Root-associated fungal microbiota of nonmycorrhizal Arabis alpina and its contribution to plant ...

51 Pages·2017·4.84 MB·English

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Root-associated fungal microbiota of nonmycorrhizal P
Arabis alpina
and its contribution to plant
phosphorus nutrition Y
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JulianaAlmarioa,b,1,GangaJeenaa,b,JörgWunderc,GregorLangena,b,AlgaZuccaroa,b,GeorgeCouplandb,c, M
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andMarcelBuchera,b,2 C
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aBotanicalInstitute,CologneBiocenter,UniversityofCologne,50674Cologne,Germany;bClusterofExcellenceonPlantSciences,UniversityofCologne,
50674Cologne,Germany;andcDepartmentofPlantDevelopmentalBiology,MaxPlanckInstituteforPlantBreedingResearch,50829Cologne,Germany
EditedbyLuisHerrera-Estrella,CenterforResearchandAdvancedStudies,Irapuato,Guanajuato,Mexico,andapprovedSeptember1,2017(receivedfor
reviewJune9,2017)
Most land plants live in association with arbuscular mycorrhizal to account for these cross-kingdom interactions to be complete.
(AM)fungiandrelyonthissymbiosistoscavengephosphorus(P) Here, we integrate these concepts and study the role of root-
fromsoil.Theabilitytoestablishthispartnershiphasbeenlostin associatedfungiotherthanAMinplantPnutrition.
someplantlineagesliketheBrassicaceae,whichraisesthequestion In some plant lineages, AM co-occurs with other mycorrhizal
ofwhatalternativenutritionstrategiessuchplantshavetogrowin symbioseslikeectomycorrhiza (woodyplants),orchid mycorrhiza
P-impoverishedsoils. To understandthecontribution ofplant–micro- (orchids), andericoid mycorrhiza (Ericaceae)(10).These associ-
biotainteractions,westudiedtheroot-associatedfungalmicrobiome ations can also promote plant nutrition; however, they have not
of Arabis alpina (Brassicaceae) with the hypothesis that some of its beendescribedinBrassicaceae.Endophyticmicrobescanpromote
componentscanpromoteplantPacquisition.Usingampliconsequenc- plantPacquisitionbydifferentprocessesincludingPsolubilization
ingofthefungalinternaltranscribedspacer2,westudiedtherootand and mineralization (11) or transfer of P in the form of soluble
rhizosphere fungal communities of A. alpina growing under natural orthophosphate. P transfer to their hosts was considered a hall-
andcontrolledconditionsincludinglow-Psoilsandidentifiedasetof mark of mycorrhizal fungi until recently. Two studies on binary
15fungaltaxaconsistentlydetectedinitsroots.Thiscohortincludeda root–fungusinteractionsshowedthattwoendophytes—theAsco-
HelotialestaxonexhibitinghighabundanceinrootsofwildA.alpina mycete Colletotrichum tofieldiae (12) and the Basidiomycete
growinginanextremelyP-limitedsoil.Consequently,weisolatedand Serendipita indica (syn. Piriformospora indica) (13)—are able
subsequentlyreintroducedaspecimenfromthistaxonintoitsnative totransferPtotheirnonmycorrhizalhostArabidopsisthaliana,
P-poorsoilinwhichitimprovedplantgrowthandPuptake.Thefun- promotingitsgrowthunderlow-Pconditions.S.indicawasalso
gusexhibitedmycorrhiza-liketraitsincludingcolonizationoftheroot
demonstratedtoparticipateinPuptakeofmaizeplantsdepending
endosphereandPtransfertotheplant.Genomeanalysisrevealeda
on the expression of a fungal high-affinity phosphate transporter
linkbetweenitsendophyticlifestyleandtheexpansionofitsrepertoire
(14).ThesestudiesprovidedproofofconceptforPtransferfrom
ofcarbohydrate-activeenzymes.Wereportthediscoveryofaplant–
fungi to nonmycorrhizal hosts; however, the ecological relevance
fungus interaction facilitating the growth of a nonmycorrhizal
of these interactions remains unclear as it is not known whether
plantundernativeP-limited conditions, thus uncoveringa previ-
theseendophytescanpromoteplantPuptakeundernativelow-P
ouslyunderestimatedroleofrootfungalmicrobiotainPcycling. soil conditions, and only C. tofieldiae was shown to be a natural GY
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Brassicaceae|microbiome|fungalendophyte|Helotiales|nutrient inhabitantofA.thalianaroots.Descriptiveandfunctionalstudies BIOL
transfer NT
Significance A
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P
Comparablewiththehumanmicrobiota,millionsofmicrobes
Most terrestrial plants live in symbiosis with arbuscular my-
colonizeplantsandformcomplexcommunitiesonplantsur-
corrhizal (AM) fungi and rely on this association to scavenge
facesandinplanttissues.Theinteractionsbetweentheplantand
the macronutrient phosphorus (P) from soil. Arabis alpina
itsmicrobiotarangefromparasitism (detrimentaltothehost) to
thrivesinP-limitedalpinehabitats,although,likeallBrassica-
mutualism(mutuallybeneficial),andtheiroutcomecanbepivotal
ceaespecies,itlackstheabilitytoestablishanAMsymbiosis.
for plant performance. Plant-associated microbes can influence
By studying the fungal microbiota associated with A. alpina
plant fitness by modulating plant growth, root architecture, nu-
trient acquisition, or drought and disease resistance (1–3). Thus, rootsweuncovereditsassociationwithabeneficialHelotiales
fungus capable of promoting plant growth and P uptake,
the plant microbiota can be seen as an extension of the plant
genome in the sense that it can increase the plant’s adaptation therebyfacilitatingplantadaptationtolow-Penvironments.
capacity(4).Thisisillustratedbythearbuscularmycorrhizal(AM)
Authorcontributions:J.A.,A.Z.,G.C.,andM.B.designedresearch;J.A.,G.J.,andJ.W.
symbiosis established between land plants and Glomeromycota
performedresearch;J.A.andG.J.contributednewreagents/analytictools;J.A.,G.J.,
fungi,whichisthoughttohavefacilitatedtheadaptationofplants andG.L.analyzeddata;andJ.A.andM.B.wrotethepaper.
to a terrestrial life (5). It is estimated that 80% of the vascular Theauthorsdeclarenoconflictofinterest.
plant species (6) receive phosphorus (P) and other nutritional
ThisarticleisaPNASDirectSubmission.
elementsfromthesefungiinexchangeforphotosynthates(7).The
FreelyavailableonlinethroughthePNASopenaccessoption.
ability to form an AM symbiosis has been lost independently in
Datadeposition:ThesequencereportedinthispaperhasbeendepositedintheNational
severalfloweringplantlineagesincludingtheBrassicaceaefamily
CenterforBiotechnologyInformationunderBioprojectPRJNA386947andPRJNA378526.
throughthelossofessentialsymbiosisgenesduringevolution(8).
SeeCommentaryonpage11574.
Given the beneficial effect of AM fungi on plant P uptake, the
1Presentaddress:DepartmentofPlantMicrobeInteractions,MaxPlanckInstitutefor
question of whether nonmycorrhizal species thrive due to the PlantBreedingResearch,50829Cologne,Germany.
exploitation of alternative P-mining strategies forms the basis of 2Towhomcorrespondenceshouldbeaddressed.Email:[email protected].
currentresearch(9).Inthecontextoftheplantholobiont,i.e.,the
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
plantandallitsmicrobialpartners,modelsofplantnutritionneed 1073/pnas.1710455114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1710455114 PNAS | PublishedonlineOctober2,2017 | E9403–E9412
onthefungalmicrobiotaofnonmycorrhizalplantsareneededto four European A. alpina accessions (Fig. 1A and SI Appendix,
improve our understanding of the ecological relevance of these Table S1) grown side by side at the Lautaret common garden
interactionsforplantnutritionattheholobiontlevel. (GAR-Lau).Atharvesttime,theaccessionsdifferedinsizeand
Although fungi represent a prominent part of the root micro- developmental stage (SI Appendix, Fig.S2D) but shared similar
biotawheretheycanplayimportantrolesaspathogenicorbene- root and rhizosphere fungal communities with comparable di-
ficialpartners,studiesofBrassicaceaespecieshavefocusedmainly versity(ANOVAP>0.05)(SIAppendix,Fig.S2B)andstructure,
onbacterialcommunities(1,15–17).Thesestudieshaveincreased as observed in the principal coordinates analysis (PCoA) (SI
our knowledge of how root bacterial communities are shaped by Appendix, Fig. S2A) and verified by permutational multivariate
environmental,edaphic,andhost-relatedfactors.Expansionofthis analysis of variance (PERMANOVA) results (P > 0.05). This
knowledgeto fungal communities iscrucialasstudieson themy- indicated that, under the assessed seminatural common garden
corrhizalhostspeciespoplar(18),sugarcane(19),andAgave(20) conditions, host genetic variation fails to impact the root-
suggest that fungal and bacterial root communities respond dif- associated fungal community in A. alpina. This contrasted with
ferently to environmental cues. Microbiome studies focusing on studies in which a small but significant contribution of the host
taxonomical description have shown that fungi detected in plant genotypetostructuringofbacterialrootmicrobiomeswasshown
tissues are often phylogenetically related to described plant path- (1,15,26,27)inhostspeciesincludingA.alpina(16).Ourwork
ogensorsaprotrophs(3,18).Comparativegenomicsanalyseshave suggests that, unlike bacterial communities, root-associated
shownthatplantbeneficialendophyticlifestylescanemergefrom fungal communities are less or not at all affected by host geno-
plant pathogenic or saprophytic fungal lineages through genome typic differences in A. alpina. However, we cannot exclude that
modificationsofteninvolvingtheexpansionorcontractionofgene highwithin-genotypevariabilitymightbeshadingasmalleffect.
families encoding carbohydrate active enzymes (CAZymes) in-
volved in plant cell-wall degradation (21–23). Prediction of the Soil and Environmental Cues Shape A. alpina Root and Rhizosphere
ecological role of fungal root endophytes is thus challenging and FungalCommunities.We next assessed how soil and environment-
requiresmoresystematicstudiesassociatingendophyteisolation,in associated factors shape fungal communities inhabiting bulk soil,
plantatesting,andgenomicinvestigation. theA.alpinarhizosphere,andtherootendosphere(Fig.1A).Un-
Arabisalpina(Brassicaceae)isanonmycorrhizalperennialarctic- dercontrolledgreenhouseconditions(GrH)westudiedtheeffect
alpine herb growing in harsh and rocky environments (24) in- of three soils (SI Appendix, Table S2) with different geographical
cluding P-impoverished soils (this study). In recent years it has origins [Reckenholz (Rec) vs. Lautaret (Lau)] and P-fertilization
emergedasamodelforecologicalanddevelopmentalstudies,and regimes [Reckenholz soils with amended nitrogen (N) and potas-
its genome has recently been sequenced (25). The aim of the sium (K) (RecNK) vs. N, P, and K (RecNPK)]. on the fungal
present study was to explore the root fungal microbiome of communitiesassociatedwithA.alpinaaccessionPajares(PM).
