Differences in arbuscular mycorrhizal fungal communities associated with sugar maple seedlings in and outside of invaded garlic mustard forest patches

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1 Biol Invasions DOI /s ORIGINAL PAPER Differences in arbuscular mycorrhizal fungal communities associated with sugar maple seedlings in and outside of invaded garlic mustard forest patches E. Kathryn Barto Pedro M. Antunes Kristina Stinson Alexander M. Koch John N. Klironomos Don Cipollini Received: 21 August 2010 / Accepted: 28 January 2011 Ó Springer Science+Business Media B.V Abstract Garlic mustard (Alliaria petiolata) is a Eurasian native that has become invasive in North America. The invasive success of A. petiolata has been partly attributed to its production of allelopathic compounds that can limit the growth of arbuscular mycorrhizal fungi (AMF). Although such effects are well known, specific effects on the richness and community composition of AMF associated with E. Kathryn Barto and Pedro M. Antunes contributed equally to this paper. E. K. Barto D. Cipollini Department of Biological Sciences, Wright State University, 3640 Colonel Glenn Hwy, Dayton, OH 45435, USA Present Address: E. K. Barto (&) Institut für Biologie, Plant Ecology, Freie Universität Berlin, Altensteinstraße 6, Berlin, Germany barto@zedat.fu-berlin.de P. M. Antunes Department of Biology, Algoma University, Sault Ste. Marie, ON P6A 2G4, Canada A. M. Koch J. N. Klironomos Biology and Physical Geography Unit, I.K. Barber School of Arts and Sciences, The University of British Columbia Okanagan, Kelowna, BC V1V 1V7, Canada K. Stinson Harvard University, Harvard Forest, 324 North Main Street, Petersham, MA, USA woody species have not been explored. We collected sugar maple (Acer saccharum) seedlings from eight natural forest sites in Ohio and Massachusetts, containing areas either invaded or uninvaded by A. petiolata. We measured AMF root colonization of seedlings, isolated DNA from the roots and performed PCR-TRFLP analysis to assess the richness and community composition of AMF. As expected, we found reduced AMF colonization in A. petiolata invaded patches. A. petiolata did not alter the detected TRF richness, but was associated with significant changes in the composition of AMF communities in half of the sites monitored in each region. Our results suggest that although AMF colonization was reduced in A. petiolata patches, many indigenous AMF communities include AMF that are tolerant to allelopathic effects of A. petiolata. Keywords Invasion Allelopathy T-RFLP Arbuscular mycorrhizal fungi Acer saccharum Alliaria petiolata Introduction Arbuscular mycorrhizal fungi (AMF) are obligately symbiotic fungi that associate with plants in 90% of surveyed plant families (Smith and Read 2008). Many plants depend on the symbiosis with AMF for normal growth and development, since AMF provide nutrients, water, and enhanced pathogen resistance in

2 E. K. Barto et al. exchange for photosynthates (Smith and Read 2008). Although AMF are widely assumed to benefit their plant hosts, they can also have detrimental effects on growth (Wilson 1984; Johnson 1993; Klironomos 2003). In addition to directly impacting growth of their plant hosts, AMF also mediate interactions between plants, enhancing competitiveness of invasive plants in some cases (Marler et al. 1999; Stampe and Daehler 2003; Carey et al. 2004; Walling and Zabinski 2004), and reducing it in others (Stampe and Daehler 2003). Some invasive plants are also allelopathic, meaning they release noxious natural products that inhibit the growth of surrounding plants (Rice 1974). This inhibition can be direct (Lawrence et al. 1991; Ridenour and Callaway 2001; Bais et al. 2003; Dorning and Cipollini 2006), or indirect by limiting the growth of AMF (Yun and Choi 2002; Stinson et al. 2006; Callaway et al. 2008). Garlic mustard [Alliaria petiolata (M. Bieb.) Cavara and Grande, Brassicaceae] is a Eurasian native that was introduced to North America in the late 1800s and has become widespread throughout North America (Nuzzo 2002). A. petiolata can adversely impact native plant abundance and biodiversity by forming dense monocultures in the