Endophytic Insect-Parasitic Fungi Translocate Nitrogen Directly from Insects to Plants S. W. Behie et al.

Science 336, 1576 (2012); DOI: 10.1126/science.1222289

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9. R. S. DeFries et al., Proc. Natl. Acad. Sci. U.S.A. 99, 14256 (2002). 10. F. Achard, H. D. Eva, P. Mayaux, H.-J. Stibig, A. Belward, Global Biogeochem. Cycles 18, GB2008 (2004). 11. Y. Pan et al., Science 333, 988 (2011). 12. A. Baccini et al., Nature Clim. Change 2, 182 (2012). 13. M. C. Hansen et al., Proc. Natl. Acad. Sci. U.S.A. 105, 9439 (2008). 14. M. C. Hansen, S. V. Stehman, P. V. Potapov, Proc. Natl. Acad. Sci. U.S.A. 107, 8650 (2010). 15. S. S. Saatchi et al., Proc. Natl. Acad. Sci. U.S.A. 108, 9899 (2011). 16. Materials and methods are available as supplementary materials on Science Online. 17. K. Mokany, R. J. Raison, A. S. Prokushkin, Glob. Change Biol. 12, 84 (2006). 18. M. A. Cairns, S. Brown, E. H. Helmer, G. A. Baumgardner, Oecologia 111, 1 (1997). 19. FAO/IIASA/ISRIC/ISSCAS/JRC. Harmonized World Soil Database (version 1.1); FAO, Rome, Italy and IIASA, Laxenburg, Austria (2009) 20. O. Arino et al., GlobCover: ESA service for global land cover from MERIS. Geoscience and Remote Sensing Symposium (IEEE International, IGARSS 2007, 2007). 21. IPCC, IPCC guidelines for national greenhouse gas inventories (IGES, Japan, 2006; www.ipcc-nggip.iges.or.jp/ public/2006gl/index.html). 22. S. E. Page et al., Nature 420, 61 (2002). 23. G. R. van der Werf et al., Proc. Natl. Acad. Sci. U.S.A. 105, 20350 (2008). 24. A. Hooijer et al., Biogeosciences 7, 1505 (2010). Acknowledgments: Funding for this work was provided to Winrock International under contract 7150484 by the World Banks World Development Report 2010: Development and Climate Change. The findings, interpretations, and conclusions expressed in this paper are entirely those of the authors. They do not necessarily represent the views of the World Bank and its affiliated organizations or those of the Executive Directors of the World Bank or the governments they represent. Support for the forest cover loss mapping work was provided by National Aeronautics and Space Administrations Land Cover and Land Use Change and MEASURES programs under grants NNG06GD95G and NNX08AP33A. The authors would like to thank K. Mokany for providing the original data used to derive relationships between above- and belowground biomass. Forest loss data are available at http://globalmonitoring.sdstate.edu/projects/gfm. Carbon stock data are available at http://carbon.jpl.nasa.gov. Emissions data are available at www.appliedgeosolutions.com/ science-paper.html.

Supplementary Materials
www.sciencemag.org/cgi/content/full/336/6088/1573/DC1 Materials and Methods Figs. S1 to S3 Tables S1 and S2 References (2529) 15 December 2011; accepted 3 May 2012 10.1126/science.1217962

Endophytic Insect-Parasitic Fungi Translocate Nitrogen Directly from Insects to Plants

S. W. Behie,1 P. M. Zelisko,2 M. J. Bidochka1* Most plants obtain nitrogen through nitrogen-fixing bacteria and microbial decomposition of plant and animal material. Many vascular plants are able to form close symbiotic associations with endophytic fungi. Metarhizium is a common plant endophyte found in a large number of ecosystems. This abundant soil fungus is also a pathogen to a large number of insects, which are a source of nitrogen. It is possible that the endophytic capability and insect pathogenicity of Metarhizium are coupled to provide an active method of nitrogen transfer to plant hosts via fungal mycelia. We used soil microcosms to test the ability of M. robertsii to translocate insect-derived nitrogen to plants. Insects were injected with 15N-labeled nitrogen, and we tracked the incorporation of 15N into amino acids in two plant species, haricot bean (Phaseolus vulgaris) and switchgrass (Panicum virgatum), in the presence of M. robertsii. These findings are evidence that active nitrogen acquisition by plants in this tripartite interaction may play a larger role in soil nitrogen cycling than previously thought.