A.alpinaanditscontributiontoplantPacquisition,followingthe Resultsshowedthatfungalalphadiversity(relatedtothenum-
hypothesis that root-associated fungi other than AM fungi can ber of taxa per sample) estimated by the Shannon diversity in-
promoteplantPuptakeundernaturalandcontrolledlow-Pcon- dexwashighlydeterminedbythecompartmenttype(P=2.10−10,
ditions.WeusedIllumina-basedampliconsequencingofthefungal 70%ofvariance,SIAppendix,TableS3),withlowervaluesinroot
taxonomical marker internal transcribed spacer 2 (ITS2) to de- relativetorhizosphereandbulksoilcompartments(ANOVAand
scribethefungalmicrobiomeinA.alpinaroots(endosphere)and Tukey’s HSD, P < 0.05) (Fig. 1C), indicating the selection of a
therhizosphere(soilzoneimmediatelysurroundingtheroot)under reducednumberoffungaltaxaenteringtheplantroots.Similarly,
greenhouse,commongarden,andnaturalconditions.Microbiome comparingthestructureofthosefungalcommunities(taxapresent
variabilityanalysisshowedthatrootfungalcommunitiesweremore and their relative abundances) by permutational multivariate
robust in response to changing environments relative to the rhi- analysis of variance(PERMANOVA on Bray–Curtis dissimilar-
zosphere assemblages, leading to the description of a set of ities) revealed that the major source of variation was the com-
15fungaltaxaconsistentlydetectedinA.alpinaroots.Withinthis partmenttype(P=10−4,29%ofvariance,SIAppendix,TableS3).
cohort we identified a fungal taxon belonging to the Helotiales Althoughneitherthesoil’sgeographicalorigin(Reckenholzvs.
order, exhibiting high abundance in the roots of wild A. alpina Lautaret)noritsP-fertilizationregime(ReckenholzsoilswithNK
plantsgrowinginanextremelyP-limitedsoil.Successfulisolationof vs. NPK amendment) significantly impacted the overall fungal
aspecimenfromthistaxonfromA.alpinaroots,followedbyits diversity (ANOVA, P > 0.05), they did affect the structure of
reintroduction into the native P-limited soil after sterilization, fungal communities. The effect of the soil’s geographical origin
promotedA.alpinagrowthandshootPaccumulation.Invitro (PERMANOVA,P=10−4,21%ofvariance)decreasedfromthe
studiesfurtherdemonstratedthatthefungus’contributiontoplant bulksoil(67%ofvariance)overtherhizosphere(49%)totheroot
growthinvolvestransferofinorganicphosphatetoitshost.Finally, compartment(30%),whereastheP-fertilizationeffectoverallwas
fungalgenomesequencingrevealedanexpansionofitsrepertoire smaller(PERMANOVA P = 10−4, 6.3% ofvariance)and stable
ofcarbohydrate-activeenzymes,whichmaybeassociatedwithits across the three compartments (14, 13, and 15% of variance in
endophyticlifestyle. Cumulatively, these results provide evidence bulk soil, rhizosphere, and root communities, respectively) (SI
for a beneficial role of a hitherto unknown member of the root Appendix,TableS3).ThiswasevidentinthePCoAonBray–Curtis
microbiotainA.alpinagrowthperformanceinlow-Penvironments. dissimilarities where greenhouse samples from the Lautaret soil
(GrH-Lau)weregroupedseparatelyfromsamplesobtainedfrom
Results the two Reckenholz soils (GrH-RecNK and GrH-RecNPK),
Root-Associated Fungal Communities in A. alpina Were Unaffected by which clustered more closely (Fig. 1B). This indicated that, under
HostGeneticVariation.Our current understanding of the structure greenhouse conditions, the compartment type, the soil’s geo-
of the root microbiome in nonmycorrhizal Brassicaceae species graphicalorigin,andtoalesserextentitsfertilizationregime,all
rests primarily on bacterial communities, and information on the participatedintheshapingofroot-associatedfungalcommunities.
factors shaping root-associated fungal consortia is scarce. We Collectively,theseresultssuggestedthatfungalcommunitiesthat
studied fungal communities associated withA. alpina roots byse- wereaccommodatedintherootendospherewerelessdiverseand
quencingthefungalITS2withprimersITS9/ITS4astheyshoweda lessaffectedbysoilchangesthanextraradicalconsortia.
better recovery of low-abundance fungal diversity in comparison We then assessed whether these fungal communities were af-
withotherprimersinapilotexperiment(SIAppendix,Fig.S1). fected by the plant growing environment and compared fungal
We assessed the effect of plant intraspecific variation on the communities established under controlled greenhouse conditions
structureoftheroot-associatedfungalcommunitybycomparing (GrH-Lau) with those established in the common garden under
E9404 | www.pnas.org/cgi/doi/10.1073/pnas.1710455114 Almarioetal.
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A B Condition: Compartment: Root P
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GrH-RecNK Rhizosphere
GrH-RecNPK Root
Greenhouse (GrH). Botanical institute, Cologne,Germany. GAR-Lau
WILD-Gal.2013 P
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GrH n=6 n=6 n=6 P RPhCoi z1o (2s2p %h)er %)e ENTA
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Alpine garden (GAR). Jardin du Lautaret,western Alps, France. o 2 (14 PCo 2 (21 SEECOM
% %)
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GAR
n=5 n=5 n=6 n=6 PCo 1 (29 %)
Bulk soil
Alpine natural site (WILD). Col du galibier, western Alps, France.
Sampling in 2013 and 2014.
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CoGGnrrHdH-i-RtRieoeccnNNPKK GGrAHR-L-Laauu WWIILLDD--GGaall..22001134 GrH-RecNKGrH-RecNPKGrH-LauGAR-LauWILD-Gal.2013WILD-Gal.2014 GrH-RecNKGrH-RecNPKGrH-LauGAR-LauWILD-Gal.2013WILD-Gal.2014 GrH-RecNKGrH-RecNPKGrH-LauGAR-LauWILD-Gal.2013WILD-Gal.2014 OLOGY
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Fig.1. ComparisonofthefungalcommunitiescolonizingA.alpinarootsandrhizosphereundergreenhouse(GrH),commongarden(GAR),andnatural A
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(WILD)conditionsindifferentsoils(RecNK,RecNPK,Lau,andGal).(A)Experimentalsetupshowingthedifferentplantgrowingconditions.Thegeographic P
originofthedifferentA.alpinaaccessionsisindicatedinparentheses.MoreinformationaboutthesoilsandtheaccessionsisgiveninSIAppendix,Tables
S1andS2,respectively.Thenumberofbiologicalreplicatespercondition(n)isindicated.(B)Principalcoordinatesanalysisonfungalcommunitydifferences
(Bray–Curtisdissimilarities)inthedifferentcompartmentsandconditions.(C)FungalalphadiversityestimatedbyShannon’sdiversityindex.Lettersa–cin-
dicatesignificantdifferencesbetweenconditionswithineachcompartment(ANOVAandTukey’sHSD,P<0.05).(D)Meanrelativeabundanceofthemajor
fungalordersinthedifferentconditionsandcompartments:bulksoil,rhizosphere,androot.AsthefourA.alpinaaccessionsstudiedexhibitedsimilarfungal
communitiesinthegardenexperiment(SIAppendix,Fig.S2),combinedresultsforthefouraccessionsareshownunderthe“GAR-Lau”condition.
alpinesummerconditions(GAR-Lau)inthesamesoil(Lau)using diversity(Fig.1C)anddifferentcommunitystructure(PERMANOVA
A.alpinaPMashostplant.Fungalcommunitiesestablishedinthe P=10−4,20%ofvariance)inrootandrhizospherefungalcommu-
same soil but under the two contrasting environments, i.e., dif- nitiesfromwildA.alpina(WILD-Gal)(SIAppendix,TableS3).This
feringinaltitudeandclimate,exhibitedsimilardiversity(ANOVA was observable in the PCoA with greenhouse and common garden
P>0.05,Fig.1C)butdifferedinstructure(PERMANOVAP= samplesfromtheLautaretsoil(GrH-LauandGAR-Lau)clustering
0.002, 10% of variance). The effect of the environment type on close and separating from samples from wild growing A. alpina
fungal community structure increased from bulk soil (PERMA- (WILD-Gal) (Fig. 1B). Collectively, these results showed that
NOVAP=0.1)overrhizospheresoil(PERMANOVAP=0.002, underalpineconditionsroot-associatedfungalcommunitiesstill
21%ofvariance)totherootcompartment(PERMANOVAP= divergedaccording tothe soiltype.
