Influence of commercial inoculation with Glomus intraradices on the structure and functioning of an AM fungal community from an agricultural site

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1 DOI /s y REGULAR ARTICLE Influence of commercial inoculation with Glomus intraradices on the structure and functioning of an AM fungal community from an agricultural site Pedro M. Antunes & Alexander M. Koch & Kari E. Dunfield & Miranda M. Hart & Ashleigh Downing & Matthias C. Rillig & John N. Klironomos Received: 26 July 2008 / Accepted: 6 October 2008 # Springer Science + Business Media B.V Abstract The use of commercial arbuscular mycorrhizal (AM) inoculants is growing. However, we know little about how resident AM communities respond to inoculations under different soil management conditions. The objective of this study was to simulate the application of a commercial AM fungal inoculant of Glomus intraradices to soil to determine whether the structure and functioning of that soil s resident AM community would be affected. The effects of inoculation were investigated over time under disturbed or undisturbed soil conditions. We predicted that the introduction of an infective AM fungus, such as G. Responsible Editor: F. Andrew Smith. P. M. Antunes (*) : M. C. Rillig Institut für Biologie, Freie Universität Berlin, Altensteinstr. 6, D14195 Berlin, Germany pedro.antunes@fu-berlin.de P. M. Antunes : A. M. Koch : M. M. Hart : A. Downing : J. N. Klironomos Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada K. E. Dunfield Department of Land Resource Science, University of Guelph, Guelph, ON N1G 2W1, Canada intraradices, would have greater consequences in disturbed soil. Using a combination of molecular (terminal restriction length polymorphism analysis based on the large subunit of the rrna gene) and classical methods (AM fungal root colonization and P nutrition) we found that, contrary to our prediction, adding inoculant to soil containing a resident AM fungal community does not necessarily have an impact on the structure of that community either under disturbed or undisturbed conditions. However, we found evidence of positive effects of inoculation on plant nutrition under disturbed conditions, suggesting that the inoculant interacted, directly or indirectly, with the resident AM fungi. The inoculant significantly improved the P content of the host but only in presence of the resident AM fungal community. In contrast to inoculation, soil disturbance had a significant negative impact on species richness of AM fungi and influenced the AM fungal community composition as well as its functioning. Thus, we conclude that soil disturbance may under certain conditions have greater consequences for the structure of resident AM fungal communities in agricultural soils than commercial AM fungal inoculations with G. intraradices. Keywords Arbuscular mycorrhizal fungi. Commercial inoculation. Community ecology. LSU rdna. Glomus intraradices. Soil disturbance. T-RFLP. Maize growth

2 Introduction Potential economic benefits have led to an increasing intentional movement of arbuscular mycorrhizal (AM) fungi through the production and application of commercial AM inoculants (Gianinazzi and Vosatka 2004). However, the ecological consequences of these practices are still unknown. Problems that may result from commercial AM fungal introductions have recently been identified (Schwartz et al. 2006). These problems may be caused by potentially invasive AM fungal isolates (i.e., capable of, directly or indirectly, suppressing resident AM fungal populations) and/or through the introduction of pathogens associated with the inoculants. AM fungal inoculants often contain a single fungal isolate of a species in the Glomus genus (Glomus intraradices is the most common) and are generally applied to soils already containing a resident AM fungal community. The purpose of inoculation is to enhance the soil s inoculum potential to improve plant productivity. However, the introduction of a novel fungal isolate may also alter the structure of the resident AM fungal community through either positive (i.e., facilitation) or negative (i.e., competition) interactions (Callaway and Walker 1997). We know little about how resident communities respond to such introductions. Moreover, consequences of inoculations on the structure of resident communities may lead to shifts in functional outcomes. Previous work supports the potential for niche complementarity in the AM fungi (Gustafson and Casper 2006; Jansa et al. 2008). Traits that provide an organism with