plant-supported rhizosphere microbial community would have been detrimental for the development of the soil nitrifying community. It was, thus, expected that the nitrification rates (actual and potential) close to the roots (root compartments) would have been lower than those far from plant roots (root-free compartments). MATERIALS AND METHODS Following a 3-week microbial equilibration pe-
riod, P. lanceolata was grown up for 13 weeks in mesh-divided compartmentalized microcosms (a 40-μm stainless mesh was used as a separator of the compartments). Three treatments were established: nonmycorrhizal P. lanceolata, P. lanceolata inoculated with Glomus intraradices B.B/E, and P. lanceolata inoculated with Gigaspora margarita BEG 34. Each treatment replicated eight times. A single P. lanceolata seedling was sown to one of the compartments called the “root compartment”, whereas the “root-free compartment” remained inaccessible to plant roots but accessible to AM extraradical mycelial growth. The soil sterilization procedure was carried out through γ irradiation to minimize sterilization-linked modification of the soil properties (Alef and Nannipieri, 1995). Nevertheless, a spike of mineralized microbial biomass at the beginning of the experiment was unavoidable. To restore the prokaryotic microbial population 10 mL of microbial suspension [non-sterilized soil:saline phosphate buffer pH 7.2 = 1:10, with 0.1% (v/v) Tween 80 filtered through a Whatman #5 (2.5-μm pore size) filter] were added to each microcosm and the soil microbial community was left to equilibrate at a constant moisture content of 60% water holding capacity (WHC) for three weeks before initiation of the experiment. More details on the experiment can be found in Veresoglou et al. (2011b). At the day of harvest a 5 g equivalent dry weight (EDW) subsample of soil from each microcosm was stored in the freezer (−20 ◦ C) and was later used for + assessment of residual NO− 3 -N and NH4 -N. A further 10 g of soil from the root compartment was used to determine nitrification potential one day after harvest according to Hart et al. (1994). Five gram of soil was brought to 60% WHC with sterile distilled water and was left to incubate in 50 mL centrifuge tubes for three weeks to determine, through the differences between − the initial and final levels of NH+ 4 -N and NO3 -N, the rates of mineralization (Beck, 1983). Similarly, in another 50 mL centrifuge tube, 0.5 mL of 10 g L−1 ammonium sulphate [(NH4 )2 SO4 ] solution were added to 5 g of soil and water content was adjusted to 60% WHC. Then the sample was left to incubate for two weeks to assess actual nitrification rates through startand end-point differences in the NO− 3 -N content (Beck, 1979). Details for both the mineralization and actual nitrification assays were presented by Alef and Nannipieri (1995). The main difference between actual nitrification rates and nitrification potential rates lies on the fact that assaying of nitrification potential further
ARBUSCULAR STATUS PREVENTS SUPPRESSION OF NITRIFICATION
involves adjustments of nutrient availability and pH so that AO may nitrify to the maximum of their ability. Moreover, while nitrification potential assessment involves four assessments of NO− 3 -N status within 26 h, the end-point assessment of NO− 3 -N for the actual nitrification assay is commonly carried out following 2week incubation. Neither assay prevents N loss through denitrification as the denitrification rates in the presence of abundant NH+ 4 -N are considerably lower than those of nitrification. As a result, when AO densities are low, negative rates may be retrieved and the assays should solely be used for inter-treatment comparisons. The end-point potassium chlorate (KCl) extracts from the root-compartment samples that were assayed for nitrification were further used for assessment of nitrite (NO− 2 -N) status to verify absence of nitrification. The + NO− 3 -N and NH4 -N concentrations were assessed in 60 g