undisturbed forest understory, a habitat typically inaccessible to invaders (McCarthy 1997; Stinson et al. 2006). Allelopathic effects of A. petiolata can be direct, as in the case of inhibition of seed germination by putative allelochemicals (Vaughn and Berhow 1999; Roberts and Anderson 2001; Prati and Bossdorf 2004; Barto et al. 2010), but recent research has focused on indirect allelopathic effects, including inhibition of AMF in competing plants. A. petiolata cannot associate with AMF so any allelopathic effects against AMF are not expected to negatively affect A. petiolata, thereby minimizing autotoxicity. Many woody species are thought to be dependent on AMF, especially during seedling stages, and mycorrhizal colonization and growth of Acer saccharum, Acer rubrum, and Fraxinus americana seedlings were suppressed by A. petiolata (Stinson et al. 2006). This strongly suggests that A. petiolata is allelopathic, possibly through both direct and indirect mechanisms. Mycorrhizal inoculum potential is reduced in soils invaded by A. petiolata (Roberts and Anderson 2001; Stinson et al. 2006), but reductions in AMF species richness appear to be less common, at least in herbaceous plants (Burke 2008). Mycorrhizal dependent woody species are generally more sensitive to allelopathic inhibition by A. petiolata than less dependent herbaceous plants, especially at the seedling stage. However, whether A. petiolata-induced reductions in AMF abundance also affect the community structure of AMF associated with woody species remains unclear. Reductions in AMF abundance could arise in different ways. First, all AMF may be inhibited equally, without effects on overall richness or composition. Second, only A. petiolatasensitive AMF may disappear, thereby lowering local AMF richness. Third, sensitive AMF may be replaced by resistant isolates, resulting in an altered AMF community composition, without necessarily changing AMF richness. To address these possibilities, we examined whether and how the percentage of root colonization, richness and community composition of AMF associating with a mycorrhizal dependent woody plant species were affected by A. petiolata invasion in the field. Materials and methods Site selection and sampling We sampled AMF communities in Acer saccharum Marsh. (sugar maple) seedlings in eight sites across two regions. We focused on A. saccharum because it associates with, and relies on, AMF, and its growth is suppressed by A. petiolata indirectly through suppression of AMF (Stinson et al. 2006). Sites were located in Massachusetts (MA) [MA1 ( N, W), MA2 ( N, W), MA3 ( N, W), MA4 ( N, W)] and Ohio (OH) [OH1 ( N, W), OH2 ( N, W), OH3 ( N, W), OH4 ( N, W)]. The distance between sites in each state ranged from 0.3 to 32.1 km. In each site, a total of 10 A. saccharum seedlings of similar size were individually collected from each of five A. petiolata invaded, and five uninvaded areas in late summer. Seedlings smaller than 30 cm in height were collected by carefully digging them out of the ground while retaining some soil around the roots. All A. saccharum seedlings collected in invaded patches were growing within 15 cm of a first year A. petiolata plant. Only A. saccharum seedlings at least 30 m away from the nearest A. petiolata plant were considered to be growing in an uninvaded area. All collected

3 Differences in arbuscular mycorrhizal fungal communities A. saccharum seedlings were at least 1 m apart to minimize the chance that seedlings would be sharing the same hyphal network. Approximately 100 mg fresh weight of root tissue from each sample was isolated and freeze dried for subsequent DNA extraction and terminal restriction fragment length polymorphism (T-RFLP) analysis to assess AMF richness (Gollotte et al. 2004; Johnson et al. 2004). The remaining roots were stained with Chlorazol Black E and mycorrhizal colonization, as abundance of hyphae (total colonization), arbuscules, and vesicles, was quantified in 100 intersections using the magnified intersects method (McGonigle et al. 1990). T-RFLP analysis DNA was extracted from freeze-dried plant roots using a DNeasy 96 Plant Kit (Qiagen Inc., Mississauga, ON, Canada) and amplified using a