the capacity to cope with environmental stresses are by and large selected for commercial purposes (Dodd and Thomson 1994). As such, inoculant isolates can likely occupy broad realized niches and thus have the potential to compete with local species. It has been suggested that highly infective fast growing AM fungal species, such as many Glomus spp., become more abundant under conditions of environmental stress (e.g., agroecosystems) (Helgason et al. 1998; Oehl et al. 2004) such as tillage disturbance (Jansa et al. 2003). In contrast, others found little effects of tillage on AM fungal communities (Schalamuk et al. 2006). At the population level, a study on G. intraradices did not find any significant tillage treatment effects on genetic diversity (Koch et al. 2004), suggesting that this species may be relatively more tolerant to disturbance than other AM fungi, particularly those that rely on spores as their main source of propagation. In contrast, Jansa et al. (2002) found that G. intraradices was not favoured by tillage. The main objective of this study was to simulate the introduction of a commercial inoculant of G. intraradices in an agricultural soil and test whether the structure and functioning of the resident AM fungal community was affected under contrasting soil disturbance conditions. Materials and methods Soil and growing conditions The experiment was conducted in a glasshouse at the University of Guelph, ON, Canada (43 31 N, W) between May and September 2006 under ambient light conditions, 24.7:18.2 C mean day: night temperatures, and 55.1:72.1% mean day: night relative humidity. The substrate selected to support plant growth was a fine sandy loam soil collected on May 3rd from the top 20 cm near the buffer strip (~10 m section of maple trees in between fields) of a conventionally farmed maize field on a farm near Belwood (43 45 N, W). The field had been under soybean in The area near the buffer strip is not farmed every year and it contained a plant community when we collected the soil. The soil was broken up mechanically and passed through a 4 mm sieve before use. To normalize the growth conditions across treatments, the bulk of all pots was filled with the pasteurized field soil. Only a small portion of the same soil unpasteurized was used as the resident AM fungal inoculum in the appropriate treatments and the non-mycorrhizal microbial fraction was equalized across treatments (see below). Pasteurization consisted of gradually raising the soil s temperature to 90 C over a period of 60 min in an electric unit, and then cooling it gradually. This method, which is not as aggressive as autoclave-based sterilization, effectively destroys AM fungi (McGonigle and Miller 1996). Soil samples (n=3, mean±s.e.m.) analysed after pasteurization contained 0.7±0.20 mg NO 3 N kg 1, 24± 0.4 mg NH 4 N kg 1, 28±0.9 mg NaHCO 3 extractable P kg 1, 199±1.2 mg CH 3 COONH 4 extractable K, 244±4.1 CH 3 COONH 4 extractable Mg and 7.3 ph (1:1 in water).

3 Experimental design and preparation Experimental units were arranged in a fully randomized manner using a factorial design where one factor was AM fungal inoculation (Inoculant, Resident, Resident + Inoculant and Control), the second factor was soil disturbance (Disturbed and Undisturbed), and the third factor was harvest time (three growth periods, each of 3 weeks). Each treatment combination was replicated four times. All experimental units were prepared on May 9th by packing 3 L pots (96 pots in total) with 2 kg of pasteurized soil to a bulk density of approximately 1.3 g cm 3. On top of this layer of pasteurized soil each treatment was prepared as follows: 1) Inoculant 16 g of AM fungal inoculant MYKE PRO SG2 (produced by Premier Tech Biotechnologies, Rivière-du-Loup, Quebec, Canada, for the purpose of being used in agricultural systems) containing a single isolate of G. intraradices isolated in Quebec, covered by 1.3 kg of pasteurized soil; 2) Control 16 g of autoclaved (121 C for 30 min) MYKE PRO covered by 1.3 kg of pasteurized soil; 3) Resident 16 g of autoclaved (121 C for 30 min) MYKE PRO and 150 g of unpasteurized soil topped with 1.15 kg of pasteurized soil; and 4) Resident + Inoculant 16 g of MYKE PRO and 150 g of unpasteurized soil topped with 1.15 kg of pasteurized soil. The amount of commercial AM fungal inoculant added to each pot was calculated based on a rate of approximately 7.5 kg ha 1, as recommended by the producer. To correct for differences in non-am microbial communities, each experimental unit received a 5 ml filtered washing comprised of extract