L−1 KCl:soil extracts of 5:1 (v/v) through ion chromatography (Dionex DX 100). By contrast, NO− 2 content was assessed colometrically (Keeney and Nelson, 1982). For statistical analysis, NH+ 4 -N values had to be log transformed and absolute values of actual nitrification rates were square root-transformed maintaining their sign to conform with the normality criterion. For the figures the back-transformed values are presented. RESULTS AND DISCUSSION Inorganic N in the microcosms was available mainly − in the form of NH+ 4 -N as NO3 -N levels were very low (Fig. 1). With regards to NH+ 4 -N availability the highest level was recorded in the root-free compartment of the non-mycorrhizal microcosms whereas lower levels were recorded in the root compartments of AM-inoculated microcosms compared to the respec-
227
tive non-mycorrhizal microcosms (Fig. 1). Nitrification potential rates, which represent a measure of potential ammonia oxidizing activity, were close to detection limits of the assay and, consequently, it was not possible to detect significant effects (Fig. 2). On the other hand, as expected, mineralization assay revealed non-significant differences amongst treatments (Fig. 2). This facilitated comparisons because it strengthened the assumption that no significant differences in inorganic-N-input in the microcosms existed. The most important result was obtained with regards to actual nitrification rates (Fig. 2). In the root-free compartments of all microcosms and the root compartments of AM-inoculated microcosms a similar level of nitrification was detected. However, in the root compartment of non-mycorrhizal microcosms the nitrification rates assessed were negative (Fig. 2). To verify the low actual nitrification rates in the root compartment of non-mycorrhizal plants, the intermediate product of nitrification (NO− 2 -N) was measured in the end-point KCl extracts from the root-compartment samples that had been assayed for nitrification. In the mycorrhizal samples NO− 2 -N levels were above detection limits. By contrast NO− 2 -N levels in the non-mycorrhizal samples were below (Fig. 3). The residual NO− 2 -N was significantly different (P < 0.001) between the three treatments. Results indicated that actual nitrification rates of the soil in the root compartment of non-mycorrhizal treatment were lower than those in the other treatments. It is well known that actual nitrification rates may be detrimentally affected by low inorganic phosphorus (P) availability or pH changes (Hart et al., 1994). Nevertheless, inorganic P availability has been known to be lower in the rhizosphere of mycorrhizal
− Fig. 1 Residual NH+ 4 -N and NO3 -N contents at harvest in the microcosms treated with non-mycorrhiza (NM), Glomus intraradices (Gi), and Gigaspora margarita (Gm). Means marked by the same letter within a given compartment are not significantly different according to least significant difference at P = 0.05 by F test. *Significant compartment effect for the NM treatment at P = 0.05. Vertical bars indicate standard errors.
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Fig. 2 Nitrification potential rates, mineralization rates, and actual nitrification rates at harvest in the microcosms treated with non-mycorrhiza (NM), Glomus intraradices (Gi), and Gigaspora margarita (Gm). Means marked by the same letter within a given compartment are not significantly different according to least significant difference at P = 0.05 by F test. *Significant compartment effect for the NM treatment at P = 0.05. Vertical bars indicate standard errors.
Fig. 3 Residual NO− 2 -N levels in the KCl extracts obtained from the end-point actual nitrification tubes from the root compartment of the microcosms treated with nonmycorrhiza (NM), Glomus intraradices (Gi), and Gigaspora margarita (Gm). Means marked by the same letter are not significantly different according to least significant difference at P = 0.05 by F test. Vertical bars indicate standard errors.