0.003, 30% of variance) (SI Appendix, Table S3). This strongly
suggested that root-associated communities were affected to a StabilityofRootandRhizosphereFungalCommunitiesAcrossVarying
greater extent by environmental changes than bulk soil commu- Growth Conditions. We next performed a general analysis in-
nities,whichremainedlargelyunaffected. cludingalloftheexperimentstoassesshowfungalcommunities
Comparatively, common garden and wild A. alpina plants were affected by the plant growing condition. Six plant growing
growingundersimilaralpinesummerconditionsbutindifferent conditions were considered based on the environment and the
soils(GAR-Lauvs.WILD-Gal)alsoshoweddifferenceswithlower soil in which the plants grew and included the confounding
Almarioetal. PNAS | PublishedonlineOctober2,2017 | E9405
effects of two different environments (greenhouse conditions: capturedonlyapartofthecommunities’differences.Interestingly,
GrH and alpine summer conditions: GAR and WILD) and four therewasasignificantinteractionbetweenthecompartmenttype
soils(Lau,RecNK,RecNPK,andGal).SinceA.alpinaaccessions andtheplantgrowingcondition(PERMANOVA,P=10−5,16%,
harbored similar fungal communities (SI Appendix, Fig. S2), SIAppendix,TableS3),suggestingthatroot,rhizosphere,andbulk
samples from the different accessions were grouped under the soil fungal communities responded differently to varying plant
sameplantgrowingcondition,GAR-Lau. growing conditions. Indeed, PERMANOVA analysiswithin each
In all plant growing conditions, A. alpina root-associated (i.e., compartment showed that the effect of the plant growing condi-
root and rhizosphere) fungal communities were dominated by tiononthefungalcommunitysteadilydecreasedfromthebulksoil
ascomycetes (58% of the fungal reads) belonging mostly to the (P=10−5,83%)overtherhizosphere(P=10−5,59%)totheroot
orders Helotiales, Hypocreales, Pleosporales, and Sordariales. (P = 10−5, 49%) (SI Appendix, Table S3), suggesting that root
Basidiomycetes (18%), unclassified fungi (14%), zygomycetes fungal communities were more robust relative to extraradical as-
(4.4%), and chytridiomycetes (4.1%) were less abundant. As semblages. In sum, this analysis at a wide scale, including con-
expectedforanonmycorrhizalplant,glomeromycetesthatinclude trasting environments and soils, showed that, although the
theAMfungiwererarelydetected(0.04%).WhiletheHelotiales microhabitattype(bulksoil,rhizosphere,orrootcompartment)is
(24% of the fungal reads in roots) and Cantharellales (16%) the main driver of fungal alpha diversity, the plant growing con-
orders were enriched in root samples, Mortierellales (6% of dition is the main factor structuring root-associated fungal com-
the fungal reads in the rhizosphere), Sordariales (4.2%), and munities,i.e.,determiningthetaxapresentandtheirabundances.
anunclassifiedbasidiomycetetaxon(4.1%)wereenrichedinthe Moreover, itsuggestedthat,compared with rhizosphere commu-
rhizosphere(pairedttestP< 0.05)(Fig.1D). nities, fungal communities living within A. alpina roots were less
Comparisonoffungalcommunitiesattheoperationaltaxonomic affectedbychangesintheplantgrowingcondition.
unit(OTU)levelshowedagainthatthecompartmenttypewasthe
main driver of fungal alpha diversity (P = 2.10−16, 72% of vari- Fungal Taxa Consistently Found in A. alpina Roots. Following the
ance) with a bigger effect than the plant growing condition postulatethatcommonlyoccurringorganismsplaycriticalrolesin
(P=10−16,9.4%)(Fig.1C)(SIAppendix,TableS3).Comparisonof theirhabitat,weaimedatidentifyingfungaltaxathatconsistently
fungalcommunitystructurebyPCoAshowedaseparationbetween colonized A. alpina roots, hypothesizing that they promote plant
rootandsoil(rhizosphereandbulksoil)samplesmainlyalongthe growthand/orPuptake.Weidentified15highlyconservedfungal
firstaxis(18%ofvariance)andaseparationbetweenplantgrowing OTUswithahighprevalenceinroots(i.e.,presentinatleast85%
conditionsmainlyalongthesecondaxis(14%ofvariance)(Fig. of the root samples) (Fig. 2A). It comprised one zygomycete
1B).Withinthethreecompartments(root,rhizosphere,andbulk (Mortierella elongata, OTU00045), one basidiomycete (Ceratoba-
soil) fungal communities clustered according to the geographic sidiaceae sp., OTU00008), and 13 ascomycetes belonging to the
origin of the soil: samples from soil Gal (WILD-Gal) separated Helotiales(4OTUs),thePleosporales(4OTUs),theHypocreales
from soil Lau (independently of the environment) and from (3 OTUs), the Sordariales (1 OTU), and one unclassified order
ReckenholzsoilsRecNK andRecNPK,whichclustered together (OTU00015). On average, this cohort represented 43% of the
(Fig.1B).PERMANOVAanalysisindicatedthatthemajorsource fungalreadsdetectedinA.alpinarootswithvaluesrangingfrom
ofvariationincommunitystructurewastheplantgrowingcondi- 10 to 93% (Fig. 2B). Of these 15 highly conserved root OTUs,
tion (P = 10−5, 32% of variance) and not the compartment type 7wereenrichedinplantrootsincomparisonwiththerhizosphere
(P=10−5,16%)(SIAppendix,TableS3).Thiscontrastedwiththe (paired t test, P < 0.05). They included two unclassified species
PCoA (Fig. 1B), which hinted to a stronger effect of the com- belongingtothePleosporales(OTU00007,92%prevalence,1.1%
partment type. This can be explained by the fact that the PCoA relativeabundance)andtheCeratobasidiaceae(OTU00008,90%,
A B GrHRecNK GrHRecNPK GrHLau GARLau WILDGal.2013 WILDGal.2014
Mortierellales
Cantharellales
s Helotiales
t
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n
i Incertae sedis
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.
el
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Hypocreales
Fig.2. FungaltaxaconsistentlyfoundinA.alpinaroots(>85%prevalenceacrossallrootsamples).(A)Maximum-likelihoodphylogenetictreeofthehighly
conservedrootOTUs.TherepresentativeITS2sequencesfromtheOTUswerealignedusingMuscle(28)andusedfortreeinferenceinPhyML(29)withaGTR+
I+γmodelwithoptimizedparameters.Fungalordersaredepictedwithdifferentcolors;whitecirclesindicatetheaveragerelativeabundance(Rel.abu.)of
theOTUinrootsamples.Root(Ro)-orrhizosphere(Rz)-enrichedOTUsareindicated(comparisonrootvs.rhizosphererelativeabundance,pairedttest,P<
0.05).OTUswith100%prevalenceareshowninboldfacetype.(B)Relativeabundanceofthe15highlyconservedrootOTUsineachrootsample.Thedataare
giveninDatasetS4.
E9406 | www.pnas.org/cgi/doi/10.1073/pnas.1710455114 Almarioetal.
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6.5%);Alternariaembellisia(OTU00010,88%,2.8%);Dactylonectria A P
torresensis (OTU00002, 100%, 11%); and three Helotiales in-
cluding Tetracladium maxilliforme (OTU00004, 100%, 8.5%), a
Cadophora OTU (OTU00033, 98%, 3%), and an unclassified
OTU (OTU00005, 86%, 7%) (Fig. 2A). None of these highly
conservedrootOTUswasrelatedto(i)AM,ectomycorrhizal,or Y
R
orchidmycorrhizalfungiknowntofacilitateplantnutrientuptake Water H.K. F229 NTA
orto(ii)fungalendophytesS.indica(syn.P.indica)orC.tofiel- ME
dFenioacueo,rmwophfaitcshhseewsereicrOeoiTddUemsscyrbcibeoelrodrnhtgiozeadtlraftuonnstfgheiresPuHctheoloantsoianOlmeidsyicooodrdrerenhrdizrkoannlopmwlananitutsos. B C eight (mg) ********* SEECOM
w
butalsoplantpathogenssuchasRhynchosporiumsecalis.Threeof h
tahbeusnedfaonucreHeesploetciiaallelsyOunTdUerssnhaotuwreadlceonnridcihtimonesnt(Finigr.o2oBts).andhigh oot fres
h
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Helotiales Fungus F229 (OTU00005) Promotes A. alpina Growth and W) ******
Shoot P Accumulation. Helotiales OTU00005 exhibited a high D
relativeabundanceintherootsofwildA.alpinaplantsfromCol Kg
g/
du Galibier (45% in WILD-Gal.2013 and 23% in WILD- m
Gal.2014samples,SIAppendix,Fig.S5B),wherethehostplants ot P (
grew on an extremely low-P soil (soil Gal, 3.7 mg/kg plant- ho
S
available P) while maintaining high shoot P concentration (SI
Water H.K.F229
Appendix,Fig.S5A).WesubsequentlyidentifiedinourCologne
Culture Collection of Root-Associated Fungi (CORFU) seven D
isolates that were recovered from A. alpina collected at Col du
GalibierandbelongedtothisOTU(DatasetS1).Thefull-length
ITS sequences of the isolates shared 99–100% similarity, and
their ITS2 regions showed 99–100% similarity to the represen-
W
tativeITS2sequenceofOTU00005.Blastanalysisrevealedthat
D
the ITS sequences of these isolates werehighly similar toother
g
Helotiales root endophytes isolated from the Brassicaceae spe- /
q
ciesMicrothlaspiperfoliatumgrowinginthesouthofSpain(30), B
K
thusreflecting arecurrent presence ofthese Helotialesfungi in
P
rootsofBrassicaceae(SIAppendix,Fig.S3). 3
3
Toaddressthesignificanceoffungalrootcolonizationforplant
P uptake, the fungus with CORFU identifier F229 (hereafter
namedF229),belongingtoOTU00005,wasselectedforfurtherin
planta experiments in gnotobiotic Murashige and Skoog (MS)
agar systems. F229 promoted growth of A. alpina F1gal and PM
rootsunderlow-Pconditionsandleftplantsunaffectedinhigh-P GY
O
conditions (SI Appendix, Fig. S4). In contrast, another six fungal OL
isolatesnotbelongingtoOTU00005(CORFUF226,F248,F247, Fig.3. FungusF229(OTU00005)increasesA.alpinagrowthandPcontent BI
F91,F83, F222), screened indifferentexperiments, all exerted a undernativelow-PsoilconditionsandiscapableofhyphalPtransfertothe ANT
rootinvitro.(A)A.alpinaF1galgrowthinsterilesoilmicrocosmuponwater L
negativeeffectonplantrootand/orshootgrowthinatleastoneof P
addition(Water),additionofheat-killedfungus(H.K.),andinoculationwith
the P conditions (SI Appendix, Fig. S4). Fourteen days post- F229(F229)(1.32±0.8×104propagulespermicrocosm)at28dpi.(Scalebars,
inoculation (dpi) on its natural host A. alpina F1gal, F229
1cm.)(B)Inter-andintracellularfungalrootcolonizationinsterilesoilmi-
asymptomaticallycolonizedtheplantroots(SIAppendix,Fig.S6B crocosmsvisualizedbyconfocalmicroscopyafterstainingthefungalcellwall
and C) with equal colonization at low-P (100 μM P MS agar, withWGA-Alexa(green,a–d),theplantcellwallwithpropidiumiodide(red,
89.6%ofcolonizedroots)and high-P(1,000 μM,88.6%ofcolo- a–c),andthecellularmembraneswithFM4-64(purple,d).(Scalebars,30μm.)
nizedroots)conditions(χ2testP=0.87).However,underlow-P (C)EffectofF229inoculationonshootfreshweightandshootPconcentration
conditions, fungal inoculation significantly increased root length insterilesoilmicrocosms.Theexperimentwasrepeatedfourtimesincluding
(+12%, t test P = 0.02) and root surface area (+19%, t test P = the Water and F229 treatments and three times including also the H.K.