from a mixture of the unpasteurized soil and the AM inoculum (Ames et al. 1987). Soil disturbance treatments Maize seeds [Zea mays L. hybrid IC 192, a cross of CG 102 and CG 108 inbred lines (Lee et al. 2006)] were surface-sterilized (50% alcohol for 5 min), rinsed with deionised water, and placed in moist sterilized (autoclaved at 121 C for 15 min) vermiculite for 72 h for germination. Three seedlings were planted into each pot. The gravimetric water content of the soil was adjusted to approximately 200 mg H 2 Og 1 dry soil, and then maintained by irrigating with deionised water every 2 days. Three weeks after plant emergence all shoots were excised (see Fig. 1). Then, half the pots were randomly selected for the disturbed treatment. Soil disturbance was done by removing and passing the soil through a 4 mm sieve. All root material separated on the sieve was cut into pieces with a length of approximately 2 cm and mixed into the soil. The soil was repacked in the pots to the original density of 1.3 g cm 3. Three surface- Fig. 1 Method used to impose disturbed versus undisturbed soil treatments over time (i.e., 12 weeks in total)

4 sterilized and pre-germinated maize seeds were added to each pot (all treatments). Plants were harvested 3 weeks after emergence, dried at 65 C for 48 h, and the dry weight determined. Shoot material pooled into three separate samples, each corresponding to 3, 2 (randomly selected) and one pots for harvests 1, 2 and 3, respectively, was ground in a Wiley mill model 3 (Thomas Scientific, Swedsboro, NJ), digested by dry ashing and analysed for P (Richards and Carter 1993). The P content of each plant was calculated by multiplying shoot biomass (average of the appropriate randomly selected pots) by their P concentration. The root systems were carefully washed out of soil and a small portion of roots was placed in a 1.5 ml microcentrifuge tube and immediately stored in a freezer set at 80 C for terminal restriction fragment length polymorphism (T-RFLP) analysis (see below). A sub-sample of root was stained (Brundrett et al. 1984) before being examined for AM colonization (McGonigle et al. 1990). Two additional 3-week cycles of maize starting on July 14th, and August 17th, respectively, were carried out with half the pots containing soil that continued to be sieved before each cycle and half containing soil that was left undisturbed. To correct for nitrogen losses at the end of the second cycle, an aqueous solution of ammonium-nitrate was applied in a 50 ml volume to each pot at a rate of 25 mg N kg 1 dry soil. T-RFLP analyses We used T-RFLP to fingerprint AM fungal communities by analysing gene polymorphism in a ~380 bp length section of the large subunit (LSU) rdna (Mummey and Rillig 2006, 2007). DNA was extracted from plant roots across all experimental units belonging to Resident and Resident+Inoculant treatments (i.e., 24 samples per treatment; eight after each growth cycle) across harvests using a DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, Canada). A nested PCR protocol was used on all of these samples to amplify DNA from the AM fungi. The fungal community was amplified with LR1/FLR2 primers (Van Tuinen et al. 1998; Trouvelot et al. 1999). The PCR product was then used as template for a second PCR using the 5 -labelled primer pair FLR3-FAM/FLR4-VIC (Applied Biosystems, Foster City, CA, USA) to amplify AM fungi (Gollotte et al. 2004). Both PCRs were comprised of a 30 µl reaction mix containing final concentration of 1 Green GoTaq Reaction Buffer (Promega, Madison, WI, USA), 1.7 mm MgCl 2, 0.13 mm of each dntp, 0.33 mm of each primer and 1.25 u GoTaq DNA Polymerase, and 1.5 µl of template DNA. Products of the first PCR were diluted 1/100 for the second PCR. Both PCRs consisted of an initial denaturation step at 93 C for 3 min followed by 35 cycles (93 C for 1 min, 58 C for 1 min, 72 C for 1 min) and a final extension step of 10 min at 72 C in a Mastercycler ep thermocycler (Eppendorf, Hamburg, Germany). PCR product sizes were verified by gel electrophoresis with a 1 kb GeneRuler TM DNA ladder (Fermentas, Burlington, ON, Canada) as standard. After the second PCR, products were purified using a QIAquick cleanup kit (Qiagen Inc.) and the amount of DNA in each sample was subsequently determined using a NanoDrop ND-1,000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The amount of DNA per sample (19.1± 0.86 ng DNA µl 1 across all samples) was standardized to an amount previously determined as optimal for sequencing before being separately digested with the restriction enzymes