itrogen gas, although it constitutes 78% of the atmosphere, is unavailable to plants as a source of nitrogen unless it is fixed by microbial symbionts (e.g., Rhizobium) or freeliving microbes (e.g., Azotobacter) (1). In many natural as well as agricultural settings, nitrogen is the limiting nutrient for plant growth. The current model of the soil nitrogen cycle relies heavily on nitrogen-fixing bacteria to furnish plants with usable nitrogen (some is fixed by lightning strikes) (2). However, there are some examples in which plants have evolved mechanisms to scavenge nitrogen from insects. Carnivorous plants are able to obtain substantial amounts of nitrogen from insects they ingest. Pitcher plants (families

1 Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1, Canada. 2Department of Chemistry, Brock University, St. Catharines, Ontario L2S 3A1, Canada.

*To whom correspondence should be addressed. E-mail: mbidochka@brocku.ca

Nepenthaceae and Sarraceniaceae) trap insects in a deep cavity filled with liquid, and insect-derived nitrogen can constitute up to 70% of the plant nitrogen content (3). In one known case of fungusmediated transfer of insect-derived nitrogen to plants, the ectomycorrhizal fungus Laccaria bicolor transfers nitrogen from soil-dwelling collembola to white pine (Pinus strobus) whose roots it colonizes (4). The ability of L. bicolor to transfer insectderived nitrogen was specific to white pine, and generally L. bicolor associates with roots of pine and spruce in temperate forests (5, 6). Nonetheless, these findings suggest that a more general example of insect-derived nitrogen transfer via fungal mycelia to plants may exist. Metarhizium spp. are ubiquitous soil-dwelling insect-pathogenic fungi that are found in a variety of ecosystems worldwide (7), occur in soils up to 106 propagules per gram (8), and can infect more than 200 species of insects (9). Insects contribute substantial VOL 336 SCIENCE

amounts of nitrogen to soil. Each square meter of habitat can provide 0.4 to 4 g (by weight) of available insect nitrogen (see supplementary text). During a routine survey of plant root symbionts, we found that Metarhizium spp. formed endophytic associations with many plant species (10, 11). Endophytes live internally within the plant, and the host plant may benefit from the interaction (12). Here, we hypothesized that Metarhizium can parasitize and kill a soil-born insect, then transfer the insect-derived nitrogen to plants via fungal mycelia and endophytic association. We used 15N-labeled waxmoth (Galleria mellonella) larvae as a model prey insect and used this model in the experimental design to measure Metarhizium-mediated translocation of 15 N to the foliage of haricot bean (Phaseolus vulgaris) or switchgrass (Panicum virgatum). 15 N-labeled waxmoth larvae were added to microcosms in which the roots of the plants were separated from each insect by means of a 30-mm mesh (fig. S1). The insects were infected by Metarhizium 48 hours after 15N injection and then placed into the microcosm, and the amount of 15N transfer to plant tissues was determined during a 1-month period. After 14 days, in the presence of Metarhizium, insect-derived nitrogen constituted 28% and 32% of the nitrogen content in haricot bean and switchgrass, respectively; this represented significantly greater 15N incorporation than in the presence of uninfected 15Nlabeled waxmoth larvae [Fig. 1, factorial analysis of variance (ANOVA), P < 0.01]. After 28 days, insect-derived nitrogen constituted 12% and 48% of bean and switchgrass nitrogen content, respectively, in the presence of Metarhizium; this again represented significantly greater 15N incorporation than in the presence of uninfected 15N-labeled waxmoth larvae (Fig. 1; factorial ANOVA, P < 0.05). Similar results were observed when the plant seeds were first inoculated with conidia of Metarhizium and subsequently formed a root endophytic association. We therefore concluded that

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Fig. 1. Percentage of plant nitrogen derived from waxmoth larvae by an endophytic, insectpathogenic fungus. Two plant species were used: haricot bean (Phaseolus vulgaris) (A to C) and switchgrass (Panicum virgatum) (D to F). Shown are results obtained with Metarhizium robertsii [(A) and (D)], with Aspergillus flavus [(B) and (E)], and without fungus [(C) and(F)].Nitrogen source: solid circles, waxmoth larvae; open circles, no waxmoth larvae. The amount of insect-derived nitrogen in the plant issues was determined with a NOI-5 emission spectrophotometer. Nitrogen content was calculated as 100 %15N in seedling leaves. Results were analyzed using t tests. Error bars represent 1 SE; N = 6. Results are the means of three separate trials done in duplicate.