0.001) while leaving shoot biomass (t test P > 0.05) and shoot P treatment,withthreetofourmicrocosmspertreatment;similarresultswere
concentration(ttestP>0.05)unchanged(SIAppendix,Fig.S6A oShbotaoitnewde,igahntdwcoasmmpieleadsurreesdulotsnfirnodmividthuealfopularnetsxp(ner≥im5e6n)twshareeresahsoawllnohfetrhee.
andB).Aneutraleffectonrootgrowthwasapparentunderhigh-P shootsfromonemicrocosmwerepooledtomeasureshootPcontentbyICP-
conditions (1,000 μM P, 14 dpi) (SI Appendix, Fig. S6A). Simi- MS(n≥9).Asterisksindicatesignificantdifferencesbetweenthetreatments
larly, a second isolate (CORFU F240), also assigned to basedontheMann–Whitneytest(P<0.05).(D)Invitrotransferof33Por-
OTU00005 and exhibiting 100% ITS sequence similarity with thophosphatetotheplantbyF229.TheF229andA.alpinaF1galplantswere
F229, also promoted root growth of A. alpina F1gal under low-P grown onlow-P(100μM P)orhigh-P(1,000μM P)MS medium in a two-
conditions(SIAppendix,Fig.S6D). compartmentsystem.33PwasaddedtothefungalHC,andafter7(experi-
ment3),10(experiment2),or15(experiment1)days,33Pincorporationinto
WenextassessedtheeffectofF229onthegrowthofitsnatural
theplantshootgrowingintheRHCwasmeasuredbyscintillationcountingof
host A. alpina F1gal under native P-limited soil conditions (Fig.
individual plants.Nofunguswasaddedtothefungalcompartmentinthe
3A). At 28 dpi in gnotobiotic microcosms filled with autoclaved
mockinoculatedtreatments.Barsrepresentindividualsamples.
soilfromtheColduGalibier(soilGal),F229fullycolonizedplant
roots(100%ofcolonizedroots)inter-andintracellularly(Fig.3B,
a–d). Vital staining of plant and fungal membranes indicated vi- partners. While addition of heat-killed fungal suspension nega-
abilityofhostandfungalcellsduringintracellularaccommodation tively affected plant growth, fungal inoculation translatedinto
(Fig. 3B, d), indicating a biotrophic interaction between the 52%highershootbiomass(Mann–WhitneytestP=3.10−13)and
Almarioetal. PNAS | PublishedonlineOctober2,2017 | E9407
61%highershootPconcentration(Mann–WhitneytestP=2.10−4) TheEndophyticLifestyleofF229isAssociatedwiththeExpansionof
compared with the water control (Fig. 3C). The shoot P levels itsCAZymeRepertoire.We aimed at identifying genomic charac-
were stilllower than observed intheir wild-growing counterparts teristicsassociatedwiththeF229endophyticbiotrophiclifestyle.
(WILD-Galtreatments,SIAppendix,Fig.S5).Wecouldimagine A five-gene phylogenetic analysis on F229 and 50 other asco-
two possible reasons explaining this discrepancy: (i) The young mycetes with available genome information confirmed the
plantsinthemicrocosmsaccumulatedlessPintheirvacuoles,the placement of the fungus within the Leotiomycetes class and
primaryintracellularcompartmentsforinorganicphosphate,than theHelotialesorder(SIAppendix,Fig.S10).Theclassificationof
the fungus at the family or genus level was not possible as the
themucholderwildplantsor(ii)differencesinpropertiesofsoil
taxonomywithintheHelotialesorderisstillunclear.Theclosest
inmicrocosmsrelativetosoilatColduGalibierlimitedPuptake,
relatives(with sequencedgenome)ofF229areplantpathogens
whichwaspartiallyalleviatedthroughrootcolonizationwithF229.
Marssonina brunnea f. sp. multigermtubi and Rhynchosporium
In sum, these results corroborate the hypothesis of a beneficial
species R. secalis, R. commune, and R. agropyri, suggesting that
effect of this endophyte on host growth and P acquisition under
the endophytic lifestyle of F229 could have evolved from an
nativelow-Psoilconditions.
ancestralplantpathogeniclifestyle(Fig.4A).Similarly,theITS-
based phylogenetic analysis including more fungal isolates sug-
F229TranslocatesPtoItsHostinVitro.Fungi can promote plant P
gests that F229 belongs to a lineage of root endophytic fungi
acquisition by different mechanisms like P solubilization, P min-
that diverged from related pathogenic Rhynchosporium and
eralization, or hyphal P transfer. We wanted to know whether
Pyrenopeziza species (SI Appendix, Fig. S3), but more Helotiales
F229iscapableofhyphaltransportofradiolabeled33Ptoitshost.
genomesareneededtoproperlyassessthishypothesisusingmore
Usingatwo-compartmentagarosesystemlimitingradiotracerdif-
robustphylogeneticanalyses.
fusion, we observed that 33P added to the hyphal compartment
The Helotiales order encompasses fungi with contrasting life-
(HC) could be traced to the plant shoot in the root and hyphal
styles including plant beneficial fungi, plant pathogens, and sap-
compartment(RHC),withbothcompartmentsconnectedonlyby
rotrophs(Fig.4A).Weusedacomparativegenomicsapproachon
fungal hyphae crossing the physical barrier (Fig.3D). Fungal col-
CAZymerepertoirestoidentifygenomicsignaturesassociatedwith
onization was restricted to the root as the fungus was never de- abiotrophiclifestyleandplant-beneficialeffectswithinthisorder.
tectedintheplantshoot(stemorleaves,SIAppendix,Fig.S7).33P Comparison of CAZyme class profiles of 11 Helotiales fungi
translocationacrossthediffusionbarrierwasblockedbyBenomyl revealed large similarities between plant beneficial fungi that
(SIAppendix,Fig.S8),acompoundthat inhibitsmicrotubulefor- clusteredtogether(Fig.4B).TheclusterincludingF229,thepoplar
mation and intracellular transport in fungi (31), which indicated endophyte Phialocephala scopiformis, and the ericoid mycorrhiza
thathyphalPtransportwasanactivefungalprocessratherthanme- O.maiuswascharacterizedbyahighernumber(ttestP<0.01)of
diated by diffusion. Moreover, hyphal 33P translocation to the plant modulesofglycosidehydrolases(GH)(averagenumberof427in
shoot was detectable as early as 7 d after 33P addition and was plant-beneficial fungi, 349 in F229, and 256 in the other fungi),
independentfromlow-orhigh-Pconditions.Thissuggeststhatin carbohydrate-binding modules (CBM) (103, 109, and 77), carbo-
F229 33P transfer to the host is not regulated by P availability, hydrate esterases (CE) (194, 160, and 105), glycosyltransferases
whichstandsincontrasttowhatwasshownforC.tofieldiae(12). (GT)(128,122,and102),andauxiliaryactivities(AA)(137,135,
These data suggest that plant growth promotion by F229 under and 83), indicating an overall larger CAZyme repertoire in the
low-PsoilconditionsinvolveshyphaltransferofPintoitshost. genomes of plant beneficial Helotiales in comparison with plant
pathogenicandsaprotrophicHelotiales(DatasetS3).Comparison
TheF229GenomeEncodesTwoHigh-AffinityPhosphateTransporters. of CAZyme family profiles showed similar results (SI Appendix,
Cellular uptake of nutrients, maintenance of cellular nutrient Fig. S11). Twenty-two CAZyme families were significantly more
homeostasis,andiontransferacrosscellular(endo)membranesin abundant in plant beneficial fungi in comparison with the other
fungus–plant symbioses involves high- and low-affinity ion trans- fungi (t test P < 0.01) (Fig. 4C). Notably, this concerned families
porters. To obtain insight into the molecular mechanisms un- associatedwithplantcell-walldegradation,actingonhemicellulose
derlyingtransportofinorganicphosphateinF229,weperformed (GH31, GH29, CE1, CE7, CE10), or in the transformation of
genome-wide analysis to identify fungal phosphate transporters. lignocellulosic compounds (AA7). Three GHs associated with
PacBiosequencingoftheF229genomeproducedafinalassembly fungalcell-walldegradation(GH20,GH72,GH76)werealsomore
of39contigswithanestimatedgenomesizeof∼85Mb(Dataset abundantinplantbeneficialfungi.ComparisonofCAZymeclasses
(SI Appendix, Fig. S12A) and selected CAZyme families (SI Ap-
S2). The final genome version showed a high level of complete-
pendix, Fig. S12B) across 51 ascomycetes genomes showed no
ness with a high coverage of core fungal (98.7%, FUNYBASE
clustering corresponding to fungal lifestyle differences, indicating
geneset)andeukaryoticgenes(99.2%,ClusterofEssentialGenes
that the observations made within the Helotiales are lineage-
geneset).Weaimedtoidentifyhomologsofsixproteinsthatplay
specific. Collectively, these data suggest that the endophytic be-
aroleinphosphatetransportintheyeastSaccharomycescerevisiae.
haviorofF229isassociatedwiththeenlargementofitsCAZyme
One is the major proton-coupled high-affinity phosphate trans-
repertoire and particularly with protein families associated with
porterPho84,threetransportphosphatewithlowaffinityintocells
plantcell-walldegradation.
