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the root cells (Fig. 1b, d). AM fungi store plant-derived carbon in vesicles in the form of lipids and use them for energy when the plant is not actively photosynthesizing. The host plant cannot access lipids within vesicles, so their production represents a complete transfer of carbon from plant host to fungus. Most roots do not contain all AM fungal structures. Even on plants colonized by AM fungi, not all roots show signs of colonization. On some roots, a multitude of fungal structures are evident. On others, those structures are nowhere to be found. We were assessing AM fungi as part of a larger project comparing soil communities under maize (corn, Zea mays) and diverse planted tallgrass prairie (Bach and Hofmockel 2015) when we began to see some unexpected patterns. G. Narvaez and K. Murray, undergraduate students at the time, noticed that prairie roots seemed to have a lot of vesicles, while corn roots had extensive hyphal infection. We looked into the literature and found little to explain the pattern we were observing. Intrigued, we began recording not only the rate of AM fungal infection, but also the rates of vesicle and hyphal presence. Roots were collected monthly in the 2012 growing season from three systems: maize monoculture, planted prairie, and fertilized planted prairie. The prairie systems were planted with 31 species
The dynamic life of arbuscular mycorrhizal fungal symbionts One of the most fascinating biological interactions lies just beneath our feet. Arbuscular mycorrhizal fungi (AM fungi) from the phylum Glomeromycota form a text-book example of symbiosis with more than 80% of plant species. Yet, few people have the opportunity to observe AM fungi directly. Most AM fungi living within a root have three distinct body structures that can be observed under a microscope: hyphae, arbuscules, and vesicles. Hyphae are thin, wispy projections that reach out from the root and absorb nutrients such as phosphorous from the soil (Fig. 1a, c). Hyphae transport nutrients back to roots through arbuscules that extend into the root cells. Arbuscules are highly branched networks that exchange soil nutrients for carbohydrates produced by the host plant during photosynthesis. AM fungi are also able to store lipids in vesicles, which are small, round structures within
FIG. 1. Arbuscular mycorrhizal fungi (AM fungi) growing in roots from (a, b) maize and (c, d) tallgrass prairie. AM fungi within maize roots produce numerous hyphae (a), which extend out from the root tissue to absorb soil nutrients. In contrast, AM fungi within prairie roots produce few hyphae (c) and numerous small round structures called vesicles (d) to store lipids for their own energy when the plant is not actively photosynthesizing. AM fungi associated with maize roots produce few vesicles (b).
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including C4 grasses, C3 grass, legumes, and forbs (Jarchow and Liebman 2013). At the time of sampling, all prairie systems had robust cover of C4 grasses, but it was not possible to isolate roots by plant functional group or species. We acknowledge AM fungal colonization rates vary across prairie plant species (Hetrick et al. 1988). There are a number of reasons why we might expect plant–AM-fungi interactions to differ in these systems. Both maize and prairie communities benefit from mutualisms with AM fungi (Plenchette et al. 1983, Bauer et al. 2012). Nitrogen fertilization, such as that applied to the maize and fertilized prairie systems in our study, can alter the strength of AM fungi-conferred benefits on host plants (Johnson 1993, Hoeksema et al. 2010). Similarly, AM fungi respond strongly to the presence of different plant hosts (Eom et al. 2000), and within ecosystems, nitrogen and phosphorus fertilization affect AM fungal abundance and community composition. In addition, AM fungal communities and their interactions with plants can also shift with time, even within a growing season (Bentivenga and Hetrick 1992, Johnson et al. 2003, Mandyam and Jumpponen 2008). However, no studies have considered vesicle and hyphal infection rates separately. Changes in these structures likely reflect changes in the AM-fungi–plant symbiosis with different land uses. After counting a representative sample of all the roots, we observed two key findings. First, the overall rate of AM fungi hyphal colonization on roots in the maize system was six times greater than the prairie, and more than three times greater than fertilized prairie (P < 0.0001, ANOVA). In contrast, overall vesicle presence on roots was three times greater in prairie and fertilized prairie roots compared with maize (P < 0.0001). Secondly, hyphal colonization rates peaked in August for all three cropping systems (P = 0.0006, Fig. 2a), and root vesicle infection in fertilized prairie and maize increased as the growing season progressed, but peaked in July and August for prairie (P = 0.0003; Fig. 2b).