Alu I and MboI (Invitrogen Inc., Burlington, ON, Canada). The restriction digestion, comprised of a 20 µl reaction mix containing 8 µl of purified PCR product (total of 60 ng DNA), 1 X REact 1 and two buffer (AluI and MboI, respectively) and 2 U of enzyme, was incubated for 4 h at 37 C. T- RF sizes in each sample were determined using an ABI 3730 DNA Analyzer (Applied Biosystems) with LIZ-500 (Applied Biosystems) as the size standard. Data analysis A 3-way factorial ANOVA was conducted for AM fungal root colonization responses and 2-way ANOVAs for each harvest time were performed on P content responses. ANOVA assumptions of normality and homogeneity of variances were confirmed using the Shapiro Wilk s W and the Levene s test, respectively. The Dunnett s test was used to compare AM dependency group means against non-am control group means. P content data corresponding to the second and third harvests were log-transformed and AM fungal colonization data were arcsine transformed to satisfy ANOVA assumptions. Untransformed data were used to calculate treatment means for tables and plots. Where appropriate, Least Square

5 Means contrasts within effects were performed. Means were compared using the Tukey s honest significance difference (HDS) test (P<0.05). Statistical analyses were performed using JMP version 7.0 (2007, SAS Institute Inc.). Community analysis consisted of determining the profile (sizes, peak heights and areas) of T-RFs in each sample using GeneMapper software v (Applied Biosystems). The Microsoft-Exel macro Treeflap (Rees et al. 2004) edu.au/~cwalsh/treeflap.xls) was used to convert fragment sizes to the nearest integer, aligning them with their respective peak heights side by side in two columns. We then developed an R script (R Development Core Team 2007; available upon request) that standardized the amount of total fluorescence among sample profiles (Dunbar et al. 2001). The number of T-RFs per sample (i.e., T-RF richness; indicative of AM fungal ribotype richness) was compared between the Resident and Resident+Inoculant treatments across harvests using the General Linear Model (Type VI sums of squares upon log-transformation). Only T- RF richness was considered in this study; not the height or area of peaks, because the latter parameters may not be indicative of relative abundance of ribotypes. The influence inoculation, disturbance and harvest time on community composition was tested by distance-based redundancy analysis (db-rda, Legendre and Anderson 1999). Bray Curtis coefficients of similarity were first calculated between samples and used to compute principal coordinates (PCoA) in PrCoord 1.0 (included in the software package CANOCO version 4.51, Biometris, Wageningen, The Netherlands). When necessary, eigenvectors were corrected for negative eigenvalues using the procedure of Lingoes (see Legendre and Legendre 1998) and then all the PCoA axes were exported to Canoco and treated as species data. To test the effects of the groups of factors, they were entered as dummy binary-variables. In CANOCO one group of factors was entered as the explanatory variables in the model while the other groups of factors were entered as covariables. The significance of such models was tested with a Monte-Carlo test based on 999 permutations. The results of the ordination of the AM fungal community composition were displayed as PCA ordination diagrams. Results Effect of inoculation on AM fungal colonization and P content of maize AM fungal root colonization significantly increased from the first harvest to the last harvest across all treatments, except for the Inoculant treatment under disturbed soil conditions (Fig. 2). Colonization did not significantly differ between the Resident and Resident+inoculant treatments (supported by the Least Square Means contrast within inoculation by disturbance effect across harvest times; F 1,66 =0.01, P<0.91). Therefore, internal root colonization was unaffected by the introduction of the inoculant in soil with the resident AM community. However, disturbance did significantly affect root colonization Fig. 2 Proportion of total AM colonization (hyphal, arbuscular and vesicular) of maize roots inoculated either with Inoculant (Myke Pro), the soil s resident AM community or both (Resident+Inoculant), and harvested at the end of each of three consecutive three-week periods of growth (1-white, 2-grey and 3-black bars) under disturbed (no pattern) and undisturbed (diagonal pattern) soil conditions. Bars represent the mean (n= 4 per treatment) ± 1 s.e. Bars with the same letter are not significantly different (Tukey s HSD test P<0.05)