(Pho87, Pho90, Pho91), and the fifth and sixth proteins (Pho
88andPho89)areutilizedunderspecializedconditions(32).Two Discussion
genes intheF229 genome(g8711.t1 and g16086.t1)encodepro-
Lowavailabilityofphosphateisamajorfactorconstrainingplant
teins sharing high similarity with S. cerevisiae Pho84 and with
growth, performance, and metabolism in many natural and ag-
fungal high-affinity phosphate transporters involved in P trans- riculturalsoilsworldwideduetothepoorsolubilityandmobility
location from fungus to plant. One gene (g3490.t1) encodes a of soil P. The AM fungi have been shown to benefit plant pro-
protein sharing similarity with Pho87, Pho90, and Pho91, and a ductivityduetotheircontributiontoplantnutrition,especiallyin
last gene (g6261.t1) encodes a protein sharing similarity with nutrient-poorsoils(10).ThepredominantfunctionofAMfungi
+
Pho88. The gene encoding the Na -dependent high-affinity is attributed to increased host plant phosphate uptake as a
Pho89 could not be identified (SI Appendix, Fig. S9). In sum, consequence of a phosphate transport mechanism (7). Brassi-
these results show that the F229 genome encodes a set of phos- caceaespecieslacktheabilitytoestablishanAMsymbiosis,and,
phate transporters potentially enabling phosphate uptake and to fully comprehend how these plants thrive in P-limited habi-
translocationtoitshostplant. tats, it is required that we improve our understanding of their
E9408 | www.pnas.org/cgi/doi/10.1073/pnas.1710455114 Almarioetal.
S
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A P
Sordariomycetes
0.2
Botrytis cinerea
es Sclerotinia sclerotiorum
Leotiomycet HRPROMARGshehhhliaadcilyyyraoriosnnnoeltscocccdaiooachhhenrellooonoeyipsssdnznshppporae oasoooy bnslprrreaiiira uuu.n mu rsmmmFsnccai2ons oisac2uiepdeog9saie*cmrfoosamrplmisyurinsie Helotiales EECOMMENTARY
Blumeria graminis S
Xyl. Xylona heveae
Dot. CZylamdoosseppotroiurima tfruitlivcuim
Pez. Tuber melanosporum
B C
*
b
FuPPPPSUnlalllngaaaapknnnnarnttttol o ppbbtlwriaaeeofnettnnphheeshoofftgiigccyeeiilaannell---NEEnreicdcorooipdtrh omyptyehcorrhiza CAZyme familiesabundanceMax.Min. R. commune.56. MbR. secalis.49. MbR. agropyri.52. MbA. sarcoides.34. MbS. sclerotiorum.38. MM. brunnea.52. MbB. cinerea.43. MbG. lozoyensis.40. MbHel. sp. F229.85. MbP. scopiformis.49. MbO. maius.46. Mb
Lifestyle
48 50 50 42 42 36 54 54 71113123 CE10
24 21 21 11 13 14 14 26 33 37 34 CE1
* 14 12 14 7 22 21 26 40 39 41 47 AA7
CAZyme classesabundance Max.Min. S. sclerotiorum.38. MbM. brunnea.52. MbA. sarcoides.34. MbR. agropyri.52. MbR. secalis.49. MbR. commune.56. MbB. cinerea.43. MbG. lozoyensis.40. Mb O. maius.46. MbP. scopiformis.49. MbHel. sp. F229.85. Mb 111221001 111221001 011221001 111122001 100111001 000213001 010111101 220121001 332333101 323433112 356433212 CGGGCGCGCEEBBHHHTT71MM2212241329046736
Lifestyle 2 3 2 2 3 1 2 3 5 5 3 CBM13
2 3 2 4 5 1 5 3 5 8 9 GH92
229205261287269277250272 530404349 GH 13 14 14 10 12 12 10 13 16 17 15 GH76
80 74 73 82 78 75 67 84 10298109 CBM 7 7 7 4 6 4 5 5 13 11 13 GH31
90 99 97107108110101106 136126122 GT 7 7 7 9 8 6 10 6 10 11 13 GH47
75 79 44 89 79 86 86125 116160135 AA 7 6 7 7 4 5 7 6 7 15 15 GH78
93 85 83114111117107127 207214160 CE 6 6 7 4 5 5 5 6 8 7 8 GT90 Y
G
5 20 2 9 9 9 12 13 19 3 23 PL 7 7 7 7 6 7 6 8 8 9 8 GH72 O
L
6 6 6 11 6 7 5 8 10 14 23 GH109 O
8 9 8 11 5 8 6 7 9 15 19 GT32 BI
T
N
A
Fig.4. TheendophyticlifestyleofF229isassociatedwiththeexpansionofitsCAZymerepertoire.(A)Maximum-likelihoodphylogenetictreeinferredfromfive PL
housekeepinggenes(28S,18S,Rpb1,Rpb2,EF1alpha).Bootstrapvalues>0.75areindicatedwithablackdot.Laccariabicolorsequenceswereusedfortree
rooting.Helotialeswithplantbeneficial,plantpathogenic,orsaprophyticlifestylesareindicated;thekeyisgiveninB.ThefulltreeisshowninSIAppendix,Fig.
S10.(B)ComparativeanalysisofCAZymerepertoiresinthegenomeofF229andrelatedHelotialeswithplantbeneficial,plantpathogenic,orsaprophyticlife-
styles.HierarchicalclusteringontheabundanceofCAZymeclasseswithintheHelotiales.AA,auxiliaryactivities;CBM,carbohydrate-bindingmodule;CE,car-
bohydrateesterase;GH,glycosidehydrolase;GT,glycosyltransferase;PL,polysaccharidelyase.(C)HierarchicalclusteringontheabundanceofselectedCAZyme
familieswithintheHelotiales.Onlyfamiliesshowingasignificantlyhigherabundanceinplant-beneficialfungiareshown(ttests,P<0.01).InBandC,thecolor
scaledepictsstandardizedvaluesforeachmodule.Fungalgenomesizesareindicatedaftertheirname.F229isshowninboldfacetypewithanasterisk.
fungal microbiota. Here we close this gap by investigating havetheabilitytocrosstheselectionfiltersimposedbythehost.
structural and functional properties of the fungal microbiota Thishypothesisissupportedbypreviousstudiesonrootmicrobial
associated with the roots of the nonmycorrhizal Brassicaceae communities that have shown a similar diversity pattern in bac-
speciesA.alpina.WehavechosenA.alpinabecauseitnaturally terialassemblagesinA.alpina,A.thaliana,andrice(1,15,16,26)
grows in low-P habitats, and, in contrast to short-lived annuals and in fungal communities of Agave (20). Furthermore, the mi-
likethemodelspeciesA.thaliana,itsperennialpatternofgrowth crohabitat type also affected the structure of these fungal com-
and development gives longer time periods for microbial com- munities,i.e.,thetaxapresentandtheirrelativeabundances(Fig.
munitiestoestablishinandaroundthe roots. 1B). Overall, the fungal microbiome associated with roots (root
and rhizosphere) of nonmycorrhizal A. alpina was dominated by
VariabilityandStabilityoftheA.alpinaRootFungalMicrobiome.Our ascomycetes,aswasshownformycorrhizalpoplarandAgave(18,
study of the factors shaping root-associated fungal communities 20), and which is likely to be predetermined by the majoritarian
showed that fungal alpha diversity is determined mainly by the presence ofascomycetes insoil(33). Still,root endosphere com-
microhabitat type, i.e., bulk soil, rhizosphere, or root compart- munities systematically differed from rhizosphere communities
ment, dropping dramatically in the root (Fig. 1C). This obser- locatedmillimetersapart(Fig.1B);theywereenrichedinHelot-
vation further sustains the view that colonization of the root ialesandCantharellalesfungianddepletedinMortierellalesand
endosphereisrestrictedtoareducednumberoffungaltaxathat Sordariales(Fig.1D).Surprisingly,therewasnoclearsimilarityin
Almarioetal. PNAS | PublishedonlineOctober2,2017 | E9409
the pattern of enriched and depleted fungal orders in A. alpina indicating a plant growth-promoting effect of twoHelotiales iso-
and mycorrhizal Agave, poplar, and sugarcane roots (18–20), lates(F229andF240,OTU00005)invitro(SIAppendix,Fig.S6)
suggestinga low level of conservation offungal microbiome pat- in combination with the high relative abundance of the corre-
ternsacrossdistantlyrelatedplanthostspecies. sponding OTU inA.alpina plants growing under P-poornatural
Addingtothestrongmicrohabitateffect,thesoilgeographical conditions (SI Appendix, Fig. S5) suggests that this taxon could
provenance was the second largest driver of fungal community facilitate plant P uptake in its natural environment. Further
structure under controlled greenhouse conditions. Soil fungal studies are needed to investigate more A. alpina natural pop-
communitiesshowstrongbiogeographicalpatternsshapedbylocal ulations at different locations to define the biogeographic distri-
climatic and edaphic factors (33–35) and thus strongly diverge butionofthisbeneficialplant–fungusassociation.