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It seems logical that AM fungi interacting with perennial plants during the growing season would invest in long-term storage of lipids in vesicles as a source of energy during months when the plants are dormant. As soon as the perennial plant emerges and begins photosynthesizing in the spring, the AM fungi is available to form a symbiotic relationship. This long-term relationship benefits both the plant and the fungus. In contrast, an annual plant like maize cannot provide a long-term relationship. AM fungi must colonize new root growth every year and likely invest more energy in producing spores to overwinter and germinate as the new crop establishes in the spring. Previous work has demonstrated that soil fertility and nutrient availability affects AM fungi production of extraradical structures including hyphae (Johnson et al. 2003), which may be driven by AM fungal community differences (Egerton-Warburton et al. 2007) and/or a shift from mutualism to parasitism (Johnson 1993). In our work, cropping system was more important than fertilization as a predictor of hyphae and vesicle production. Given the importance of the AM fungal mutualisms to most prairie species and previously documented declines in mutualist quality in corn fields (Johnson et al. 1992), a predominance of parasitic fungi in prairie systems seems unlikely. Instead, we suggest that such differences could result from phenotypic differences among AM fungi in response to annual or perennial plants, or from differences in life histories among the AM fungi species occurring in various cropping systems, including their relative investment in hyphae and vesicles. The seasonal changes we observed were also compelling because they imply that, within a growing season, AM fungi are changing how much they invest in soil exploration and nutrient uptake (through hyphae) and storage of energy for future use (vesicles). Previous work has shown differences in AM fungi colonization rates across a growing season, including investment in vesicle
FIG. 2. Arbuscular mycorrhizal fungi (a) hyphae and (b) vesicle colonization rates on the roots of maize (corn, black line), planted tallgrass prairie (gray line), and fertilized tallgrass prairie (red line) across a growing season. Corn roots produced more numerous hyphae and prairie roots produced more numerous vesicles. Vesicle presence varied between sample times differently in each system. Hyphae presence peaked in August in all systems.
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and hyphal colonization in response to N fertilization in tallgrass prairie (Bentivenga and Hetrick 1992, Johnson et al. 2003, Mandyam and Jumpponen 2008). Our observation of sustained vesicle investment in fertilized prairie and minimal vesicle production in heavily fertilized maize in contrast to a peak in vesicle colonization rates in unfertilized prairie in July supports this seasonal narrative, although overall colonization rates differed substantially between the maize and fertilized prairie roots. This observation spurs several questions about whether AM fungi are investing in different growth structures in response to plant growth stage, carbon allocation, and soil conditions, or if these patterns reflect different AM fungal communities. Overall, our results suggest that multiple aspects of environmental change, including fertilization, changes in land-use, and shifts in plant communities, may lead to changes in mycorrhizal mutualisms, which will be reflected in changes in the relative production of fungal structure within and around plant roots. Further research is needed to understand the functional importance of these changes and the consequences of these changes for maintaining mutualistic interactions. ACKNOWLEDGMENTS Funding for undergraduate research provided by the Iowa State University Biogeosciences Research Experience for Undergraduate Program (K. Murray) and the Iowa State Science with Practice program (G. Narvaez-Rivera). Roots were collected at Comparison of Biofuel Systems research site, which is supported by a grant from the Agriculture and Food Research Initiative of the USDA National Institute of Food and Agriculture (NIFA), grant number #20116700330364 (K. S. Hofmockel), and J. T. Bauer was supported by grant number 2016-67012-24680 from the USDA-NIFA. LITERATURE CITED Bach, E. M., and K. S. Hofmockel. 