Comparative Analysis of Prokaryotic Communities Associated with Organic and Conventional Farming Systems

RESEARCH ARTICLE Compa a i e Analysis o P oka yo ic Communi ies Associa ed wi h O ganic and Con en ional Fa ming Sys ems Eliza e a Pe shina 1,2 *, Ja i Valkonen 3 , Päi i Ku ki 4 , Eka e ina I ano a 1,5 , E geny Chi ak 1 , Ilia Ko igo 1 , Nykolay P o o o 1 , E geny And ono 1,2 1Labo a o y o mic obiological moni o ing and bio emedia ion o soils, All-Russia Resea ch Ins i u e o Ag icul u al Mic obiology, Sain -Pe e sbu g, Russia, 2Sain -Pe e sbu g S a e Uni e si y, Sain -Pe e sbu g, Russia, 3Depa men o Ag icul u al Sciences, Uni e si y o Helsinki, Helsinki, Finland, 4Na u al Resou ces Ins i u e Finland, Mikkeli, Finland, 5Labo a o y o biology and biochemis y o soils, V.V. Dokuchae Soil Science Ins i u e, Moscow, Russia *pe shina.eliza e a@yandex. u Abs ac One o he mos impo an challenges in ag icul u e is o de e mine he e ec i eness and en i onmen al impac o ce ain a ming p ac ices. The aim o p esen s udy was o de e - mine and compa e he axonomic composi ion o he mic obiomes es ablished in soil ollow- ing long- e m exposu e (14 yea s) o a con en ional and o ganic a ming sys ems (CFS and OFS acco dingly). Soil om uncla ed o es nex o he ields was used as a con ol. The analysis was based on RT-PCR and py osequencing o 16S RNA genes o bac e ia and a chaea. The numbe o bac e ia was signi ican ly lowe in CFS han in OFS and woodland. The highes amoun o a chaea was de ec ed in woodland, whe eas he amoun s in CFS and OFS we e lowe and simila . The mos common phyla in he soil mic obial communi ies analyzed we e P o eobac e ia (57.9%), Acidobac e ia (16.1%), Ac inobac e ia (7.9%), Ve - ucomic obia (2.0%), Bac e oide es (2.7%) and Fi micu es (4.8%). Woodland soil di e ed om c oplands in he axonomic composi ion o mic obial phyla. C oplands we e en iched wi h P o eobac e ia (mainly he genus Pseudomonas), while Acidobac e ia we e de ec ed almos exclusi ely in woodland soil. The mos p onounced di e ences be ween he CFS and OFS mic obiomes we e ound wi hin he genus Pseudomonas, which signi ican ly (p<0,05) inc eased i s numbe in CFS soil compa ed o OFS. O he di e ences in mic o- biomes o c opping sys ems conce ned mino axa. A highe ela i e abundance o bac e ia belonging o he amilies Oxalobac e iaceae,Ko ibac e iaceae,Nakamu ellaceae and gen- e a Rals onia,Paenibacillus and Pedobac e was ound in CFS as compa ed wi h OFS. On he o he hand, mic obiomes o OFS we e en iched wi h p o eobac e ia o he amily Comamonadaceae (gene a Hylemonella) and Hyphomic obiaceae, ac inobac e ia om he amily Mic ococcaceae, and bac e ia o he gene a Geobac e ,Me hylo ene a,Rhizobium (mainly Rhizobium leguminosa um) and Clos idium. Thus, he ields unde OFS and CFS did no di e g ea ly o he composi ion o he mic obiome. These esul s, which we e also PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 1/16 OPEN ACCESS Ci a ion: Pe shina E, Valkonen J, Ku ki P, I ano a E, Chi ak E, Ko igo I, e al. (2015) Compa a i e Analysis o P oka yo ic Communi ies Associa ed wi h O ganic and Con en ional Fa ming Sys ems. PLoS ONE 10(12): e0145072. doi:10.1371/jou nal. pone.0145072 Edi o : A. Ma k Ibekwe, U. S. Salini y Lab, UNITED STATES Recei ed: July 25, 2015 Accep ed: No embe 28, 2015 Published: Decembe 18, 2015 Copy igh : © 2015 Pe shina e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion ile. Funding: Financial suppo o he Ag o- Bio echnology ocused on Roo -Mic obe Sys ems Bal ic Sea egion ne wo k (AB-RMS) was ecei ed om No den/No dFo sk (Au ho s: Ja i Valkonen, Päi i Ku ki):h p://www.no d o sk.o g/en. Financial suppo o bioin o ma ics da a analysis was ob ained om Russian Science Founda ion (RSF) g an 14-26- 00094: "Analysis o he gene ic and e olu iona y po en ial o he soil mic obiome o imp o e plan p oduc i i y and soil e ili y" (Au ho s: E geny Chi ak, con i med by clus e analysis, indica ed ha mic obial communi ies in he ield soil do no necessa ily di e la gely be ween con en ional and o ganic a ming sys ems. In oduc ion Soil mic oo ganisms can se e as bioindica o s o an h opogenic s ess expe ienced by he soil du ing ag icul u al use [1]. Fo a long pe iod o ime, biologically aluable soil mic oo ganisms ha e been s udied by isola ion and cul i a ion in labo a o y [2]. The nex -gene a ion sequenc- ing echnologies ha e in ensi ied explo a ion o soil mic obial di e si y and allowed o iden i y biological indica o s, no only among he mic obes ha can be cul u ed in i o, bu also among he bac e ia and a chaea which canno be cul u ed [3]. One o he mos impo an challenges o mode n ag icul u e is o de e mine he e ec i e- ness and en i onmen al impac o sys ems based on o ganic o con en ional a ming (OFS and CFS espec i ely). O ganic a ming is conside ed ecologically iendly and o ha e less damag- ing e ec s on he ecosys em, whe eas con en ional ag icul u e is hough o cause signi ican changes in biocenoses due o he in ensi e inpu s o syn he ic e ilize s [4,5,6]. P oduc i i y in CFS is gene ally highe han in OFS, bu he nega i e impac on he en i onmen associa ed wi h he use o a pa icula ype o a ming sys em is deba ed [7,8,9]. The DOK (sho o he Ge man wo ds dynamic, o ganic and con en ional, espec i ely) expe imen is one o he mos comp ehensi e s udies on he long- e m e ec s o di e se ag i- cul u al echniques on he ecosys em [10]. S udying he soil mic obial di e si y by py osequen- cing and analysis o he axonomic ma ke s o bac e ia and ungi Ha mann e al. (2014) ound ha o ganic e ilize amendmen s had a posi i e e ec on he composi ion o mic obial communi ies and on he α-di e si y pa ame e s. O ganic ma e inpu s inc eased he ichness and dec eased e enness indices [10]. This e ec has been ound also in o he s udies [11,12]. On he o he hand, signi ican inc ease in ichness can lead o posi i e o neu al e ec s on e enness in sys ems wi h o ganic e ilize amendmen [13,14,15]. The luc ua ions in α-di e - si y pa ame e s a e o en explained by p edominance o he copio ophic mic oo ganisms, whose g ow h is s imula ed by o ganic e ilize s [10,16]. Bu his s a emen is equi able only in he sho - ime expe imen s, pa icula ly i was shown ha copio ophic bac e ia a e empo a - ily s imula ed by he addi ion o o ganic e ilize s o soil. In he long- un, unde s able condi- ions, he a io o oligo ophic o copio ophic bac e ia may be g ea e in OFS han in CFS [17]. Indeed, axonomic analyses indica e a he phylum le el ha P o eobac e ia and Fi micu es end o domina e in he o ganic a ming sys ems, while Ac inobac e ia, and o a lesse ex en Acidobac e ia, p edomina e in con en ionally managed c oplands and na u al en i onmen s [18,19]. High abundance o he plan g ow h-p omo ing bac e ia (PGPB), mainly he gene a Rhizobium,B ady hizobium,Meso hizobium,Bu kholde ia,S eno ophomonas,Pseudomonas, Sphingomonas and