from the “everything is everywhere” postulate suggested for mi-
crobial communities (36). The soil’s P-fertilization regime also EndophyticHelotialesFungusF229PromotesGrowthandPAcquisitionin
impacted the structure of fungal communities, albeit to a lesser A. alpina. While most root-associated microbes compete with the
extent than the soil geographical origin (Fig. 1B). This indicated plant and with each other for essential nutrients, some may have
thatlong-termPfertilizationoftheRecNPKsoil(37)shiftedsoil the potential to positively affect plant nutrition and growth. Hel-
fungalcommunities.Differencesinrootcommunitiescouldbethe otiales fungal isolate F229 belonging to OTU00005 was isolated
consequence of these changes, but we cannot exclude that they from A. alpina roots growing at the Col du Galibier natural site
could be linked to changes in the plant nutritional status associ- characterized by low-P availability in soil. The fungus exhibited
ated with fertilization. Such changes could lead to differences in biotrophic endophytic growth as it asymptomatically colonized
root-associatedcommunitiesbyalteringtherootexudationprofile plant roots inter- and intracellularly (Fig. 3B) rather than killing
and/ormorphology,similarlytotheAMsymbiosisthatisconfined root cells during the infection process, coinciding with the root
to conditions in which the plant is P-starved (7, 37). Even when enrichmentofthecorrespondingOTU(Fig.2A).F229wasableto
growing in the same soil, fungal communities described under translocatePtotheplantunderhigh-andlow-PconditionsonMS
controlledgreenhouseconditionsdifferedfromthoseestablished agar (Fig. 3D). However, no increase in shoot P content was ob-
under alpine summer conditions (Fig. 1B). This environment ef- servedunderthoseconditions(SIAppendix,Fig.S6).Oneplausible
fect increased from the bulk soil to the root compartment, sug- explanationisthatasobservedforAMsymbiosis,translocationofP
gesting that root-associated communities were more responsive
bythefungusdoesnotnecessarilytranslateintoincreasedPcontent
to environmental change than bulk soil communities, which
in the plant since the plant can tune-down the direct P uptake
remainedroughlyalike.Lownighttemperatures(prevalentunder
pathway and use the mycorrhizal pathway instead (41). When
alpine summer conditions) affect plant defense mechanisms as
reintroduced into its native low-P soil fungus, F229 successfully
showninArabidospsis(38),whichcoulddirectlyimpactendophytic
colonized plant roots and enhanced shoot growth and shoot P
fungalcommunities.
concentration (Fig. 3C) through an active process (Fig. 3C). Our
Our analysis including all of the plant growing conditions
results stay in accordance with a role of F229 in extending the
studied showed that at this wide scale it was no longer the mi-
potentialrangeofplantnutrientabsorptioninlow-Psoilsandpo-
crohabitattype,i.e.,thecompartmenttype,buttheplantgrowing
tentially also in P-rich habitats. In addition to increasing the ab-
condition that was the main factor shaping fungal communities.
sorptive surface area of the host plant root system, hyphal P
ThisisconsistentwithwhathasbeendescribedinAgave(20)and
translocation mediated through the activity of phosphate trans-
poplar(18),wheretheplantbiogeographywasthemajorsourceof
portersencodedinthefungalgenomewouldenableaccesstosoilP
variation in fungal and bacterial communities. Interestingly, root
sourcesotherwiseunavailabletotheplant(Fig.3DandSIAppen-
communities were less affected by the plant growing condition
dix,Fig.S9).Wecannotexclude,however,Pdeliveryfromfungus
than soil communities (rhizosphere and bulk soil) (Fig. 1B) as
tohostasaconsequenceoflysisoffungalcells,amechanismthat
observed inAgave (20). This pointed to the existence of a set of
was proposed for nutrient transfer in orchid mycorrhizae (42).
fungal taxa consistently colonizing A. alpina roots in contrasting
Overall, our work on F229 (P transfer) and studies on S. indica
growthconditions.Ourstudyrevealedahighlyconservedsetof15
(P transfer) (13, 14), C. tofieldiae (P transfer) (12), Heteroconium
OTUs that was dominated by ascomycetes (Fig. 2A) and repre-
chaetospira(Ntransfer),(43)andMetarhiziumrobertsii(Ntransfer)
sentedupto93%ofthefungalreadsinA.alpinaroots(Fig.2B).
(44) provide accumulating evidence that fungus-to-plant nutrient
SevenOTUsofthiscoremicrobiomeweresignificantlyenriched
transfer,generallyassignedtoclassicalmycorrhizalsymbioses(10),
inthe root endospherein comparisonwith the rhizosphere (Fig.
ismorecommonthanpreviouslythought.
2B), suggesting not only that these taxa were able to cross the
Plant-colonizing fungi rely on hydrolytic enzymes including
selectionbarrierimposedbytheroot,butalsothattheyreacheda
higher abundance within the root endosphere, implying some CAZymes for degradation of the plant cell wall and penetration
degreeofadaptationtothisniche.Althoughmostoftheidentified into thehosttissue (45, 46),and changes inthe CAZymereper-
OTUscouldnotbeclassifiedatthespecieslevel,wecouldidentify toires have been associated with lifestyle changes in plant-
the A. embellisia and D. torresensis species. Both Alternaria and associated fungi (21, 22). The evolution from pathogenic ances-
Dactylonectriageneraareknowntocontainahighnumberofplant tors toward the beneficial endophytic lifestyle of F229 was
pathogenic species albeit with no evidence of pathogenicity in accompaniedbytheenlargementofitsCAZymearsenal(Fig.4).
A. alpina. Interestingly, three closely related Helotiales OTUs This contrasts with observations of ectomycorrhizal fungi where
were identified as highly conserved, root-enriched, and highly the transition from a saprophytic to an endophytic lifestyle was
abundantinwild-growingplants(Fig.2A).WhileHelotialesfungi associatedwithareductionofthenumberofgenesencodingplant
represented24%oftheA.alpinarootmicrobiome,theywerenot cell-wall–degrading enzymes (21, 22). This discrepancy has been
commonly found in root microbiomes of the mycorrhizal hosts noted in other root endophytes including mycorrhizal fungi (21,
Agave (20), poplar (18), or sugar cane (19). However, they were 45,47).Oneexplanationisthatthearsenalofenzymespotentially
foundtodominatetherootmicrobiomeofmycorrhizalEricaceae involved in plant cell-wall degradation is a genomic indicator of
speciesgrowingundersimilarcoldandnutrient-limitedconditions saprophyticgrowthinplantdebrisinsoil,makingthesefungiless
as A. alpina (39), suggesting that this could be a specificity of dependentontheirhostforphotosyntheticallyderivedcarbon.
plantsgrowinginsuchharshenvironments.TheHelotialesorder Inconclusion,bystudyingthefungalmicrobiotaassociatedwith
is not well studied, and its phylogeny is still obscure. Although A.alpinaroots,wehaveuncoveredabeneficialHelotialesfungus
Helotiales fungi have often been isolated from plant roots, their capable of promoting plant growth and P uptake and thereby
ecological functions remain largely unknown (40). Our results potentiallyfacilitatingplantadaptationtolow-Penvironments.
E9410 | www.pnas.org/cgi/doi/10.1073/pnas.1710455114 Almarioetal.
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MaterialsandMethods experimentalconditions,amyceliumsuspensionwasusedforinoculation. P
Aftergrowingthefungusfor4wkonmaltyeastpeptoneagar,thefungal
Plant Growth and Sample Collection. We analyzed the fungal communities
myceliumwasrecoveredfromthesurfaceoftheagar,weighted,anddiluted
colonizingtherootsandrhizosphereof60A.alpinaplantsgrowing(i)under
to250mg/mLwithsterilewater,and∼30glassbeadspermilliliter(Ø1.7–
GrHinthreedifferentsoils(Lau,RecNK,andRecNPK;soilcharacteristicsare
2.1mm)(CarlRoth)wereaddedbeforegrindingtwiceat6,200×gfor10sin
given in SI Appendix, Table S2); (ii) under alpine summer conditions in a
aPrecellysinstrument(BertinTechnologies).Themyceliumwassubsequently
commongardenintheFrenchAlps(GAR);and(iii)atanaturalsiteinthe Y
washedtwicethroughadditionofninevolumesofwaterandcentrifugation R
FrenchAlps(WILD)over2y(2013and2014)(Fig.1A).FourA.alpinaaccessions A
(PM,E3, S2,and F1gal) originating fromdifferentEuropeanlocationswith at700×gfor2min.Thefinalpelletwasresuspendedinsterilewater,and ENT
themycelialconcentrationwasadjustedto10mg/mL.Fungus-treatedmi- M
different soil characteristics (SI Appendix, Table S1) were included in the M
commongardenexperimenttoassessthecontributionoftheplantgenotype corfomcoyscmelsiuwmerpeeirnopcout)la,theedatw-kitilhle1d0cmonLtorofltshriescineoivceudla1t0iomnLsuosfpethnissiosuns(p1e0n0smiong CO
tothestructuringoftheroot-associatedfungalcommunity.Rootandrhizo- afterautoclaving,andwatercontrolsreceived10mLofsterilewater.Plate SEE
spherecompartmentsfromfivetosixreplicatesperconditionwerecollected.
dilution series were made with the inoculum suspension, and colony
Therhizospherewassampledasthesoiltightlyadheringtoroots,androot
counting after 4 d indicated a level of inoculation of 1.32 ± 0.8 × 104
samples were enriched in endophytic fungi by detaching surface-adhering
propagulesperpot.Soilhumiditywasadjustedto70%ofthewater-holding
fungi through sonication (1). Three unplanted bulk-soil samples were in-
capacity without further watering. A. alpina F1gal seeds were surface-
cludedineachexperiment.ThedetailedprocedureisgiveninSIAppendix,
sterilized as described in SI Appendix, Materials and Methods, and strati-
MaterialsandMethods.Ineachtreatment,surface-sterilizedrootswereused
fiedfor1wkat4°Conmoiststerilefilterpaper,and10seedsweresub-
to recover fungal root endophytes deposited in CORFU (Dataset S1). The
sequently placed on the soil surface in each microcosm 2 d after fungal
methodisdescribedinSIAppendix,MaterialsandMethods.