plants due to an expansion of the P-depletion zone (Smith and Read, 2008). In agreement to this idea, Veresoglou et al. (2011b) had demonstrated, in the specific experimental system, that total P content was higher for mycorrhizal plants. Assessment of soil pH of representative soil samples also indicated no modification of soil pH status (unpublished data). Based on these results, apparently, the recorded differences in nitrification rates should have reflected a decline in AO abundance in the non-mycorrhizal root compartment. Because the experimental approach adopted excluded bacterial predators from the microcosms, the microbial densities at harvest should have represented the peak microbial densities in the experiment. AO are known to be poor competitors for soil NH+ 4 (Boll-
mann et al., 2002). Hence, higher levels of residual inorganic N were recorded for the non-mycorrhizal root compartment when compared to the respective mycorrhizal treatments (Fig. 1) and were coupled to insignificant differences in N mineralization (N inputs). The low actual nitrification rates that were recorded for the root compartment of non-mycorrhizal P. lanceolata thus signified that AO should have encountered intense competition for NH+ 4 reserves early in the experiment and could not proliferate. The author attributes this result to: 1) the fact that AM plants are known to exhibit a growth depression in the early stages of experimentation (Smith and Read, 2008). As a result, their requirements in inorganic N at the specific stage should have been lower than the plants in the nonmycorrhizal treatment and AO growth in the AMinoculated microcosms encountered less strong competition for pools of NH+ 4 ; and 2) the allocation from the AM-inoculated plants of carbon to the AM fungi that would have otherwise been secreted in the form of exudates and delivered to the bacterial community, intensifying competition for N. A decline in sugar exudation following AM colonization is commonly reported in studies (Jones et al., 2004). By contrast, in the non-mycorrhizal microcosms no such mechanism existed to alleviate competition for soil N and AO had to cease growth. The results agree with those of AmoraLazcano et al. (1998), where they verified the better establishment of AO in the sterilized soil planted with AM-inoculated seedlings. CONCLUSIONS With regards to the original hypothesis limited
ARBUSCULAR STATUS PREVENTS SUPPRESSION OF NITRIFICATION
differences were recorded between the two compartments but there were differences between mycorrhizal and non-mycorrhizal root compartments. The results showed that the specific experimental approach was not suitable for the study of slow-growing microbes in the presence of AM fungi. This raises the question whether a longer equilibration period should be adopted. In the absence of bacterial predators, extensive equilibration might be expected to lead to further immobilization of nutrients, as an increasing part of the bacterial community would swift to an inactive state. It is, thus, highly likely that elongation of the equilibration period could deteriorate nutrient limitation of the soil. Future experimentation is required to address whether identified problems may be resolved with the addition of protozoa or nematodes. ACKNOWLEDGEMENTS I would like to thank Dr. L. SHAW from University of Reading, UK and Dr. R. SEN from Manchester Metropolitan University, UK for valuable feedback. I also acknowledge the contribution of four anonymous reviewers in improving the quality of the manuscript. REFERENCES Alef, K. and Nannipieri, P. 1995. Methods in Applied Soil Microbiology and Biochemistry. Academic Press, San Diego, USA. Amora-Lazcano, E., V´ azquez, M. M. and Azc´ on, R. 1998. Response of nitrogen-transforming microorganisms to arbuscular mycorrhizal fungi. Biol. Fert. Soils. 27: 65–70. Beck, T. H. 1979. Die nitrifikation in B¨ oden (sammelreferat). Z. Pflanzenernaehr. Bodenkd. 142: 344–364. Beck, T. H. 1983. Die N-Mineralisierung von B¨ oden im Laborbrutversuch. Z. Pflanzenernaehr. Bodenkd. 146: 243–252. Bollmann, A., B˚ ar-Gilissen, M. J. and Laanbroek, H. J. 2002. Growth at low ammonium concentrations and starvation response as potential factors involved in niche differentiation among ammonia-oxidizing bacteria. Appl. Environ. Microb. 68: 4751–4757. Cavagnaro, T. R., Jackson, L. E., Scow, K. M. and Hristova, K. R. 2007. Effects of arbuscular mycorrhizas on ammonia oxidizing bacteria in an organic farm soil. Microb. Ecol. 54: 618–626. Chapin III, F. S., Matson, P. A. and Mooney, H. A. 2004. Principles of Terrestrial Ecosystem Ecology. Springer, New York.