Rhodoplanes, has been documen ed among he p o eobac e ia in OFS [13,16,18,2]. Fi micu es in c oplands a e ep esen ed by bac e ia capable o deg ading a ious complex o ganic ma e ials and include, e.g., he gene a Bacillus,Clos idium,Epulopiscium, Paenibacillus and Solibacillus [10]. These da a ob ained by he mode n molecula echniques a e pa ially consis en wi h he da a ob ained using bac e ial cul i a ion echniques [20]. Mic obial dynamics associa ed wi h ce ain land-use p ac ices mus be conside ed oge he wi h he spa ial and empo al a ia ions in mic obial composi ion, occu ing in soil as a esul o plan g ow h and seasonal changes. Spa ial luc ua ions in soil mic obial communi ies de i e om he unequal dis ibu ion o he o ganic compounds wi hin indi idual soil agg ega es o Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 2/16 Ilia Ko igo, Eliza e a Pe shina, E geny And ono ): h p://g an . sc . u/ o ms? id=2W -G10J V8- 3i8XBU2cLb00. Discussion chap e was p epa ed oge he wi h he esea ch scien is s pa icipa ing in Russian Science Founda ion (RSF) g an 14-26- 00079: "Bio-physical and chemical diagnosis o soil o ganic ma e quali y o he de elopmen o scien i ic and heo e ical basis o Ag obio echnology" (Au ho : Eka e ina I ano a, h p://g an . sc . u/ o ms? id=1IgCW10Jq DX3i8WD72cLb00.) The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . ho izons [21,22,23] and on he di e en dis ances om he plan oo s [24,25]. As i was shown by an Diepeningen and co-wo ke s he composi ion o soil mic obial communi y oscil- la ed, depending on he dis ance emaining om he oo . These wa elike pa e ns we e de ec ed bo h o oligo ophic and copio ophic bac e ia bo h in OFS and CFS soils bu we e signi ican ly s onge in con en ional c oplands [24]. One o he main ad an ages o employing he py osequencing echniques in biodi e si y s udies is he imp o emen o knowledge abou he impac o ag icul u e on uncul u able mic oo ganisms. The mos p onounced e ec on soil mic obiome e ealed in he DOK expe i- men was he impac o o ganic e ilize s o he abundance o Acidobac e ia in soil. In i o cul i a ion me hods o his bac e ial phylum a e lacking o mos o i s membe s, excep in a e a emp s o de ine he ole o hese bac e ia in he ag icul u al sys ems managed wi h o ganic e ilize s amendmen s [26]. Among acidobac e ia, gene a Cand.Solibac e and Cand. Ko ibac e ha e been ound exclusi ely associa e wi h CFS, whe eas Chlo acidobac e ia and he RB25 g oup ha e been ound associa ed wi h OFS in p e ious s udies [10]. The aim o his s udy was o compa e long- e m impac s o OFS and CFS on mic obial di e si y in soil. In he expe imen al s a ion Ka ila (Mikkeli, Finland) whe e OFS and CFS ha e been ca ied ou in adjacen ields o 14 yea s. The aim was o compa e he axonomic s uc- u e o mic obiomes in OFS and CFS and o iden i y mic obes speci ically inhabi ing hese ecosys ems. Ma e ials and Me hods Soil sampling Field expe imen s we e ca ied ou unde pe mission o he Na u al Resou ces Ins i u e Fin- land ( o me ly MTT Ag iFood Resea ch Finland). The ield s udies did no in ol e endange ed o p o ec ed species. Sampling was done a once om CFS and OFS ields in he expe imen al s a ion Ka ila (Mikkeli, Finland) du ing he season o ac i e plan g ow h in July 2011. The ields had been clea ed om pine-sp uce o es in he beginning o 20 h cen u y and he e o e soil samples we e collec ed om he pine-sp uce o es nex o he ields included o compa ison (Table 1). The soil ype was a coa se ine sand in bo h sampling ields. Acco ding o US soil axonomy soil was sandy Aquic Haploc yod. Soil samples we e aken om he op soil laye (10 cm) using soil d ill (Ø 1 cm). A each sampling si e h ee ci cles (Ø 1 m) we e ma ked and 10 soil subsamples we e aken om inside each ci cle and combined. Hence, h ee samples ( eplica es) we e ob ained o analysis om each ype o soil (woodland, CFS and OFS). The dis ance be ween he sampling si es was 45 m in a e age. All samples we e immedia ely anspo ed o he labo a o y and Table 1. Summa y o he cul i a ion his o y o he ields sampled in he expe imen al s a ion “Ka ila” (Mikkeli, Finland). Fo de ails, see S1 Table. Yea CFS OFS 1928 The o es was cu down 1997–2010 Applica ion o he CFS, egula inpu o mine al e ilize s Applica ion o he OFS, egula o ganic e iliza ion wi h cow slu y and g een manu e 1997–2006 Sowing o sp ing ce eals, black cu an (in one pa o he field) C op o a ion in 4 s eps (1997–2010): 1)sp ing ce eal wi h ley 2) 3 yea s o clo e -g ass ley 3) sp ing ce eal 4) e ch-oa s 2007 Ba e allow, glyphosa e was used 2008–2010 Ley wi h oa s doi:10.1371/jou nal.pone.0145072. 001 Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 3/16 s o ed a -70°C. Coo dina es o he sampling si es we e he ollowing: woodland soil sample 1 (N61°40'32.46", E27°13'53.70"), 2 (N61°40'32.04", E27°13'55.80") and 3 (N61°40'31.86", E27° 13'57.54"); OFS soil sample 1 (N61°40'29.64", E27°13'40.44"), 2 (N61°40'30.00", E27°13'44.40") and 3 (N61°40'30.42", E27°13'48.96"); and CFS soil sample 1 (N61°40'38.22", E27°13'50.04"), 2 (N61°40'37.50", E27°13'51.24") and 3 (N61°40'36.30", E27°13'53.16"). The cul i a ion his o y o he ields in Ka ila is p esen ed in Table 1. De ails o he cul i a- ion p ac ices du ing he las h ee g owing seasons p io o sampling a e p o ided in S1 Table. A he ime o sample collec ion imo hy g ass (Phleum p a ense) and meadow escue (Fes uca p a ensis) we e g own as a mix u e in bo h sampled ields (OFS and CFS). Besides analysis o he mic obiome, he soil samples om OFS, CFS and woodland we e subjec ed o ag ochemical analyses (Table 2). DNA ex ac ion DNA was ex ac ed om 0.2 g o soil using Powe Soil DNA Isola ion Ki (Mobio Labo a o ies, Solana Beach, CA, USA), which included a bead-bea ing s ep, acco ding o he manu ac u e ’s speci ica ions. Homogeniza ion o he samples was pe o med using Fa sP ep (MP Biomedi- cals, San a Ana, CA, USA). The pu i y and quan i y o DNA we e es ed by elec opho esis in 0.5× TAE bu e on 1% aga ose. DNA concen a ions we e measu ed a 260 nm using SPECTROS a Nano (BMG LABTECH, O enbe g, Ge many). The a e age DNA yield was 2–5μg DNA wi h he concen a ion o 10–50 ng/μl. Quan i a i e PCR analyses Rela i e abundances o bac e ial and ungal small subuni RNA gene copies we e analyzed by quan i a i e PCR (qPCR) ( eac ion olume 25 μl) using iQ™SYBR G een Supe mix (BIO RAD, He cules, USA) and 10 ng o sample DNA. Fo bac e ia, he o wa d p ime Eub338 and e e se p ime Eub518 we e used [27]. The o wa d p ime a c915 and he e e se p ime a c1059 we e used o a chaea [28]. To es ima e bac e ial and a chaeal small-subuni RNA gene abundances, s anda d cu es we e gene a ed using a 10- old se ial dilu ion o a plasmid con aining a ull-leng h copy o 16S RNA gene belonging ei he o he Esche ichia coli o FG-07 s ain o Halobac e ium salina um (cou esy o G. Ju gens, Uni e si y o Helsinki). All qPCR eac