metarhizium transfers nitrogen from insect cadaver to plant

8/12/2019 Metarhizium Transfers Nitrogen From Insect Cadaver to Plant

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DOI: 10.1126/science.1222289, 1576 (2012);336Scienceet al.S. W. Behie

Insects to PlantsEndophytic Insect-Parasitic Fungi Translocate Nitrogen Directly fro

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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 Administrati

Land Cover and Land Use Change and MEASURES program

under grants NNG06GD95G and NNX08AP33A. The autho

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/DC1Materials 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 FungiTranslocate Nitrogen Directlyfrom Insects to PlantsS. W. Behie,1 P. M. Zelisko,2 M. J. Bidochka1*

Most plants obtain nitrogen through nitrogen-fixing bacteria and microbial decompositionof plant and animal material. Many vascular plants are able to form close symbiotic associationswith endophytic fungi. Metarhizium is a common plant endophyte found in a large numberof 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 insectpathogenicity of Metarhiziumare coupled to provide an active method of nitrogen transferto plant hosts via fungal mycelia. We used soil microcosms to test the ability of M. robertsiito translocate insect-derived nitrogen to plants. Insects were injected with 15N-labeled nitrogen,

and we tracked the incorporation of

15

N into amino acids in two plant species, haricot bean(Phaseolus vulgaris) and switchgrass (Panicum virgatum), in the presence of M. robertsii. Thesefindings are evidence that active nitrogen acquisition by plants in this tripartite interactionmay play a larger role in soil nitrogen cycling than previously thought.

Nitrogen 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 free-

living 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 ni-

trogen from insects. Carnivorous plants are able

to obtain substantial amounts of nitrogen from

insects they ingest. Pitcher plants (families

Nepenthaceae and Sarraceniaceae) trap insects in

a deep cavity filled with liquid, andinsect-derived

nitrogen can constitute up to 70% of the plant ni-

trogen content (3). In one known case of fungus-

mediated transfer of insect-derived nitrogen to

plants, the ectomycorrhizal fungusLaccaria bi-

colortransfers nitrogen from soil-dwelling col-

lembola to white pine (Pinus strobus) whoseroots

it colonizes (4).

The ability ofL. bicolorto transfer insect-

derived nitrogen was specific to white pine, and

generallyL. bicolorassociates with roots of pine

and spruce in temperate forests (5,6). Nonethe-

less, these findings suggest that a more general

example of insect-derived nitrogen transfer via fun-

gal mycelia to plants may exist.Metarhizium spp.

are ubiquitous soil-dwelling insect-pathogenic

fungi that are found in a variety of ecosystems

worldwide (7),occurin soils upto 106propagules

per gram (8), and can infect more than 200 spe-

cies of insects (9). Insects contribute substantial

amounts of nitrogen to soil. Each square me

of habitat can provide 0.4 to 4 g (by weight)

available insect nitrogen (see supplement

text).

During a routine survey of plant root sybionts, we found thatMetarhizium spp. form

endophytic associations with many plant sp

cies (10, 11). Endophytes live internally with

the plant, and the host plant may benefit fro

the interaction (12). Here, we hypothesized t

Metarhizium can parasitize and kill a soil-bo

insect, then transfer the insect-derived nit

gen to plants via fungal mycelia and endophy

association.

We used 15N-labeled waxmoth (Galle

mellonella) larvae as a model prey insect a

used this model in the experimental design

measureMetarhizium-mediated translocation15

N to the foliage of haricot bean (Phaseovulgaris) or switchgrass (Panicum virgatu15N-labeled waxmoth larvae were added to m

crocosms in which the roots of the plants w

separated from each insect by means of a 30-m

mesh (fig. S1). The insects were infected

Metarhizium 48 hours after 15N injection a

then placed into the microcosm, and the amou

of 15N transfer to plant tissues was determin

during a 1-month period. After 14 days, in

presence ofMetarhizium, insect-derived nitrog

constituted 28% and 32% of the nitrogen cont

in haricot bean and switchgrass, respectively; t

represented significantly greater 15N incorpo

tion than in the presence of uninfected 15

labeled waxmoth larvae [Fig. 1, factorial analy

of variance (ANOVA),P< 0.01]. After 28 da

insect-derived nitrogen constituted 12% and 4

of bean and switchgrass nitrogen content, resp

tively, in the presence ofMetarhizium; this ag

represented significantly greater15N incorporat

than in the presence of uninfected 15N-label

waxmoth larvae (Fig. 1; factorial ANOVA, P

0.05). Similar results were observed when

plant seeds were first inoculated with conidia

Metarhiziumand subsequently formed a root e

dophytic association. We therefore concluded t

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

*To whom correspondence should be addressed. E-mail:[email protected]