inoculation. The microcosms were closed and placed in a phytochamber
(VersatileEnvironmentalTestChamber;Sanyo)with16-h/8-hday/nightcy-
ShootPMeasurementsbyInductivelyCoupledPlasmaMassSpectrometry.For
clesat22/18°Cand70%relativehumidity.Themicrocosmswererandom-
determinationofshootPconcentrationbyinductivelycoupledplasmamass
izedeveryotherday.After28d,plantswereharvestedindividually,shoot
spectrometry (ICP-MS), shoot samples were dried for 2 d at 65 °C before
weight was measured, and all of the shoots from one microcosm were
digestion.Formatureplants(i.e.,∼3moold;GrH,GAR,andWILDexperi-
pooledtomeasureshootPcontentbyICP-MSasdescribedabove.Oneroot
ments),samplesweredigestedusingamicrowavesystem(Multiwave3000;
systempermicrocosmwascollectedformicroscopyanalysisoffungalcolo-
AntonPaar).Approximately0.3gofdryhomogenizedplantmaterialwas
nization.MicroscopyanalyseswereconductedasdescribedinSIAppendix,
digestedusing4mLofHNO3(66%vol/vol)and2mLofH2O2(30%vol/vol). Materials and Methods. The experiment was repeated four times; three
The microwave program included a power ramp of 10 min followed by experiments included the “heat-killed”’ treatment, with three to five mi-
30minat1,400Wandafinal15minofcoolingdown.Finalsolutionswere
crocosms per treatment. Due to a reduced germination rate, on average
diluted1:5withdeionizedwaterbeforeanalysis.Foryoungplants(i.e.,1mo sevenplantspermicrocosmcouldbesampled(n=10–39pertreatmentper
old; MS agar and sterilized soil experiments) plant material was digested
experiment).Datanormalitywaschecked,andmeanswerecomparedwith
using500μLofHNO3(66%)at100°Cfor20min.Finalsolutionswerediluted the Mann–Whitney test (P < 0.05). The impact of fungal inoculation on
1:10withdeionizedwaterbeforeanalysis.Solutionblankswereincluded.
A.alpinagrowth was alsostudiedonMS agar; the detailed procedure is
ThePconcentrationwasdeterminedusinganAgilent7700ICP-MS(Agilent giveninSIAppendix,MaterialsandMethods.
Technologies)followingthemanufacturer’sinstructions.
33PTranslocationExperiments.Invitrohyphaltransferof33Porthophosphate
Fungal Microbiome Analysis. For fungal community description, DNA was totheplantbyF229wasstudiedasdescribedinref.12.Abicompartment
extractedfromeachcompartment,i.e.,root,rhizosphere,andbulksoil,and systemwasestablishedbyplacingtwosmallroundpetriplates(Ø3.8cm)
usedforfungalITS2PCRamplificationwithprimersITS9/ITS4(SIAppendix, constituting the HC into a square petri plate that was filled with low-P
TableS4).TaggedampliconsweresequencedusinganIlluminaMiseqplatform (100 μM P) or high-P (1,000 μM P) MS agar up to the rim of the small
producing 2 × 300 paired-end reads, and data analysis was conducted in plate,whichservedastheRHC.Rootswereprecludedfromgrowingintothe
Mothur(48).Thefinal3′388.918high-qualityfungalreadswereclusteredus- HCbyregularlymovingroottipsbeforerootingrowth,thusmaintainingthe
ingdenovo OTU pickingat97% sequence similarity.Afterdiscarding low physicalbarrierbetweenbothcompartments(Fig.3D).Four-week-oldfun- GY
abundance(<50reads)andnonfungalOTUs,2.966OTUswereobtained,and gal potato dextrose agar plates were used to inoculate the HC by trans- LO
O
eachOTUwastaxonomicallyclassifiedusingtheUNITEdatabaseinMothur. ferringa0.5-cm3agarplugcontainingfungalhyphae.A.alpinaF1galseeds BI
Onaverage,38.405finalfungalreadsand567OTUswereobtainedpersample weresurface-sterilized, stratified, andallowedto germinateonlow-PMS NT
(SIAppendix,TableS5).HighlyconservedrootOTUs(>85%prevalenceacross agar as described in SI Appendix, Materials and Methods. After 2 wk of LA
P
allrootsamples)aregiveninDatasetS4.ThedetailedprocedureisgiveninSI fungalgrowthinthebicompartmentsystem,two1-wk-oldA.alpinaF1gal
Appendix,MaterialsandMethods.Therawsequencingdatahavebeende- seedlingsweretransferredtotheRHC.NofunguswasaddedtotheHCin
posited at the National Centerfor Biotechnology Information (NCBI) Short themocktreatments.PlateswereclosedwithMicroporetape,placedver-
ReadArchiveunderBioprojectPRJNA386947. ticallyinaphytochamber(Sanyo,16h/8hday/nightcyclesat22/18°Cand
70%relativehumidity),andincubatedforanother2wk.Whenfungalhy-
StatisticalMethodsUsedforMicrobiomeStudies.Analyseswereconductedin phaehadcrossedthephysicalbarrierbetweenbothcompartments,350kBq
R3.2.3.TheOTUrelativeabundanceswerecalculatedandtransformedusing ofcarrier-free33P-labeledH3PO4(∼3pmol;HartmannAnalytik)wasadded
a log10 (x + 1) formula. Bray–Curtis dissimilarities between samples were to the HC. Plates were incubated horizontally in the phytochamber, and
calculatedusingthe“vegdist”functionoftheveganpackage(49)andused after7(experiment1),10(experiment2),or15d(experiment3),plantshoots
for principal coordinates analysis using the “dudi.pco” function of the weresampled,driedovernightat65°C,digestedwith500μL66%HNO3at
ADE4 package (50). Fungal alpha diversity was estimated in each sample 100°Cfor20minwithadditionof250μLH2O2,andheatedat100°Cfor1min.
using the Shannon diversity index (H) calculated in Mothur. Means were Thesolutionwasdiluted1:10,and500μLwasmixedwith4.5mLofscintilla-
comparedwithANOVAfollowedbyTukey’sHSD(P<0.05).PERMANOVAon tionmixture(Rotiszintecoplus;Roth)andusedfordetectionof33Psignals
Bray–Curtis dissimilarities was conducted to study the effect of different with a scintillation counter (Beckman Coulter LS 6500). Between 5 and
factorsonthestructureoffungalcommunitiesusingthe“Adonis”function 36plantswereanalyzedpertreatmentperexperiment.TheeffectofBenomyl
oftheveganpackage(atP<0.05).Aspreviouslyperformedinastudyon on fungal 33P translocation across the compartments was studied in low-P
metalbioaccumulationinplants(51),wecalculatedaP-accumulationfactor (100μMP)MSagarintheabsenceoftheplantbysamplinga1-cm2agar
(P concentration in the plant shoot divided by the plant-available P con- piecefromtheRHC7dafteradditionof33PtotheHC(SIAppendix,Fig.S8A).
centrationinthesoil).Plant-availablePinthesoil(SIAppendix,TableS2) ForBenomyl-treatedsamples,theHCwascoveredwith1mLofaBenomyl
was measured using the ammonium-acetate EDTA extraction method (InstituteofOrganicIndustrialChemistry,Warsaw)solutionof3μg/mL(wt/vol),
(AAE10)bytheLaboratoryforSoilAnalysis(Thun,Switzerland),andshootP andplateswereleftopentodryfor1hbeforeadditionof33PtotheHC.The
wasmeasuredbyICP-MSasindicatedabove. restoftheexperimentwasconductedasdescribedabove.Theexperimentwas
repeatedthreetimeswith5–20replicates.
EffectofF229InoculationonA.alpinaGrowthinSterile-SoilMicrocosms.For
sterile-soilmicrocosms,250gofsoilGal(lowplant-availableP:3.7mg/kg,SI Phylogenetic Analysis of F229 and Related Ascomycetes. The F229 genome
Appendix,TableS2)wasputinto500-mLglassjars(Weck)andautoclaved (NCBIBioProjectPRJNA378526)wassequencedandannotatedasdescribedin
twicewitha48-hinterval.SincefungusF229didnotsporulateunderour SIAppendix,MaterialsandMethods.Amultigenephylogeneticanalysiswas
Almarioetal. PNAS | PublishedonlineOctober2,2017 | E9411
conductedon51ascomycetegenomesincludingF229.Aphylogenetictree anddifferencesintheabundanceofparticularCAZymesbetweengroupswere
wasinferredfromfivehousekeepinggenes:28S,18S,Rpb1,Rpb2,andEF1α. assessedwithttests(P<0.01).
Foreachgene,thenucleotidesequencesretrievedfromthegenomeswere OtherexperimentalproceduresaredescribedinSIAppendix,Materials
aligned with MUSCLE (28), and informative positions were selected using andMethods.
Gblockswithrelaxedparameters(52).Alignmentswereconcatenatedand
used to compute a maximum-likelihood tree using PhyML (29) with the ACKNOWLEDGMENTS.WethankJanineAltmüllerandChristianBeckeratthe
GTR+I+γmodelandtheSH-aLRT method forbranch-support(1,000itera- Cologne Center for Genomics (https://portal.ccg.uni-koeln.de) for help with
tions)calculationusingSeaview(53). ampliconsequencing;theMaxPlanckGenomeCenterCologne(mpgc.mpipz.
mpg.de) for fungal genome sequencing; the Station Alpine Joseph Fourier
ComparativeAnalysisoftheAbundanceofCAZymesinF229andRelated (Jardin Botanique Alpin du Lautaret, https://www.jardinalpindulautaret.fr);
Stefan Wötzel for plant cultivation; René Flisch (Agroscope Reckenholz) for
Ascomycetes.TheabundanceofCAZymesintheF229genomewascom-
providingsoil;NinaGerlach,SabineMetzger,StefanieJunkerman,andtheCo-
paredwiththatof51otherascomycetesgenomeswithplant-beneficial,plant-
logneBiocenterMassSpectrometryPlatformforICP-MSanalysis;andE.Kemen
pathogenic, saprophytic, and plant unrelated lifestyles. In each genome,
andA.Rondeletforvaluablediscussionsandcommentsonthemanuscript.This
CAZymemoduleswereidentifiedasdescribedinSIAppendix,Materialsand workwassupportedbyfundsfromtheClusterofExcellenceonPlantSciences
Methods,andtheirabundanceswerecompared(DatasetS3).CAZymeprofiles (J.A.,M.B.,G.J.,A.Z.,andG.C.);thePlantFellowsProgramme(FP7MarieCurie
werecomparedbyhierarchicalclustering(Euclideandistanceswithaverage Actions, www.plantfellows.ch/; Grant GA-2010-267243 to J.A.); and the
linkagemethod)inMorpheus(https://software.broadinstitute.org/morpheus/), DFGPriorityProgram1529Adaptomics(J.W.).