ions we e un in iplica e. The eac ion was ca ied ou in iCycle (BIO RAD, He cules, USA) using he ollowing: 94°C o 15 min, ollowed by 40 cycles o 94°C o 30 s, 50°C o 30 s and 72°C o 30 s. Mel ing cu e analyses we e done o e i y ha he ampli ied p oduc s we e o he expec ed size. Fungal and bac e ial gene copy numbe s we e Table 2. Ag ochemical p ope ies o soil samples. CFS OFS WOOD Ca (mg/kg) 1247,33±129,90 1087,00±86,31 242,33±23,13 P (mg/kg) 14,70±1,79 10,50±1,22 3,33±0,26 K (mg/kg) 135,73±17,25 69,76±8,92 72,70±9,67 Mg (mg/kg) 146,67±28,18 117,33±11,21 45,13±5,07 pH 6,67±0,09 5,77±0,07 4,43±0,03 Conduc i i y 0,83±0,07 0,63±0,15 0,37±0,03 To al N 0,27±0,02 0,34±0,03 0,21±0,01 To al C 4,80±0,05 5,49±0,09 5,63±0,12 CFS–con en ional a ming sys em, OFS–o ganic a ming sys em, WOOD–woodland. doi:10.1371/jou nal.pone.0145072. 002 Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 4/16 es ima ed using a eg ession equa ion o each assay ela ing he cycle h eshold (C ) alue o he known numbe o copies in he s anda ds. S a is ical analysis o he qPCR da a was ca ied ou using one-way ANOVA in STATISTICA10 En e p ise (www.s a so .com). S a is ical signi icance was es ed by Fische ’s leas signi ican di e ence (LSD) and Bon e oni adjus ed p- alues. Ba -coded py osequencing o bac e ial and a chaeal communi ies The pu i ied DNA empla es we e ampli ied wi h uni e sal mul iplex p ime s F515 5’- GTGCCAGCMGCCGCGGTAA-3’and R806 5’-GGACTACVSGGGTATCTAAT-3’[29] a - ge ing he a iable egion V4 o bac e ial and a chaeal 16S RNA genes. Each mul iplex p ime con ained he adap e , 4-bp key (TCAG), 10-bp ba code and p ime sequences. The expec ed leng h o he ampli ica ion p oduc was 400 bp. Pu i ica ion, pooling and py osequencing o he amplicons we e pe o med wi h eagen s acco ding o manu ac u e ’s ins uc ions (Roche, B an o d, USA). Py osequencing was ca ied ou using GS Junio sys em (Roche). Bioin o ma ics o he py osequencing-de i ed da ase The aw sequences we e p ocessed using QIIME e . 1.8.0 [30]. To educe sequencing e o s, he mul iplexed eads we e i s il e ed o quali y and g ouped acco ding o ba code sequences. Sequences we e omi ed om he analysis i hey we e less han 200 bp, had a qual- i y sco e less han 25, con ained unco ec able ba codes, p ime s, ambiguous cha ac e s o a homopolyme leng h equal o g ea e han 8 bp. All non-bac e ial ibosomal sequences and chime as we e also emo ed om he da abase. In o al, 17 311 sequences we e ob ained wi h an a e age o 1923 sequences pe lib a y. The da ase was subjec ed o he no maliza ion p oce- du e esul ing in 1100 sequences pe sample. The minimum, median and maximum leng hs o sequences we e 200, 355 and 313 bp, espec i ely. Simila sequences we e clus e ed in o ope a- ional axonomic uni s (OTUs) wi h a minimum iden i y o 97% using de no o and closed e e - ence algo i hms. A ep esen a i e se o sequences was chosen by selec ing he mos abundan sequence om each OTU. Rep esen a i e sequences om each OTU we e subjec ed o RDP naï e Bayesian RNA Classi ie [31] wi h a con idence le el o 80% and aligned using PyNas [32] and G eengenes da abase [33]. Aligned sequences we e used o build a dis ance ma ix wi h a dis ance h eshold o 0.1 and phylogene ic ee necessa y o downs eam analysis. Sequence da a we e a chi ed in SRA da abase wi h accession SUB473223. To compa e mic obial communi ies he alpha and be a di e si y analyses we e pe o med. To es ima e alpha di e si y, he indices o ichness (obse ed species, ChaoI) and e enness (PD_whole ee, Shannon e enness, Simpson index) we e calcula ed. The - es was pe o med o e i y he obse ed di e ences. Fo be a di e si y he weigh ed Uni ac me ics [34] was used o calcula e he amoun o dissimila i y (dis ance) be ween he compa ed bac e ial com- muni ies. The esul s we e p esen ed in PCoA analysis using QIIME e . 