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Falkengren-Grerup, U. 1995. Interspecies differences in the preference of ammonium and nitrate in vascular plants. Oecologia. 102: 305–311. Govindarajulu, M., Pfeffer, P. E., Jin, H. R., Abubaker, J., Douds, D. D., Allen, J. W., Bucking, H., Lammers, P. J. and Shachar-Hill, Y. 2005. Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature. 435: 819–823. Hart, S. C., Stark, J. M., Davidson, E. A. and Firestone, M. K. 1994. Nitrogen mineralization, immobilization, and nitrification. In Weaver, R. W., Angle, S., Bottomley, P., Bezdiecek, D., Smith, S., Tabatabai, A., Wollum, A., Mickelson, S. H. and Bigham, J. M. (eds.) Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, WI. pp. 985–1018. Jones, D. L., Hodge, A. and Kuzyakov, Y. 2004. Plant and mycorrhizal regulation of rhizodeposition. New Phytol. 163: 459–480. Keeney, D. R. and Nelson, D. W. 1982. Nitrogen— inorganic forms. In Page, A. L., Miller, R. H. and Keeney, D. R. (eds.) Methods of Soil Analysis Part 2—Chemical and Microbiological Properties. American Society of Agronomy, Soil Science Society of America, Madison, WI. pp. 643–698. Prosser, J. I. 2007. The ecology of nitrifying bacteria. In Bothe, H., Ferguson, S. J. and Newton, W. E. (eds.) Biology of the Nitrogen Cycle. Elsevier, Amsterdam. pp. 223–243. Raven, J. A., Wollenweber, B. and Handley, L. L. 1992. A comparison of ammonium and nitrate as nitrogen sources for photolithotrophs. New Phytol. 121: 19–32. Shaw, L. J., Beaton, Y., Glover, L. A., Killham, K. and Meharg, A. A. 1999. Re-inoculation of autoclaved soil as a non-sterile treatment for xenobiotic sorption and biodegradation studies. Appl. Soil Ecol. 11: 217–226. Smith, S. E. and Read, D. J. 2008. Mycorrhizal Symbiosis. 3rd Edition. Academic Press, Amsterdam. Tanaka, Y. and Yano, K. 2005. Nitrogen delivery to maize via mycorrhizal hyphae depends on the form of N supplied. Plant Cell Environ. 28: 1247–1254. Veresoglou, S. D., Sen, R., Mamolos, A. P. and Veresoglou, D. S. 2011a. Plant species identity and arbuscular mycorrhizal status modulate potential nitrification rates in nitrogen-limited grassland soils. J. Ecol. 99: 1339– 1349. Veresoglou, S. D., Shaw, L. J. and Sen, R. 2011b. Glomus intraradices and Gigaspora margarita arbuscular mycorrhizal associations differentially affect nitrogen and potassium nutrition of Plantago lanceolata in a low fertility dune soil. Plant Soil. 340: 481–490. Wang, B. and Qui, Y. L. 2006. Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza. 16: 299–363.
Arbuscular Mycorrhiza Prevents Suppression of Actual Nitrification Rates in the (Myco-)Rhizosphere of Plantago lanceolata∗1 S. D. VERESOGLOU∗2 Faculty of Agriculture, Laboratory of Ecology and Environmental Protection, Aristotle University of Thessaloniki, 541 24 Thessaloniki (Greece) (Received June 9, 2011; revised January 5, 2012)
ABSTRACT The vast majority of herbaceous plants engage into arbuscular mycorrhizal (AM) symbioses and consideration of their mycorrhizal status should be embodied in studies of plant-microbe interactions. To establish reliable AM contrasts, however, a sterilized re-inoculation procedure is commonly adopted. It was questioned whether the specific approach is sufficient for the studies targeting the bacterial domain, specifically nitrifiers, a group of autotrophic, slow growing microbes. In a controlled experiment mycorrhizal and non-mycorrhizal Plantago lanceolata were grown up in compartmentalized pots to study the AM effect on nitrification rates in the plant rhizosphere. Nitrification rates were assayed following an extensive 3-week bacterial equilibration step of the re-inoculated soil and a 13-week plant growth period in a controlled environment. Under these specific conditions, the nitrification potential levels at harvest were exceptionally low, and actual nitrification rates of the root compartment of non-mycorrhizal P. lanceolata were significantly lower than those of any other compartment. It is then argued that the specific effects should be attributed to the alleged higher growth rates of non-mycorrhizal plants that are known to occur early in the AM experiment. It is concluded that the specific experimental approach is not suitable for the study of microbes with slow growth rates. Key Words:
ammonia oxidizers, nitrification potential, plant-microbe interaction, root compartment
Citation: Veresoglou, S. D. 2012. Arbuscular mycorrhiza prevents suppression of actual nitrification rates in the (myco-)rhizosphere of Plantago lanceolata. Pedosphere. 22(2): 225–229.
