Plant Soil DOI 10.1007/s11104-008-9806-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. 2008
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 e-mail:
[email protected] 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
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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, 80° 13’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, 80°15’W). The field had been under soybean in 2005. 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 NO3−N kg−1, 24± 0.4 mg NH4−N kg−1, 28±0.9 mg NaHCO3−extractable P kg−1, 199±1.2 mg CH3COONH4−extractable K, 244±4.1 CH3COONH4−extractable Mg and 7.3 pH (1:1 in water).
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Experimental design and preparation Experimental units were arranged in a fully randomized manner using a 4×2×3 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 Fig. 1 Method used to impose disturbed versus undisturbed soil treatments over time (i.e., 12 weeks in total)
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 H2O g−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-
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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 MgCl2, 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 GeneRulerTM 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. TRF 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
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Means contrasts within effects were performed. Means were compared using the Tukey’s honest significance difference (HDS) test (P