nested PCR approach. We used LR1/FLR2 primers in the first PCR (Trouvelot et al. 1999), followed by a second PCR with 5 0 -labelled primers FLR3-FAM/ FLR4-VIC (Applied Biosystems, Foster City, CA, USA) to amplify AMF DNA (Gollotte et al. 2004). Both PCR reactions consisted of 1 9 Green GoTaq Ò Reaction Buffer (Promega, Madison, WI, USA), 1.7 mm MgCl 2, 0.13 mm of each dntp, 0.33 mm of each primer, 1.25 units GoTaq Ò DNA Polymerase, and 1 ll of template DNA in a final volume of 15 ll. The PCR conditions were 93 C for 2 min; then 35 cycles of 93 C for 1 min, 55 C for 1 min, 72 C for 1 min; then 72 C for 10 min in a Mastercycler Ò ep thermocycler (Eppendorf, Hamburg Germany). We tested the first PCR step using three concentrations of purified DNA; the original concentration, a 1:50 dilution of purified DNA, and a 1:100 dilution of purified DNA. No dilution successfully amplified DNA from every sample, so we combined the PCR products from all three attempts, and used a 1:100 dilution of the combined PCR product as the template for the second PCR. The final PCR product was purified with a Qiaquick cleanup kit (Qiagen Inc.) before quantification of DNA in each sample with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Two restriction enzymes, AluI and MboI (Invitrogen Inc., Burlington, ON, Canada) were used in digests consisting of 50 ng DNA, 19 REact Ò 1 buffer (for AluI) or REact Ò 2 buffer (for MboI), and 1 unit of enzyme in 20 ll total volume. Digestions were incubated at 37 C for 6 h before analysis on an ABI 3,730 DNA Analyzer (Applied Biosystems) with LIZ-500 as the size standard. Data analysis Terminal restriction fragment (TRF) sizes and peak heights were determined using Peak Scanner software v. 1.0 (Applied Biosystems) with a threshold of 50 absorbance units (AU) for MboI, and 100 AU for AluI, which had more background noise. Stutter peaks are PCR artifacts that appear on profiles near real peaks with up to 20% the peak area of real peaks, and a fragment length one or two base pairs less than the real peak. Each profile was scanned and stutter peaks were manually deleted, then total fluorescence of each profile was standardized (Dunbar et al. 2001). TRFs were aligned with T-Align ( ie/*talign/) using a confidence interval of 1.0 to combine fragment lengths differing by less than one base pair into the same TRF (Smith et al. 2005). TRFs found only once in the entire data set were rare, and since they may have originated from primer mismatches they were excluded from further analysis. TRF peak heights may not correlate well with the abundance of a certain AMF ribotype in the field due to several factors, including TRF s shared among different AMF, differences in gene copy number among different AMF (Corradi et al. 2007), and PCR bias during the amplification steps. We therefore coded the data as a binary matrix indicating presence or absence of each TRF in each sample, and did not analyze peak heights. Percent colonization of vesicles, arbuscules, and hyphae were analyzed using non-parametric MANO- VA and ANOVA using the PERMANOVA? add-on package of the PRIMER 6 software ( Clarke and Gorley 2006; Anderson et al. 2008). We analyzed a nested mixed model, where the random factor site was nested within the random factor region (OH or MA), and the invasion status was nested as a fixed factor within site. Analysis of TRF richness was performed by treating TRF presence/ absence data of each of the four enzyme/primer combinations as single variables in a MANOVA. Similarly, AMF colonization analysis was performed on the percentages of roots colonized by arbuscules,