6 (F 1,54 =168.8 P<0.0001) across all AM fungal treatments but we did not detect a significant disturbance x inoculation interaction (F 2,54 =2.6 P<0.08). All plants under the Control treatment were free of AM fungal colonization (data not shown). Total colonization responses were strongly correlated with those of arbuscular colonization (R 2 =0.77, P<0.0001) and, therefore, not shown. The P content of maize in response to the presence of resident AM fungi changed across harvests. Initially responses were negative or neutral, but by the second harvest they turned positive in undisturbed (Resident) and disturbed (Resident+Inoculant) soil and, by the third harvest, in undisturbed soil (both treatments) (Fig. 3). The outcome of the plant-am fungal interaction depended on the inoculation treatment (F 2,12 =8.1 P<0.02; F 2,12 =0.11 P<0.1; F 2,12 = 10.8 P<0.002 for harvests 1, 2 and 3, respectively) and on soil disturbance (F 1,12 =16.2 P<0.002; F 1,12 = 4.04 P<0.068; F 1,12 =20.3 P<0.001 for harvests 1, 2 and 3, respectively). The inoculant introduction in soil with the resident AM fungal community altered the functioning of the symbiosis, which became more beneficial under disturbed conditions (suggested by the Least Square Means contrast within inoculation by disturbance effect across harvest times between Resident and Resident+Inoculant treatments; F 1,48 = 2.86, P<0.1 and by the significant inoculation x disturbance interaction detected for the second harvest F 2,12 =6.1 P<0.02). Conversely, maize plants did not respond to the inoculant alone at any harvest, suggesting that interactions between the inoculant and the resident AM fungal community were responsible for the host plant effects in response to inoculant use observed for disturbed conditions. Biomass responses were strongly correlated with those of P content (R 2 =0.51, P<0.0001) and, therefore, not shown. AM fungal community response to inoculation Since the data obtained with each restriction enzyme/ primer combination or their union lead to similar interpretations, only those corresponding to AluI (forward primer) are shown. A total of 29 T-RFs were observed (Table 1). T-RF richness (i.e., the total number of T-RFs in individual plant root T-RFLP profiles) did not vary from harvest to harvest (F 2,35 = 1.82 P<0.18), indicating that all of the AM fungi capable of colonizing maize were able to establish in the roots within the first 6 weeks. T-RF richness also did not vary between plants either growing with the Resident or the Resident + Inoculant community (F 1,35 =0.49 P<0.49). T-RF richness across all treatments averaged 6.1 (±s.e.m. =0.40) T-RFs in roots growing in soil with the Resident community and 5.7 (±s.e.m. =0.32) T-RFs in roots growing in presence of the Resident + Inoculated AM fungal community. In contrast, T-RF richness was negatively affected by soil disturbance (F 1,35 =10.1 P<0.003). We found an average of 5.1 (±s.e.m. =0.32) T-RFs in roots growing under disturbed conditions, whereas roots from undisturbed soil contained 6.6 (±s.e.m. =0.35) T-RFs. Fig. 3 Arbuscular mycorrhizal (AM) dependency for P content of maize plants growing in soil either with Inoculant (Myke Pro), the Resident AM fungal community or both (Resident+Inoculant), and subjected to three consecutive 3-week periods of growth (1-white, 2-grey and 3-black bars). Bars represent the mean (n=3) ± 1 s.e.; asterisks represent significant differences in P content when plants were grown with versus without AM fungi after Dunnett-test analysis. The effect of soil disturbance, is represented by bars without (disturbed conditions) or with diagonal pattern (undisturbed conditions.). AM dependency was calculated as the P content of AM maize minus that of non AM plants divided by the P content of AM plants. As a baseline, the P content of non AM plants was 2.8±0.18, 3.4± 0.05, and 1.7±0.08 mg plant 1 under disturbed conditions and 3.0±0.19, 3.6±0.21 and 1.7±0.24 mg plant 1 under undisturbed conditions and for harvests 1 2 and 3, respectively. For each harvest bars with the same letter are not significantly different (Tukey s HSD test P<0.05)