22 JUNE 2012 VOL 336 SCIENCE www.sciencemag.org76

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the hyphae infected the insect and transferred ni-

trogen 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 15N-

labeled waxmoth larvae contained less than 10%

insect-derived nitrogen, which was not significant-

ly different from plants grown in the presence of

uninfected 15N-labeled waxmoth larvae (Fig.

factorial ANOVA,P> 0.05). In this experime

insects were infected byM. robertsiiorA. flav

within 2 days of placement into the microcos

Insects were alive when placed into the mic

cosm but within two days of inoculation the l

vae were dead. Six days after inoculation inse

were mummified with fungal conidia, this w

not observed in control experiments (fig. S

Fungal propagules of both species were fou

within 0.5 cm of the plant roots within 6 da(fig. S3). Liquid chromatographymass spectro

etry was used to confirm the incorporation

insect-derived 15N into plant amino acids (table S

Our results show that these plants derive

significant proportion of nitrogen from soil

sects through their endophytic associations w

the insect pathogen, Metarhizium spp. (Fig.

PossiblyM. robertsiiprovides nitrogen to the pl

in exchange forcarbon. A plant carbon transpor

Mrt (Metarhiziumraffinose transporter), has be

reported forMetarhiziumand is required for su

cessful root colonization (14). Symbiotic assoc

tion ofMetarhizium with plants may aid in pl

survival in nitrogen-limited soils as vectors for quiring nitrogen from insects.

References and Notes1. 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.

52, 1041 (2001).

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(2001).

5. D. L. Richter, J. N. Bruhn, Soil Biol. Biochem. 25, 35

(1993).

6. C. A. Buschena, R. L. Doudrick, N. A. Anderson,Can

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.

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Grasshoppers, C. J. Lomer, C. Prior, Eds. (CAB

International, Wallingford, UK, 1991), pp. 200207.

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1992), pp. 144159.

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Microbiology157 , 2904 (2011).

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(2012).

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Annu. Rev. Ecol. Syst. 29, 319 (1998).

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185 (2005).

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(2011).

Acknowledgments:Supported by a Natural Sciences andEngineering Research Council of Canada Discovery Grant

(M.J.B.). We thank M. K. Bidochka for Fig. 2.

Supplementary Materialswww.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

Fig. 2.Representationofthe transfer of the insect-derived nitrogen to plantsthrough an associationwith endophytic, insect-parasitic Metarhizium.

Metarhizium infects and

kills a soil-born insect.From the dead parasitizedinsect, fungal mycelia thenassociate endophyticallywith plant roots, throughwhich nitrogen transloca-tion occurs. The reversemay also occur; that is,endophytically associated

Metarhiziumcould para-sitize and kill an insect.

Fig. 1. Percentage ofplant nitrogen derivedfrom waxmoth larvae byan endophytic, insect-pathogenic fungus. Twoplant species were used:haricot bean(Phaseolus vul-

garis) (A to C) and switch-grass (Panicum virgatum)(D to F). Shown are resultsobtainedwithMetarhiziumrobertsii[(A) and (D)], with

Aspergillus flavus[(B) and(E)], and without fungus[(C)and(F)]. Nitrogen source:solid circles, waxmoth lar-vae; open circles, no wax-moth larvae. The amountof insect-derived nitrogenin the plant issues was de-terminedwith a NOI-5emis-sion spectrophotometer.Nitrogen content was cal-culated as 100 %15N inseedling leaves. Results

were analyzed using ttests.Error bars represent 1 SE;N= 6.Results are the meansofthreeseparate trialsdonein duplicate.

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