4 E. K. Barto et al. vesicles, and hyphae. Analyses and reported P-values are based on 4,999 permutations. The influence of A. petiolata invasion on community composition was determined 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 (part of Canoco version 4.51, Biometris, Wageningen, The Netherlands). All the PCoA axes were exported to Canoco and treated as species data. Groups of factors (region, site and invasion status) were entered as dummy binary variables and their effects tested by using one group of factors as the explanatory variables in the model while the other group of factors was entered as covariables. The significance of such models was tested with a Monte- Carlo test based on 499 permutations. Where appropriate we used forward selection of environmental variables and conducted Monte-Carlo permutation tests based on 499 permutations. The results of the ordination of the AM fungal community composition, as assessed by PCR-T-RFLP, were displayed as PCA ordination diagrams. AMF colonization (%) hyphae AMF colonization (%) arbuscules AMF colonization (%) vesicles a b c MA MA no Alliaria with Alliaria OH OH Results 0 MA OH Overall, mycorrhizal colonization of roots was altered by A. petiolata (MANOVA: F 8, 64 = 2.60, P = ) and was consistently lower in A. petiolatainvaded than in A. petiolata-free areas. Colonization also varied with site (F 6, 64 = 3.78, P = ), but was consistent across regions (F 1, 6 = 0.57, P = ). The A. petiolata-sensitivity of AMF was primarily driven by changes in root colonization of arbuscules and vesicles, since hyphal colonization was not affected by the presence of A. petiolata (arbuscular colonization: F 8, 64 = 3.17, P = ; vesicular colonization: F 8, 64 = 2.64, P = ; hyphal colonization: F 8, 64 = 1.35, P = ) and was higher overall in OH than in MA (Fig. 1). Only arbuscular colonization varied among sites (arbuscular colonization: F 6, 64 = 4.91, P = ; vesicular colonization: F 6, 64 = 0.76, P = ; hyphal colonization: F 6, 64 = 1.77, P = ). A. petiolata reductions of AMF, and especially arbuscular colonization, were stronger in OH than in MA (Fig. 1). On the other hand, A. petiolata did not affect overall TRF number, our Fig. 1 Mycorrhizal colonization of A. saccharum (mean ± SE) in sites either invaded or uninvaded by A. petiolata (N = 17 20). MA Massachusetts, OH Ohio. a Hyphal colonization, percent of root length containing hyphae. b Arbuscular colonization, percent of root length containing arbuscules. c Vesicular colonization, percent of root length containing mycorrhizal vesicles surrogate for AMF richness (F 8, 60 = 1.30, P = ). TRF numbers were also consistent among sites within a region (F 6, 60 = 0.85, P = ), but fewer TRFs were detected in MA than in OH (Fig. 2; F 1, 6 = 15.45, P = ). The composition of AMF communities varied significantly among sites in OH (Trace = 0.124; F = 1.417; P = 0.030) and MA (Trace = 0.149; F = 1.342; P = ). Using invasion status and site as environmental data and then removing the variance explained by site (using site as covariable environmental data) we found that A. petiolata invasion significantly affected AMF community composition in both OH (Trace = 0.152; F = 1.370; P = 0.022) and MA (Trace = 0.195; F = 1.409; P = 0.014). Using

5 Differences in arbuscular mycorrhizal fungal communities TRF number forward selection to determine how much of this variance was explained at each site, we found that invasion by A. petiolata significantly altered AMF community composition in two of the four sites surveyed in each region (Fig. 3). Discussion no Alliaria with Alliaria MA Fig. 2 Total number of TRFs identified in roots of A. saccharum (mean ± SE) from sites either invaded or uninvaded by A. petiolata (N = 17 20). MA Massachusetts, OH Ohio. Points indicate TRF numbers for each enzyme/ primer combination; circles and triangles represent MboI and AluI digestions, respectively, and open symbols and closed symbols represent VIC and FAM labels, respectively Although AMF richness was not affected by A. petiolata invasion, AMF colonization was suppressed in invaded sites, and exploratory ordination analysis indicated that A. petiolata-induced AMF OH community composition shifts had occurred at sites in both regions. This suggests that AMF strains sensitive to A. petiolata are being replaced by more resistant strains. These changes could be due to allelochemicals directly impacting sensitive AMF or through effects on A. saccharum, which could limit the amount of carbon available for mycorrhizal fungi. Regardless of the precise mechanism responsible for the shift in AMF community composition, the induced changes in AMF communities may also affect plant community structure in invaded habitats (van der Heijden et al. 1998), although in ways which