1. BulgarelliD,etal.(2012)RevealingstructureandassemblycuesforArabidopsisroot- 29.Guindon S, et al. (2010) New algorithms and methods to estimate maximum-
inhabitingbacterialmicrobiota.Nature488:91–95. likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst Biol 59:
2. BerendsenRL,PieterseCMJ,BakkerPAHM(2012)Therhizospheremicrobiomeand 307–321.
planthealth.TrendsPlantSci17:478–486. 30.GlynouK,etal.(2016)Thelocalenvironmentdeterminestheassemblyofrooten-
3. Porras-AlfaroA,BaymanP(2011)Hiddenfungi,emergentproperties:Endophytes dophyticfungiatacontinentalscale.EnvironMicrobiol18:2418–2434.
andmicrobiomes.AnnuRevPhytopathol49:291–315. 31.LarsenJ,ThingstrupI,JakobsenI,RosendahlS(1996)Benomylinhibitsphosphorus
4. VandenkoornhuyseP,QuaiserA,DuhamelM,LeVanA,DufresneA(2015)Theim- transportbutnotfungalalkalinephosphataseactivityinaGlomus–cucumbersym-
portanceofthemicrobiomeoftheplantholobiont.NewPhytol206:1196–1206. biosis.NewPhytol132:127–133.
5. FieldKJ,PresselS,DuckettJG,RimingtonWR,BidartondoMI(2015)Symbioticoptions 32.PerssonBL,etal.(1998)PhosphatepermeasesofSaccharomycescerevisiae.Biochim
fortheconquestofland.TrendsEcolEvol30:477–486. BiophysActa1365:23–30.
6. SmithSE,ReadDJ(2010)MycorrhizalSymbiosis(Academic,London). 33.TedersooL,etal.(2014)Fungalbiogeography.Globaldiversityandgeographyofsoil
7. BucherM(2007)Functionalbiologyofplantphosphateuptakeatrootandmycor- fungi.Science346:1256688.
rhizainterfaces.NewPhytol173:11–26. 34.Talbot JM, et al. (2014) Endemism and functional convergence across the North
8. DelauxP-M,etal.(2014)Comparativephylogenomicsuncoverstheimpactofsym- Americansoilmycobiome.ProcNatlAcadSciUSA111:6341–6346.
bioticassociationsonhostgenomeevolution.PLoSGenet10:e1004487. 35.PeayKG,KennedyPG,TalbotJM(2016)DimensionsofbiodiversityintheEarthmy-
9. LambersH,etal.(2015)PhosphorusnutritioninProteaceaeandbeyond.NatPlants1: cobiome.NatRevMicrobiol14:434–447.
15109. 36.PeayKG,BidartondoMI,ArnoldAE(2010)Noteveryfungusiseverywhere:Scalingto
10.vanderHeijdenMGA,MartinFM,SelosseM-A,SandersIR(2015)Mycorrhizalecology thebiogeographyoffungal-plantinteractionsacrossroots,shootsandecosystems.
andevolution:Thepast,thepresent,andthefuture.NewPhytol205:1406–1423. NewPhytol185:878–882.
11.RichardsonAE,SimpsonRJ(2011)Soilmicroorganismsmediatingphosphorusavail- 37.WillmannM,etal.(2013)Mycorrhizalphosphateuptakepathwayinmaize:Vitalfor
abilityupdateonmicrobialphosphorus.PlantPhysiol156:989–996. growthandcobdevelopmentonnutrientpooragriculturalandgreenhousesoils.
12.HirumaK,etal.(2016)Rootendophytecolletotrichumtofieldiaeconfersplantfitness FrontPlantSci4:533.
benefitsthatarephosphatestatusdependent.Cell165:464–474. 38.AlcázarR,ParkerJE(2011)Theimpactoftemperatureonbalancingimmunere-
13.Bakshi M, et al. (2015) WRKY6 restricts Piriformospora indica-stimulated and sponsivenessandgrowthinArabidopsis.TrendsPlantSci16:666–675.
39.TojuH,TanabeAS,IshiiHS(2016)Ericaceousplant-fungusnetworkinaharshalpine-
phosphate-inducedrootdevelopmentinArabidopsis.BMCPlantBiol15:305.
subalpineenvironment.MolEcol25:3242–3257.
14.YadavV,etal.(2010)Aphosphatetransporterfromtherootendophyticfungus
40.TedersooL,etal.(2009)Ascomycetesassociatedwithectomycorrhizas:Molecular
Piriformosporaindicaplaysaroleinphosphatetransporttothehostplant.JBiol
Chem285:26532–26544. diversityandecologywithparticularreferencetotheHelotiales.EnvironMicrobiol
11:3166–3178.
15.LundbergDS,etal.(2012)DefiningthecoreArabidopsisthalianarootmicrobiome.
Nature488:86–90. 41.sSumpipthlyStEo,pSlmanitthsiFrrAe,spJaekctoibveseonfIg(r2o0w0t3h)rMesypcoonrrsheisz.aPllafunntgPihycasinold1o3m3:i1n6a–t2e0p.hosphate
16.DombrowskiN,etal.(2017)RootmicrobiotadynamicsofperennialArabisalpinaare
42.BougoureJ,etal.(2014)High-resolutionsecondaryionmassspectrometryanalysisof
dependentonsoilresidencetimebutindependentoffloweringtime.ISMEJ11:
carbondynamicsinmycorrhizasformedbyanobligatelymyco-heterotrophicorchid:
43–55. RhizanthellananoSIMSanalysis.PlantCellEnviron37:1223–1230.
17.WagnerMR,etal.(2016)Hostgenotypeandageshapetheleafandrootmicro-
43.UsukiF,NarisawaK(2007)Amutualisticsymbiosisbetweenadarkseptateendophytic
biomesofawildperennialplant.NatCommun7:12151.
fungus,Heteroconiumchaetospira,andanonmycorrhizalplant,Chinesecabbage.
18.ShakyaM,etal.(2013)Amultifactoranalysisoffungalandbacterialcommunity Mycologia99:175–184.
structureintherootmicrobiomeofmaturePopulusdeltoidestrees.PLoSOne8:
44.BehieSW,ZeliskoPM,BidochkaMJ(2012)Endophyticinsect-parasiticfungitrans-
e76382. locatenitrogendirectlyfrominsectstoplants.Science336:1576–1577.
19.deSouzaRSC,etal.(2016)Unlockingthebacterialandfungalcommunitiesassem- 45.LahrmannU,etal.(2015)Mutualisticrootendophytismisnotassociatedwiththe
blagesofsugarcanemicrobiome.SciRep6:28774. reductionofsaprotrophictraitsandrequiresanoncompromisedplantinnateim-
20.Coleman-DerrD,etal.(2016)Plantcompartmentandbiogeographyaffectmicro- munity.NewPhytol207:841–857.
biomecompositionincultivatedandnativeAgavespecies.NewPhytol209:798–811. 46.KubicekCP,StarrTL,GlassNL(2014)Plantcellwall-degradingenzymesandtheir
21.KohlerA,etal.;MycorrhizalGenomicsInitiativeConsortium(2015)Convergentlosses secretioninplant-pathogenicfungi.AnnuRevPhytopathol52:427–451.
ofdecaymechanismsandrapidturnoverofsymbiosisgenesinmycorrhizalmutualists. 47.HacquardS,etal.(2016)Survivaltrade-offsinplantrootsduringcolonizationby
NatGenet47:410–415. closelyrelatedbeneficialandpathogenicfungi.NatCommun7:13072.
22.PeterM,etal.(2016)Ectomycorrhizalecologyisimprintedinthegenomeofthe 48.SchlossPD,etal.(2009)Introducingmothur:Open-source,platform-independent,
dominantsymbioticfungusCenococcumgeophilum.NatCommun7:12662. community-supportedsoftwarefordescribingandcomparingmicrobialcommuni-
23.FeselPH,ZuccaroA(2016)Dissectingendophyticlifestylealongtheparasitism/ ties.ApplEnvironMicrobiol75:7537–7541.
mutualismcontinuuminArabidopsis.CurrOpinMicrobiol32:103–112. 49.OksanenJ,BlanchetG,KindtR(2016)Vegan:CommunityEcologyPackage.Available
24.TorängP,etal.(2015)Large-scaleadaptivedifferentiationinthealpineperennial atcran.r-project.org/package=vegan.
herbArabisalpina.NewPhytol206:459–470. 50.DrayS,DufourA-B(2007)Theade4package:Implementingthedualitydiagramfor
25.WillingE-M,et al.(2015) GenomeexpansionofArabisalpinalinkedwithretro- ecologists.JStatSoftw22:1–20.
transpositionandreducedsymmetricDNAmethylation.NatPlants1:14023. 51.vanderEntA,BakerAJM,ReevesRD,PollardAJ,SchatH(2013)Hyperaccumulatorsof
26.EdwardsJ,etal.(2015)Structure,variation,andassemblyoftheroot-associated metalandmetalloidtraceelements:Factsandfiction.PlantSoil362:319–334.
microbiomesofrice.ProcNatlAcadSciUSA112:E911–E920. 52.CastresanaJ(2000)Selectionofconservedblocksfrommultiplealignmentsfortheir
27.PeifferJA,etal.(2013)Diversityandheritabilityofthemaizerhizospheremicrobiome useinphylogeneticanalysis.MolBiolEvol17:540–552.
underfieldconditions.ProcNatlAcadSciUSA110:6548–6553. 53.GouyM,GuindonS,GascuelO(2010)SeaViewversion4:Amultiplatformgraphical
28.EdgarRC(2004)MUSCLE:Multiplesequencealignmentwithhighaccuracyandhigh userinterfaceforsequencealignmentandphylogenetictreebuilding.MolBiolEvol
throughput.NucleicAcidsRes32:1792–1797. 27:221–224.
E9412 | www.pnas.org/cgi/doi/10.1073/pnas.1710455114 Almarioetal.