INTRODUCTION Nitrogen is the element most frequently limiting primary productivity in terrestrial ecosystems (Chapin III et al., 2004). Within the N cycle, nitrification, the − oxidation of ammonium (NH+ 4 ) to nitrate (NO3 ) via an intermediate step that generates nitrite (NO− 2 ), is of exceptional interest as it modifies the relative − availability of the two forms of N (NH+ 4 and NO3 ) that are most readily available for plant N nutrition (Falkengren-Grerup, 1995). NH+ 4 uptake for the plants is less expensive in terms of resources, but requires an extensive root system which results in lower maximum specific growth rates (Raven et al., 1992). As a result, relative availability of the two forms of inorganic N could determine the efficiency of a plant species in− vestment strategy towards either NH+ 4 or NO3 assimi∗1 ∗2
Supported by a PhD fellowship from the Chloros trust. Corresponding author. E-mail: [email protected].
− lation. Transformation of NO− 2 to NO3 , under usual − soil conditions, is faster than oxidation of NH+ 4 to NO2 − and, thus, generated NO2 is short-lived and of little ecological importance. Hence, literature on nitrification commonly addresses the transformation of NH+ 4 to , which is mediated by a group of predominantly NO− 2 autotrophic bacteria and archaea that are collectively known as ammonia oxidizers (AO) (Prosser, 2007). Generally, research on the N-cycle has overlooked the fact that the vast majority of plants engage in arbuscular mycorrhizal (AM) associations (Wang and Qui, 2006). AM fungi are believed to assist N-nutrition of host plants through assimilation of N, either exclusively (Tanaka and Yano, 2005) or preferentially (Govindarajulu et al., 2005), in the form of NH+ 4 . Thus there is a high likelihood that they can affect fitness of autotrophic ammonia oxidizers (AO) and influence ni-
226
S. D. VERESOGLOU
trification activity of soils. There have been three studies that addressed the effect of AM fungi on nitrification. Amora-Lazcano et al. (1998), in a 60 dayexperiment, detected that non-mycorrhizal maize supported lower numbers of autotrophic AO than AMinoculated maize. Cavagnaro et al. (2007) failed to detect the differences between mycorrhiza-defective mutant tomatos and their wild-type progenitors in either the composition or the density of AO bacteria. By contrast, Veresoglou et al. (2011a) in a series of experiments asserted that nitrification potential activity in the rhizosphere of mycotrophic plants consistently declined. Clearly further investigation is required to address why the results of Amora-Lazcano et al. (1998) have not been reproducible. A common source of bias, when the nitrifying community is addressed in the sterilized soils re-inoculated with bacteria, is the low growth rates of AO, which may require several days for a single doubling event even under laboratory conditions (Prosser, 2007). As a result, commonly the study of the AO community is performed under highly dynamic conditions. For example, the sequential harvesting approach in Amora-Lazcano et al. (1998) revealed that AO counts had been increasing rapidly with time till the end of the experiment. Most AM studies have proceeded with experimentation immediately after inoculating the soil with the bacterial filtrate (e.g., Amora-Lazcano et al., 1998). However, an optimization step that includes the equilibration of the soil with the bacterial community for approximately 2 weeks is optimally recommended before initiation of experiments targeting the microbial community (Shaw et al., 1999). The aim of the study was to assay the nitrification rates of the nutrient-limited sterilized soils in the rhizosphere of Plantago lanceolata inoculated with AM when an extensive 3-week equilibration step preceded initiation of the experiment. It was hypothesized that the slow growth rates of AO coupled to early encountering of competition for inorganic N with the plants and the plant-supported rhizosphere microbial community would have been detrimental for the development of the soil nitrifying community. It was, thus, expected that the nitrification rates (actual and potential) close to the roots (root compartments) would have been lower than those far from plant roots (root-free compartments). MATERIALS AND METHODS Following a 3-week microbial equilibration pe-