7 Table 1 Frequency of T-RFs found in each treatment T-RF (bp) Disturbed soil Undisturbed soil Total Resident Res. + Inoculant Resident Res. + Inoculant 1* ** Total *Numbers in this row correspond to the harvests. N=4 except for ** where N=3 due to deficient DNA amplification. Furthermore, the inoculation x disturbance interaction was not significant (F 1,35 =0.75 P<0.39), which does not support the hypothesis that the commercial inoculum would be highly competitive and alter resident AM fungal richness, especially under disturbed conditions. When looking at the similarity of the T-RF profiles as indicators of community composition we did not find that inoculation significantly affected the composition of the resident community at any harvest (Fig. 4). In contrast, harvest time significantly affected AM fungal community composition (Trace= 0.195, F-ratio=1.183, P-value=0.033) and, by the third harvest, soil disturbance had selected for significantly dissimilar communities (Trace=0.217, F-ratio=1.801, P-value=0.004). Discussion This study shows that the introduction of a commercial AM fungal inoculant in an agricultural soil did not affect the structure of the resident AM fungal community. This was the case in both disturbed or undisturbed soil conditions, which was unexpected. Indeed, we had hypothesized that G. intraradices

8 Fig. 4 Ordination diagram of principal components 1 and 2 (Axis 1 Axis 2) across harvests. The distance between the symbols (each representing an individual sample) in the diagram approximates the dissimilarity of their T-RF composition, measured by their Euclidean distance. Solid symbols correspond to samples from disturbed pots whereas open symbols to those from undisturbed pots. Circles and boxes correspond to samples from the Resident and Residents + Inoculant treatments, respectively would influence the structure of the resident AM fungal community especially under conditions of soil disturbance. Thus, our findings support the hypothesis that adding a new organism to a community does not necessarily mean that this organism will have an impact (through either competition or facilitation) on the structure of that community. We did detect, however, that adding the inoculant to the resident AM fungal community enhanced the P content of maize in disturbed soil. This indicates that the inoculant interacted, directly or indirectly, with the resident AM fungi possibly altering the intra-radical relative abundances and/or the functions of the different AM fungi. Such possibility is supported for example by the study of Jansa et al. (2008), which using real-time PCR provide direct evidence for functional complementarity among AM fungal species colonizing a single root system. G. intraradices is a common AM fungal species with a global distribution (Opik et al. 2006). The interaction of the inoculant with the resident AM fungal community likely resulted from the increase in inoculum potential or, if the isolate was not already present in the resident community, from strain specific effects. In a previous study, both genetic and functional variability were found within a population of G. intraradices from an agricultural field (Koch et al. 2006). The absence of significant inoculation effects on the structure of the resident AM fungal community should be interpreted with caution because it is known that there is often a lag-time between the introduction of non-resident biota and the occurrence of damage due to potential invasion (Sakai et al. 2001). However, according to our study, such lagtime would be greater than 12 weeks. Other reasons for interpreting this result with caution may include the limitations of the T-RFLP methodology, which may not be applicable to the entire phylum Glomeromycota and group different AM fungal species together, thereby underestimating richness. However, T-RFLP allows greater replication than other methods and is a valuable method of finding dissimilarities between communities (Avis et al. 2006; Mummey and Rillig 2007), which we were able to detect between soil disturbance treatments. The absence of both structural and functional effects of inoculation on the resident AM fungal community in undisturbed soil raises the question as to whether the inoculant was able to establish there. Future research using strainspecific methods such as mitochondrial DNA or microsatellites may help resolve this question (e.g., Croll et al. 2008). In contrast to inoculation, soil disturbance had a significant negative impact (i.e., generally leading to reductions in internal root colonization, P nutrition and T-RF richness) on the AM fungi colonizing maize roots and the continuous use of disturbance altered community structure. This is consistent with other studies in agricultural systems which show that soil disturbance reduces AM fungal colonization of roots (e.g., Antunes et al. 2006) and tends to preferentially