are difficult to predict. Nutrient transfer to the plant likely occurs in the arbuscules (Gianinazzi-Pearson et al. 1991), while vesicles are thought to be fungal storage structures (Smith and Read 2008). Arbuscules are therefore considered a sign of vitality of the symbiosis and their disruption indicates that the capacity of A. saccharum to benefit from associating with AMF was reduced in GM invaded areas. Vesicles can be produced in response to unfavorable conditions (see Smith and Read 2008) and our data indicate that the energy storage capacity of the fungi was also suppressed by A. petiolata. As in this study, Stinson et al. (2006) found reduced AMF colonization of A. saccharum grown in soils where A. petiolata had previously grown, but they grew their plants in a greenhouse using field collected soil. Collection of AXIS 2 (13%) OHIO OH2 uninvaded OH2 invaded OH4 uninvaded OH4 invaded AXIS 2 (20%) MASSACHUSETTS MA3 uninvaded MA3 invaded MA4 uninvaded MA4 invaded AXIS 1 (19%) AXIS 1(21%) Fig. 3 Ordination diagrams based on T-RFLP profiles derived from samples in sites either invaded or uninvaded by A. petiolata. Only sites for which a significant portion of the variance is explained by A. petiolata presence as determined by forward selection (P \ 0.05) are represented. Numbers in parentheses indicate the amount of variance accounted for by each principal component axis. Lines connecting sample symbols are presented only as an aid for visual delineation of treatment groups

6 E. K. Barto et al. field soil disrupts the extraradical AMF hyphal network, and reduces inoculum potential of the soil, particularly for forest soils (Jasper et al. 1991). Results of greenhouse studies may not always represent what actually occurs in the field. This study is therefore an important verification that the inhibitory effects of A. petiolata observed in many greenhouse and laboratory studies (Roberts and Anderson 2001; Prati and Bossdorf 2004; Callaway et al. 2008) are also apparent in the field. We observed large regional differences in effects of A. petiolata invasion on AMF colonization, with AMF in OH being more sensitive to allelopathic effects than AMF in MA. A. petiolata was first noted in North America in New York (Nuzzo 2002), and likely reached MA before OH (Lankau et al. 2009). According to the novel weapons hypothesis, which does explain biogeographic differences in allelopathic effects of A. petiolata (Callaway et al. 2008), species or strains naïve to the allelopathic compounds produced by A. petiolata should be more sensitive than strains with a longer exposure history. Therefore, the lower inhibition of AM root colonization in MA may indicate that AMF communities in these sites had already started shifting towards dominance of AMF that are more resistant to A. petiolata. Effects observed in OH, where invasions by A. petiolata are likely more recent, may represent the response of AMF communities that are still dominated by more A. petiolatasensitive strains. Environmental variation of abiotic factors such as soil nutrient levels, moisture, and temperature could also explain the differences in community structure we observed between sites. As for differences between invaded and uninvaded areas, a more thorough investigation of how A. petiolata affects different edaphic characteristics is needed. It is important to note that although we were able to identify differences in AMF community composition in invaded and uninvaded areas, these were sitespecific differences and communities in all invaded areas were not similar. This suggests that there are multiple trajectories for selection, and that different resistant strains will become dominant in different sites. Additional sampling of sites around the Northeast and Midwest encompassing a range of soil types and times since invasion would help resolve this issue, and determine how quickly AMF communities become resistant to the allelopathic effects of A. petiolata. Adaptation to allelopathic effects of Centaurea maculosa by native grass species has been observed in as little as years (Callaway et al. 2005), and AMF may adapt even more quickly. In fact, AMF resistant to the allelopathic effects