riod, P. lanceolata was grown up for 13 weeks in mesh-divided compartmentalized microcosms (a 40-μm stainless mesh was used as a separator of the compartments). Three treatments were established: nonmycorrhizal P. lanceolata, P. lanceolata inoculated with Glomus intraradices B.B/E, and P. lanceolata inoculated with Gigaspora margarita BEG 34. Each treatment replicated eight times. A single P. lanceolata seedling was sown to one of the compartments called the “root compartment”, whereas the “root-free compartment” remained inaccessible to plant roots but accessible to AM extraradical mycelial growth. The soil sterilization procedure was carried out through γ irradiation to minimize sterilization-linked modification of the soil properties (Alef and Nannipieri, 1995). Nevertheless, a spike of mineralized microbial biomass at the beginning of the experiment was unavoidable. To restore the prokaryotic microbial population 10 mL of microbial suspension [non-sterilized soil:saline phosphate buffer pH 7.2 = 1:10, with 0.1% (v/v) Tween 80 filtered through a Whatman #5 (2.5-μm pore size) filter] were added to each microcosm and the soil microbial community was left to equilibrate at a constant moisture content of 60% water holding capacity (WHC) for three weeks before initiation of the experiment. More details on the experiment can be found in Veresoglou et al. (2011b). At the day of harvest a 5 g equivalent dry weight (EDW) subsample of soil from each microcosm was stored in the freezer (−20 ◦ C) and was later used for + assessment of residual NO− 3 -N and NH4 -N. A further 10 g of soil from the root compartment was used to determine nitrification potential one day after harvest according to Hart et al. (1994). Five gram of soil was brought to 60% WHC with sterile distilled water and was left to incubate in 50 mL centrifuge tubes for three weeks to determine, through the differences between − the initial and final levels of NH+ 4 -N and NO3 -N, the rates of mineralization (Beck, 1983). Similarly, in another 50 mL centrifuge tube, 0.5 mL of 10 g L−1 ammonium sulphate [(NH4 )2 SO4 ] solution were added to 5 g of soil and water content was adjusted to 60% WHC. Then the sample was left to incubate for two weeks to assess actual nitrification rates through startand end-point differences in the NO− 3 -N content (Beck, 1979). Details for both the mineralization and actual nitrification assays were presented by Alef and Nannipieri (1995). The main difference between actual nitrification rates and nitrification potential rates lies on the fact that assaying of nitrification potential further
ARBUSCULAR STATUS PREVENTS SUPPRESSION OF NITRIFICATION
involves adjustments of nutrient availability and pH so that AO may nitrify to the maximum of their ability. Moreover, while nitrification potential assessment involves four assessments of NO− 3 -N status within 26 h, the end-point assessment of NO− 3 -N for the actual nitrification assay is commonly carried out following 2week incubation. Neither assay prevents N loss through denitrification as the denitrification rates in the presence of abundant NH+ 4 -N are considerably lower than those of nitrification. As a result, when AO densities are low, negative rates may be retrieved and the assays should solely be used for inter-treatment comparisons. The end-point potassium chlorate (KCl) extracts from the root-compartment samples that were assayed for nitrification were further used for assessment of nitrite (NO− 2 -N) status to verify absence of nitrification. The + NO− 3 -N and NH4 -N concentrations were assessed in 60 g L−1 KCl:soil extracts of 5:1 (v/v) through ion chromatography (Dionex DX 100). By contrast, NO− 2 content was assessed colometrically (Keeney and Nelson, 1982). For statistical analysis, NH+ 4 -N values had to be log transformed and absolute values of actual nitrification rates were square root-transformed maintaining their sign to conform with the normality criterion. For the figures the back-transformed values are presented. RESULTS AND DISCUSSION Inorganic N in the microcosms was available mainly − in the form of NH+ 4 -N as NO3 -N levels were very low (Fig. 1). With regards to NH+ 4 -N availability the highest level was recorded in the root-free compartment of the non-mycorrhizal microcosms whereas lower levels were recorded in the root compartments of AM-inoculated microcosms compared to the respec-
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tive non-mycorrhizal microcosms (Fig. 1). Nitrification potential rates, which represent a measure of potential ammonia oxidizing activity, were close to detection limits of the assay and, consequently, it was not possible to detect significant effects (Fig. 2). On the other hand, as expected, mineralization assay revealed non-significant differences amongst treatments (Fig. 2). This facilitated comparisons because it strengthened the assumption that no significant differences in inorganic-N-input in the microcosms existed. The most important result was obtained with regards to actual nitrification rates (Fig. 2). In the root-free compartments of all microcosms and the root compartments of AM-inoculated microcosms a similar level of nitrification was detected. However, in the root compartment of non-mycorrhizal microcosms the nitrification rates assessed were negative (Fig. 2). To verify the low actual nitrification rates in the root compartment of non-mycorrhizal plants, the intermediate product of nitrification (NO− 2 -N) was measured in the end-point KCl extracts from the root-compartment samples that had been assayed for nitrification. In the mycorrhizal samples NO− 2 -N levels were above detection limits. By contrast NO− 2 -N levels in the non-mycorrhizal samples were below (Fig. 3). The residual NO− 2 -N was significantly different (P < 0.001) between the three treatments. Results indicated that actual nitrification rates of the soil in the root compartment of non-mycorrhizal treatment were lower than those in the other treatments. It is well known that actual nitrification rates may be detrimentally affected by low inorganic phosphorus (P) availability or pH changes (Hart et al., 1994). Nevertheless, inorganic P availability has been known to be lower in the rhizosphere of mycorrhizal
− Fig. 1 Residual NH+ 4 -N and NO3 -N contents at harvest in the microcosms treated with non-mycorrhiza (NM), Glomus intraradices (Gi), and Gigaspora margarita (Gm). Means marked by the same letter within a given compartment are not significantly different according to least significant difference at P = 0.05 by F test. *Significant compartment effect for the NM treatment at P = 0.05. Vertical bars indicate standard errors.
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S. D. VERESOGLOU
Fig. 2 Nitrification potential rates, mineralization rates, and actual nitrification rates at harvest in the microcosms treated with non-mycorrhiza (NM), Glomus intraradices (Gi), and Gigaspora margarita (Gm). Means marked by the same letter within a given compartment are not significantly different according to least significant difference at P = 0.05 by F test. *Significant compartment effect for the NM treatment at P = 0.05. Vertical bars indicate standard errors.
Fig. 3 Residual NO− 2 -N levels in the KCl extracts obtained from the end-point actual nitrification tubes from the root compartment of the microcosms treated with nonmycorrhiza (NM), Glomus intraradices (Gi), and Gigaspora margarita (Gm). Means marked by the same letter are not significantly different according to least significant difference at P = 0.05 by F test. Vertical bars indicate standard errors.