Haricot Bean
45 40 35 30 25 20

Switchgrass D

15 10 5 0 -5 1 10 5 0 -5 1 10 5 0 -5 1 7 14 28 1 7 14 28 7 14 28 1 7 14 28 7 14 28 1 7 14 28

References and Notes

1. E. C. Cocking, Plant Soil 252, 169 (2003). 2. J. N. Galloway, Environ. Pollut. 102, 15 (1998). 3. W. Schulze, E. D. Schulze, I. Schulze, R. Oren, J. Exp. Bot. 52, 1041 (2001). 4. J. N. Klironomos, M. M. Hart, Nature 410, 651 (2001). 5. D. L. Richter, J. N. Bruhn, Soil Biol. Biochem. 25, 355 (1993). 6. C. A. Buschena, R. L. Doudrick, N. A. Anderson, Can. J. For. Res. 22, 1883 (1992). 7. R. J. St. Leger, in Parasites and Pathogens of Insects, N. C. Beckage, S. N. Thompson, B. A. Federici, Eds. (Academic Press, New York, 1993), pp. 211229. 8. R. J. Milner, in Biological Control of Locusts and Grasshoppers , C. J. Lomer, C. Prior, Eds. (CAB International, Wallingford, UK, 1991), pp. 200207. 9. D. W. Roberts, A. Hajek, in Frontiers in Industrial Mycology, G. F. Leathan, Ed. (Chapman & Hall, New York, 1992), pp. 144159. 10. M. Wyrebek, C. Huber, R. K. Sasan, M. J. Bidochka, Microbiology 157, 2904 (2011). 11. R. K. Sasan, M. J. Bidochka, Am. J. Bot. 99, 101 (2012). 12. K. Saikkonen, S. H. Faeth, M. Helander, T. J. Sullivan, Annu. Rev. Ecol. Syst. 29, 319 (1998). 13. L. R. Scully, M. J. Bidochka, Can. J. Microbiol. 51 , 185 (2005). 14. W. Fang, R. J. St. Leger, Plant Physiol. 154, 1549 (2011). Acknowledgments: Supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (M.J.B.). We thank M. K. Bidochka for Fig. 2.

Time (days)

Fig. 2. Representation of the transfer of the insectderived nitrogen to plants through an association with endophytic, insectparasitic Metarhizium. Metarhizium infects and kills a soil-born insect. From the dead parasitized insect, fungal mycelia then associate endophytically with plant roots, through which nitrogen translocation occurs. The reverse may also occur; that is, endophytically associated Metarhizium could parasitize and kill an insect.

Supplementary Materials
www.sciencemag.org/cgi/content/full/336/6088/1576/DC1 Materials and Methods Supplementary Text Figs. S1 to S3 Table S1 References (1518) 22 March 2012; accepted 2 May 2012 10.1126/science.1222289

the hyphae infected the insect and transferred nitrogen back to the plant. Plants grown in a soil microcosm containing a non-endophytic insect pathogen (Aspergillus

flavus strain 6982) (13) in the presence of 15Nlabeled waxmoth larvae contained less than 10% insect-derived nitrogen, which was not significantly different from plants grown in the presence of SCIENCE VOL 336

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uninfected 15N-labeled waxmoth larvae (Fig. 1; factorial ANOVA, P > 0.05). In this experiment, insects were infected by M. robertsii or A. flavus within 2 days of placement into the microcosm. Insects were alive when placed into the microcosm but within two days of inoculation the larvae were dead. Six days after inoculation insects were mummified with fungal conidia, this was not observed in control experiments (fig. S2). Fungal propagules of both species were found within 0.5 cm of the plant roots within 6 days (fig. S3). Liquid chromatographymass spectrometry was used to confirm the incorporation of insect-derived 15N into plant amino acids (table S1). Our results show that these plants derived a significant proportion of nitrogen from soil insects through their endophytic associations with the insect pathogen, Metarhizium spp. (Fig. 2). Possibly M. robertsii provides nitrogen to the plant in exchange for carbon. A plant carbon transporter, Mrt (Metarhizium raffinose transporter), has been reported for Metarhizium and is required for successful root colonization (14). Symbiotic association of Metarhizium with plants may aid in plant survival in nitrogen-limited soils as vectors for acquiring nitrogen from insects.

Insect-derived N in leaves (%)