1.8.0 [30]. All es i- ma es we e measu ed o he no malized da a (no maliza ion was ca ied ou up o he smalles numbe o sequences p esen in he sample). The mul iple ma ix eg ession based on Man el pe mu a ions [35] implemen ed in he phy ools R package (h p://www.phy ools.o g) was conduc ed o e eal he ela ionships be ween communi y composi ion and di e en ag ochemical p ope ies o soil. To educe ac- o space dimensionali y (by emo ing edundan a iables) we pe o med mul iple pai wise es s o Spea man ank-o de co ela ion. Signi ican dependency obse ed be ween pH and P allowed us o emo e he la e om ou ea u e se . The abundances o OTUs we e compa ed be ween samples by calcula ing he median ela- i e change alues o all g oups o iplica es. A posi i e median indica ed an inc ease in Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 5/16 abundance, whe eas a nega i e median was aken as e idence o decline o abundance. A basic pe mu a ion es was used o in e signi icance, whe eas a jackkni e-like esampling app oach was applied o es he s abili y o median es ima es. Resul s Land use e ec s on edaphic soil p ope ies The ag ochemical p ope ies o c opland soils managed acco ding o he wo di e en a ming sys ems we e a he simila , bu di e ed om he woodland soil despi e o he simila soil ype (Table 2). The woodland soil had he highes con en o o ganic ma e and C/N index, whe eas he lowes C/N alue was obse ed in CFS. Soil pH was lowes in he woodland. As o he main biogenic elemen s, woodland soil was ich in sul u and manganese, while he c oplands we e highe in magnesium, calcium and phospho us (Table 2). Rela i e quan i ies o bac e ia and a chaea es ima ed by qPCR The amoun s o he bac e ial and a chaeal biomass es ima ed by qPCR we e exp essed as he copy numbe o RNA ope ons pe g am o soil and used o compa ing he ela i e abun- dances o mic oo ganisms in he soil samples. The copy numbe o ibosomal ope ons in he genomes o mic oo ganisms a ies and is, in a e age, 4.09 o bac e ia and 1.76 o a chaea acco ding o he nDB da abase [36]. The expe imen al da a on he a e age copy numbe s o E.coli and H.salina um RNA ope ons in soil samples we e used o calcula e he abundance o bac e ial and a chaeal communi ies, espec i ely. The a e age numbe o bac e ia in soil was 8.37·10 8 o CFS, 1.56·10 9 o OFS and 2.19·10 9 o woodland (Fig 1). A chaea we e abou h ee olds o magni ude less abundan and hei a e age numbe s we e 8.15·10 5 o CFS, 2.41·10 6 o OFS, and 1.37·10 7 o woodland (Fig 1). These esul s showed ha he popula ion densi ies o bac e ia and a chaea we e lowes in CFS and highes in woodland (p<0.05). This endency was pa icula ly no iceable o a chaea, whose numbe s in he woodland we e 2 o de s o mag- ni ude highe han in he c oplands. The numbe o bac e ia in OFS was signi ican ly highe han in CFS (p <0.05), whe eas he o al coun s o a chaea did no a y be ween CFS and OFS (Fig 1). α-biodi e si y o he soil mic obial communi ies The biodi e si y wi hin each indi idual sample was es ima ed using ichness (numbe o obse ed