2015. Coupled carbon and nitrogen inputs increase microbial biomass and activity in prairie bioenergy systems. Ecosystems 18:417–427. Bauer, J. T., N. M. Kleczewski, J. D. Bever, K. Clay, and H. L. Reynolds. 2012. Nitrogen-fixing bacteria, arbuscular mycorrhizal fungi, and the productivity and structure of prairie grassland communities. Oecologia 170:1089–1098. Bentivenga, S. P., and B. A. D. Hetrick. 1992. Seasonal and temperature effects on mycorrhizal activity and dependence of cool- and warm-season tallgrass prairie grasses. Canadian Journal of Botany 70:1596–1602. Egerton-Warburton, L. M., N. C. Johnson, E. B. Allen, A. L. M. Egerton-warburton, N. C. Johnson, E. B. Allen, E. Monographs, B. Allen, L. M. Egerton-Warburton, N. C. Johnson, and E. B. Allen. 2007. Mycorrhizal community dynamics following nitrogen fertilization: a cross-site test in five grasslands. Ecological Monographs 77:527–544. Eom, A.-H., D. C. Hartnett, and G. W. T. Wilson. 2000. Host plant species effects on arbuscular mycorrhizal fungal communities in tallgrass prairie. Oecologia 122:435–444. Hetrick, B. A. D., D. G. Kitt, and G. T. Wilson. 1988. Mycorrhizal dependence and growth habit of warm-season and
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cool-season tallgrass prairie plants. Canadian Journal of Botany-Revue Canadienne De Botanique 66:1376–1380. Hoeksema, J. D., V. B. Chaudhary, C. A. Gehring, N. C. Johnson, J. Karst, R. T. Koide, A. Pringle, C. Zabinski, J. D. Bever, J. C. Moore, G. W. T. Wilson, J. N. Klironomos, and J. Umbanhowar. 2010. A meta-analysis of context-dependency in plant response to inoculation with mycorrhizal fungi. Ecology Letters 13:394–407. Jarchow, M. E., and M. Liebman. 2013. Nitrogen fertilization increases diversity and productivity of prairie communities used for bioenergy. GCB Bioenergy 5:281–289. Johnson, N. C. 1993. Can fertilization of soil select less mutualistic mycorrhizae? Ecological Applications 3:749–757. Johnson, N. C., P. J. Copeland, R. K. Crookston, and F. L. Pfleger. 1992. Mycorrhizae: possible explanation for yield decline with continuous corn and soybean. Agronomy Journal 84:387. Johnson, N. C., D. L. Rowland, L. Corkidi, L. M. EgertonWarburton, and E. B. Allen. 2003. Nitrogen enrichment alters mycorrhizal allocation at five mesic to semiarid grasslands. Ecology 84:1895–1908. Mandyam, K., and A. Jumpponen. 2008. Seasonal and temporal dynamics of arbuscular mycorrhizal and dark septate endophytic fungi in a tallgrass prairie ecosystem are minimally affected by nitrogen enrichment. Mycorrhiza 18:145–155. Plenchette, C., J. A. Fortin, and V. Furlan. 1983. Growth responses of several plant species to mycorrhizae in a soil of moderate P-fertility - I. Mycorrhizal dependency under field conditions. Plant and Soil 70:199–209. DATA AVAILABILITY Data and analytical code associated with this study are available at https://doi.org/10.5281/zenodo.1044476.
ELIZABETH M. BACH ,1,4 GISELLE NARVAEZ-RIVERA,1,5 KIRA MURRAY,1,6 JONATHAN T. BAUER2,7, AND KIRSTEN S. HOFMOCKEL 1,3,8 Manuscript received 20 July 2017; revised 31 October 2017; accepted 8 November 2017. Corresponding Editor: John Pastor. 1 Ecology, Evolution, and Organismal Biology Department, Iowa State University, 251 Bessey Hall, 2200 Osborne Drive, Ames, Iowa 50011 USA. 2 Department of Biology, Indiana University, Jordan Hall 142, 1001 East 3rd Street, Bloomington, Indiana 47405 USA. 3 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Boulevard, Richland, Washington 99354 USA. 4 Present address: Department of Biology, School of Global Environmental Sustainability, Colorado State University, 108 Johnson Hall, Fort Collins, Colorado 80523 USA. 5 Present address: Department of Anthropology, Iowa State University, 3102 Pearson Hall, Ames, Iowa 50011 USA. 6 Present address: Freestone Environmental Services, 1100 Jadwin Avenue, Suite 250, Richland, Washington 99352 USA. 7 Present address: Department of Plant Biology, Plant Biology Labs, Michigan State University, 612 Wilson Road, Room 368, East Lansing, Michigan 48824 USA. 8 Corresponding Author. E-mail:
[email protected] 