plants due to an expansion of the P-depletion zone (Smith and Read, 2008). In agreement to this idea, Veresoglou et al. (2011b) had demonstrated, in the specific experimental system, that total P content was higher for mycorrhizal plants. Assessment of soil pH of representative soil samples also indicated no modification of soil pH status (unpublished data). Based on these results, apparently, the recorded differences in nitrification rates should have reflected a decline in AO abundance in the non-mycorrhizal root compartment. Because the experimental approach adopted excluded bacterial predators from the microcosms, the microbial densities at harvest should have represented the peak microbial densities in the experiment. AO are known to be poor competitors for soil NH+ 4 (Boll-
mann et al., 2002). Hence, higher levels of residual inorganic N were recorded for the non-mycorrhizal root compartment when compared to the respective mycorrhizal treatments (Fig. 1) and were coupled to insignificant differences in N mineralization (N inputs). The low actual nitrification rates that were recorded for the root compartment of non-mycorrhizal P. lanceolata thus signified that AO should have encountered intense competition for NH+ 4 reserves early in the experiment and could not proliferate. The author attributes this result to: 1) the fact that AM plants are known to exhibit a growth depression in the early stages of experimentation (Smith and Read, 2008). As a result, their requirements in inorganic N at the specific stage should have been lower than the plants in the nonmycorrhizal treatment and AO growth in the AMinoculated microcosms encountered less strong competition for pools of NH+ 4 ; and 2) the allocation from the AM-inoculated plants of carbon to the AM fungi that would have otherwise been secreted in the form of exudates and delivered to the bacterial community, intensifying competition for N. A decline in sugar exudation following AM colonization is commonly reported in studies (Jones et al., 2004). By contrast, in the non-mycorrhizal microcosms no such mechanism existed to alleviate competition for soil N and AO had to cease growth. The results agree with those of AmoraLazcano et al. (1998), where they verified the better establishment of AO in the sterilized soil planted with AM-inoculated seedlings. CONCLUSIONS With regards to the original hypothesis limited
ARBUSCULAR STATUS PREVENTS SUPPRESSION OF NITRIFICATION
differences were recorded between the two compartments but there were differences between mycorrhizal and non-mycorrhizal root compartments. The results showed that the specific experimental approach was not suitable for the study of slow-growing microbes in the presence of AM fungi. This raises the question whether a longer equilibration period should be adopted. In the absence of bacterial predators, extensive equilibration might be expected to lead to further immobilization of nutrients, as an increasing part of the bacterial community would swift to an inactive state. It is, thus, highly likely that elongation of the equilibration period could deteriorate nutrient limitation of the soil. Future experimentation is required to address whether identified problems may be resolved with the addition of protozoa or nematodes. ACKNOWLEDGEMENTS I would like to thank Dr. L. SHAW from University of Reading, UK and Dr. R. SEN from Manchester Metropolitan University, UK for valuable feedback. I also acknowledge the contribution of four anonymous reviewers in improving the quality of the manuscript. REFERENCES Alef, K. and Nannipieri, P. 1995. Methods in Applied Soil Microbiology and Biochemistry. Academic Press, San Diego, USA. Amora-Lazcano, E., V´ azquez, M. M. and Azc´ on, R. 1998. Response of nitrogen-transforming microorganisms to arbuscular mycorrhizal fungi. Biol. Fert. Soils. 27: 65–70. Beck, T. H. 1979. Die nitrifikation in B¨ oden (sammelreferat). Z. Pflanzenernaehr. Bodenkd. 142: 344–364. Beck, T. H. 1983. Die N-Mineralisierung von B¨ oden im Laborbrutversuch. Z. Pflanzenernaehr. Bodenkd. 146: 243–252. Bollmann, A., B˚ ar-Gilissen, M. J. and Laanbroek, H. J. 2002. Growth at low ammonium concentrations and starvation response as potential factors involved in niche differentiation among ammonia-oxidizing bacteria. Appl. Environ. Microb. 68: 4751–4757. Cavagnaro, T. R., Jackson, L. E., Scow, K. M. and Hristova, K. R. 2007. Effects of arbuscular mycorrhizas on ammonia oxidizing bacteria in an organic farm soil. Microb. Ecol. 54: 618–626. Chapin III, F. S., Matson, P. A. and Mooney, H. A. 2004. Principles of Terrestrial Ecosystem Ecology. Springer, New York.
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