of A. petiolata have been found in multiple environments (Burke 2008; Koch et al. 2011), and after initial decreases in AMF richness following invasion, richness appears to begin increasing again after only 50 years of exposure to A. petiolata (Lankau 2011). Woody species appear to be more sensitive to allelopathic inhibition by A. petiolata than herbaceous species, with reductions in plant growth being more pronounced in the woody species (Stinson et al. 2006). Furthermore, we observed significant reductions in colonization of a woody species while Burke (2008) found no effect of A. petiolata invasion on colonization of three herbaceous plants. The general resilience of the AMF community is apparent in the lack of effect on AMF richness in woody and herbaceous plants, but it remains to be seen how the function of altered microbial communities in invaded stands change relative to communities in uninvaded areas. We had initially hypothesized several ways in which AMF could respond to A. petiolata invasion. With overall AMF richness being unaffected, evidence of A. petiolata-induced community changes in half of the monitored sites suggests an on-going replacement of A. petiolata-sensitive AMF by more tolerant AMF. The exact mechanisms that lead to these observed shifts in the AMF communities may be due to altered host availability in the presence of A. petiolata, or to direct or indirect allelopathic inhibition by A. petiolata, and our study was not designed to distinguish between these possibilities. However, there is growing evidence suggesting that A. petiolata impacts AMF communities in the field (Burke 2008), and similar inhibition has been seen in pot studies using soil conditioned by A. petiolata, thereby removing any non-host effects (Stinson et al. 2006). In un- or less affected sites all AMF strains appear to be affected similarly, and possibly represent areas where sensitive AMF had already been replaced. It is also possible that site characteristics relating to the underlying soil chemistry in these areas created conditions favorable for resistant AMF, removing sensitive strains before invasion occurred. Diverse plant communities could also provide root refuge for sensitive strains, lessening their exposure to A. petiolata metabolites in the soil and ensuring

7 Differences in arbuscular mycorrhizal fungal communities that sensitive and resistant strains continue to co-exist in some invaded areas. In conclusion, AMF colonization of a woody plant in the field was suppressed in sites invaded by A. petiolata. Although general AMF richness was unaffected by A. petiolata invasion, we did observe shifts in AMF community composition that concurred with A. petiolata invasion. Further studies should assess the functional capabilities of these altered AMF communities to determine whether or not their ability to enhance plant growth and provide other ecosystem services has been diminished. Acknowledgments We would like to thank Steph Enright, Dunbar Carpenter, and Sasha Mushegian for help collecting samples, Ben Wolfe for assistance in the lab, Mehrdad Hajibabaei and Isabelle Meusnier and the Biodiversity Institute of Ontario for support with T-RFLP analysis, and Dan Mummey for discussions about data analysis. Funding was provided by an Environmental Protection Agency Greater Research Opportunities Fellowship to EK Barto (# ), fellowships from the Swiss National Science Foundation to AMK (PBLAA and PAOOA ), a National Science Foundation Long Term Ecological Research Grant (DEB ) to the Harvard Forest, and a Discovery grant to JN Klironomos from the Natural Sciences and Engineering Research Council of Canada. These experiments comply with the laws of the USA and Canada. References Anderson MJ, Gorley RN, Clarke KR (2008) PERMANO- VA? for PRIMER: guide to software and statistical methods., Plymouth Bais HP, Vepachedu R, Gilroy S, Callaway RM, Vivanco JM (2003) Allelopathy and exotic plant invasion: from molecules and genes to species interactions. Science 301: Barto EK, Friese CF, Cipollini D (2010) Arbuscular mycorrhizal fungi protect a native plant from allelopathic effects of an invader. 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