species, Chao1) and e enness (Shannon e enness, Simpson) indices (Table 3). Woodland samples had he highes pe cen o co e age ( he numbe o OTUs o chao1 a io exp essed as a pe cen age) pe lib a y (82.7% in a e age). The co e age alues o OFS and CFS samples we e 65.1% and 65.8%, espec i ely. The obse ed species ichness and Simpson index o dominance we e no signi ican ly di e en be ween he samples (Table 3). Mic obial communi y composi ion A he phylum le el he e we e 22 majo bac e ial axa p esen in mos o he soils P o eobac- e ia (57.9% in a e age), Acidobac e ia (16,1%), Ac inobac e ia (7,9%), Ve ucomic obia (2,0%), Bac e oide es (2,7%) and Fi micu es (4,8%). The phyla wi h ela i e abundance less han 1% we e conside ed a e. They included C ena chaeo a,A ma imonade es, BHI80-139, Chlamydiae,Elusimic obia,Fib obac e es, GAL15, Ni ospi ae, TM6, TM7 and WPS-2. Some phyla, such as Fib obac e es, BHI80-139, TM6 and TM7, we e ound only in c oplands. The po ion o o ganisms wi h unknown axonomy anged om 0.6 o 2.1% and was he highes in CFS. Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 6/16 A he phylum le el, only mino di e ences we e ound be ween he bac e ial communi ies o CFS and OFS, whe eas he di e ences be ween woodland soil and c oplands we e mo e appa en (Fig 2). P o eobac e ia we e among he mos abundan phyla in c oplands, whe eas Acidobac e ia domina ed in he woodland soil (Fig 2). In gene al, di e en mic obial axa in woodland soil we e mo e e enly ep esen ed, including Fi micu es,Ac inobac e ia,Ni ospi a, Gemma imonade es and Chlo o lexi. The mic obiomes o c oplands and woodland soils di e ed ma kedly in he composi ion o P o eobac e ia (Fig 2). Woodland soil was domina ed by Alphap o eobac e ia and Gammap o- eobac e ia, while in c oplands Be ap o eobac e ia and Gammap o eobac e ia we e subs i u ing Alphap o eobac e ia. Bac e ia om he amilies Pseudomonadaceae and En e obac e iaceae accoun ed o mo e han 80% o he gammap o eobac e ia. The amily Pseudomonadaceae was almos exclusi ely ep esen ed by he genus Pseudomonas (Fig 3). The abundance o bac e ia o his genus a ied be ween he c oplands (16.0% in CFS and 13.2% in OFS). In he woodland soil he numbe o pseudomonads was only 5.3%. On he o he hand, he p o eobac e ial amily Sinobac e iaceae coun ed o mo e han 7.2% in he woodland soil, as compa ed wi h only 1.7% in OFS and CFS. Simila ly, be ap o eobac e ia o he genus Bu kholde ia and alphap o- eobac e ia o he amily B ady hizobiaceae and he genus Rhodoplanes we e subs an ially mo e common in he woodland soil han in c opland soils (Fig 4). Among he mos no able di - e ences in he mic obial axonomic composi ion be ween woodland and c oplands was he much highe p e alence o he phylum Acidobac e ia in woodland, in pa icula , bac e ia o he Fig 1. The numbe o bac e ia and a chaea pe g am o soil, es ima ed by quan i a i e PCR. The aw da a on he numbe o 16S RNA genes pe g am o soil, calib a ed o he E.coli and H.salina um 16S DNA copy numbe , we e ansla ed o he numbe o p oka yo ic cells pe g am o soil by use o he in o ma ion on he a e age numbe o 16S RNA copies in bac e ial and a chaeal genomes deposi ed in nDB da abase [36]. E o ba s indica e s anda d de ia ion (n = 3). doi:10.1371/jou nal.pone.0145072.g001 Table 3. Alpha-di e si y pa ame e s o soil mic obiomes. Sample ID Fa ming sys em a CFS OFS WOOD PD_whole_ ee 30,25±0,87 32,39±0,87 22,24±0,57 Shannon 6,58±0,19 6,83±0,19 6,72±0,08 Simpson 0,96±0,01 0,97±0,01 0,98±0 Chao1 b 406,53±39,8 450,97±39,8 264,57±9,05 Numbe o OTUs 267,33±11,67 293,67±11,67 218,67±3,71 Shannon e enness b 0,82±0,02 0,83±0,02 0,86±0,01 a CFS, con en ional a ming sys em; OFS, o ganic a ming sys em; WOOD, woodland. b The alpha-di e si y pa ame e s indica ed significan di e ences (p <0,05). doi:10.1371/jou nal.pone.0145072. 