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
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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
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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
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ional Jou nal o En i onmen al Sciences. 2013; 4(1): 28–41.
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