9 favour species in the Glomeraceae relative to other families in the roots of field-grown maize (Hijri et al. 2006; Jansa et al. 2003). Maize has been shown to respond positively in terms of growth and nutrition to isolates of G. intraradices (e.g., Mickelson and Kaeppler 2005; Sudova and Vosatka 2007), but in our study the inoculant G. intraradices did not affect the host s P content in absence of the resident AM fungal community. This further supports that host-plant responses to individual AM isolates may vary depending on the isolate s interactions with other AM fungi (Gustafson and Casper 2006) in combination with environmental factors (Smith and Smith 1996; Johnson et al. 1997). Since inoculation of the resident AM fungal community tended to enhance plant performance under disturbed conditions, the suggestion that AM fungal inoculation is beneficial for crop yields in tilled and severely managed soils (Schwartz et al. 2006) is supported by our study. Moreover, our study indicates that AM fungal introductions can promote plant growth benefits in such situations, without necessarily affecting the structure of resident AM fungal communities. To further investigate the potential consequences of AM fungal inoculations future research should investigate the extent to which the responses observed in this study are transferable to field conditions, consider other types of inoculum (e.g., resident, different non-resident or mixed-isolate inocula) and target plant populations or communities in vicinity of agricultural crops and/or subject to diverse types of disturbance and environmental conditions. Further research should also consider the longterm effects of commercial inoculation. However, even though commercial inoculants are already spread globally, if their use suppresses resident AM fungal populations the ethical problems of using them in field studies for scientific research need to be considered. In conclusion, soil disturbance may under certain conditions have greater consequences for AM fungal effects on plant productivity and the structure of resident AM fungal communities than certain AM fungal introductions through commercial inoculation. Acknowledgments This research was supported by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to JNK. The authors would like to thank Drs. D.L. Mummey and J.R. Powell for their suggestions regarding T-RFLP, K. Bailey for providing the soil and A. Farrow for technical support. References Ames RN, Mihara KL, Bethlenfalvay GJ (1987) The establishment of microorganisms in vesicular-arbuscular mycorrhizal and control treatments. Biol Fertil Soils 3: doi: /bf Antunes PM, de Varennes A, Zhang T, Goss MJ (2006) The tripartite symbiosis formed by indigenous arbuscular mycorrhizal fungi, Bradyrhizobium japonicum and soya bean under field conditions. J Agron Crop Sci 192: doi: /j x x Avis PG, Dickie IA, Mueller GM (2006) A dirty business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi. Mol Ecol 15: doi: /j x x Brundrett MC, Piche Y, Peterson RL (1984) A new method for observing the morphology of vesicular arbuscular mycorrhizae. Can J Bot 62: Callaway RM, Walker LR (1997) Competition and facilitation: a synthetic approach to interactions in plant communities. Ecology 78: Croll D, Wille L, Gamper HA, Mathimaran N, Lammers PJ, Corradi N, Sanders IR (2008) Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices. New Phytol 178: doi: /j x Dodd JC, Thomson BD (1994) The screening and selection of inoculant arbuscular-mycorrhizal and ectomycorrhizal fungi. Plant Soil 159: Dunbar J, Ticknor LO, Kuske CR (2001) Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rrna genes from bacterial communities. Appl Environ Microbiol 67: doi: /aem Gianinazzi S, Vosatka M (2004) Inoculum of arbuscular mycorrhizal fungi for production systems: science meets business. Can J Bot 82: doi: /b Gollotte A, van Tuinen D, Atkinson D (2004) Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species Agrostis capillaris and Lolium perenne in a field experiment. Mycorrhiza 14: doi: /s Gustafson DJ, Casper BB (2006) Differential host plant performance as a function of soil arbuscular mycorrhizal fungal communities: experimentally manipulating cooccurring Glomus species. Plant Ecol 183: doi: /s Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW (1998) Ploughing up the wood-wide web? Nature 394: doi: /28764 Hijri I, Sykorova Z, Oehl F, Ineichen K, Mader P, Wiemken A, Redecker D (2006) Communities of arbuscular mycorrhizal fungi in arable soils are not necessarily low in diversity. Mol Ecol 15: doi: /j x x

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