003 Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 7/16 amily Ko ibac e aceae (mainly Candida us Ko ibac e , Figs 3and 4), he o de Ellin6513 and Solibac e ales (Fig 4). The main bac e ial gene a ound in soil mic obiomes a e shown in Fig 3. The mos p o- nounced di e ences be ween he CFS and OFS mic obiomes we e wi hin he genus Pseudomo- nas, which we e signi ican ly (p<0,05) mo e abundan in CFS soil, as compa ed wi h OFS. O he s a is ically signi ican (p<0,05) di e ences in he axonomic composi ion be ween CFS and OFS mic obiomes we e mino and ound among he bac e ial gene a wi h equencies a ely exceeding 1% o all axa. Compa ed wi h OFS, CFS had highe ela i e abundances o he ac inobac e ia belonging o he amily Nakamu ellaceae, acidobac e ia o he amily Fig 2. Abundance a ios o he mos common bac e ial phyla in he soil in o ganic (OFS) s. con en ional (CFS) a ming sys ems, and he woodland s. a mland sys ems (wood s. FS; FS combines OFS and CFS samples). Ci cle size indica es he a e age abundance o he phylum. doi:10.1371/jou nal.pone.0145072.g002 Fig 3. Hea map compa ison o he mic obiomes in c oplands (CFS and OFS) and he woodland. Colo s ma k he a e age ela i e abundance (in numbe o sequences pe sample) o each bac e ial genus wi hin he sample. Only iden i ied gene a wi h o al coun s exceeding 5 sequences pe lib a y a e p esen ed. doi:10.1371/jou nal.pone.0145072.g003 Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 8/16 Ko ibac e aceae, p o eobac e ia o he g oups SC-I-84, Ellin6067, Oxalobac e iaceae (pa icu- la ly he genus Jan hinobac e ium) and Rals onia and bac e ia, belonging o he gene a Paeni- bacillus and Pedobac e . Mic obial communi y in OFS was en iched wi h p o eobac e ia o he Fig 4. OTUs analyzed in a boo s apped maximum likelihood phylogene ic ee and hei abundance p esen ed in a able. Pai wise es s indica ed ei he an inc ease (+) o a dec ease (–) in abundance be ween samples o he o ganic a ming sys em (OFS), he con en ional a ming sys em (CFS) and he woodland (Wood). Blank cells indica e insu icien da a. The signi icance o di e ence was assessed using a pe mu a ion es , INS indica es insigni ican di e ence. doi:10.1371/jou nal.pone.0145072.g004 Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 9/16 50. Sudhaka an M, Ramamoo hy D, Rajesh kuma S. Impac s o con en ional, sus ainable and o ganic a ming sys em on soil mic obial popula ion and soil biochemical p ope ies, Puduche y, India. In e na- ional Jou nal o En i onmen al Sciences. 2013; 4(1): 28–41. 51. Laws MT, G a es WR. Ni ogen inhibi s nodula ion and e e sibly supp esses ni ogen ixa ion in nod- ules o Alnus ma i ime J. Ame . Soc. Ho . Sci. 2005; 130(4): 496–499. 52. Lomba d N, P es a E, an Elsas JD, Simone P. Soil-speci ic limi a ions o access and analysis o soil mic obial communi ies by me agenomics. FEMS Mic obiol Ecol. 2011; 78(1): 31–49. doi: 10.1111/j. 1574-6941.2011.01140.x PMID: 21631545 Soil P oka yo ic Communi ies o Fa ming Sys ems PLOS ONE | DOI:10.1371/jou nal.pone.0145072 Decembe 18, 2015 16 / 16