595
Ba ley Rhizosphe e Bac e iome Dynamics unde O ganic and
Mine al Inpu s: The impo ance o in e c opping p edecesso
S e an Shile 1, Ma iana Pe ko a1, Vanya Popo a1, I elina Neyko a1, I an Rangelo 2
1 Depa men o Mic obiology and En i onmen al Bio echnologies, Ag icul u al Uni e si y-Plo di , 4000 Plo di , Bulga ia
2 Index 11 JSC, Plo di 4000, Bulga ia
Co esponding au ho : S e an Shile (s e an.shile @au-plo di .bg)
Copy igh : © S e an Shile e al.
This is an open access a icle dis ibu ed unde
e ms o he C ea i e Commons A ibu ion
License (A ibu ion 4.0 In e na ional – CC BY 4.0).
Resea ch A icle
Abs ac
In eg a ing in e c opping wi h g een manu e wi hin c op o a ion o e s a sus ainable
pa hway o enhance soil mic obial di e si y and o e all ag oecosys em heal h. This
s udy e alua ed he e ec s o di e se e ilisa ion p ac ices on he soil mic obiome in
a ba ley (Ho deum ulga e L.) c opping sys em ollowing he inco po a ion o an oa -
e ch (A ena sa i a and Vicia sa i a) and g een manu e inco po a ion e sus single
c opping o oa s. The ield expe imen ook place a he Ag icul u al Uni e si y o
Plo di , Bulga ia. Using high- h oughpu me agenomic sequencing, we assessed he
s uc u e and unc ions o he soil mic obial communi y. The e ilisa ion ea men s
included mine al e ilise , e micompos , a combina ion o e micompos and mine al
e ilise , and biocha , along wi h a non- e ilised con ol. The in es iga ion compa ed
wo p eceding c opping sys ems be o e ba ley cul i a ion: an oa - e ch in e c op used
as g een manu e, inco po a ed in o he soil a he ipening s age, and a con en ional
app oach whe e ba ley was sown di ec ly wi hou p io g een manu ing. All ea men s
we e conduc ed in iplica e. Sequencing allows o de ailed axonomic and unc ional
p o iling. The mos abundan bac e ial phyla iden i ied we e Ac inobac e io a,
P o eobac e ia, and Bac e oido a, wi h Ac inobac e io a showing inc eased abundance
unde e ilised condi ions. Soils ea ed wi h compos and compos -mine al mix u es
exhibi ed no ably highe alpha di e si y. Be a di e si y analyses (PCA, PCoA, UPGMA)
e ealed clea di e ences in mic obial communi y s uc u e among he ea men s.
Ce ain gene a, such as Sphingomonas, No ihe spi illum, and Ag omyces, we e
pa icula ly en iched in soils ecei ing e micompos o g een manu e. Thei p esence
sugges s enhanced mic obial unc ionali y and nu ien cycling in hose plo s.
O e all, he combina ion o g een manu e and o ganic inpu s suppo ed mo e di e se
and unc ionally ac i e mic obial communi ies, con ibu ing o he esilience and
sus ainabili y o ba ley-based c opping sys ems. This s udy ocuses on he bac e ial
componen o he soil mic obiome; nu ien cycling and soil heal h a e go e ned by he
combined in e ac ions among bac e ia, ungi, and p o ozoa.
Key wo ds: Ba ley, high- h oughpu me agenomic sequencing, mic obial di e si y,
plan -mic obe in e ac ions soil bac e iome, e micompos
Academic edi o : Susheel Bhanu Busi
Recei ed:
30 July 2025
Accep ed:
20 Oc obe 2025
Published:
18 No embe 2025
Ci a ion: Shile S, Pe ko a M, Popo a
V, Neyko a I, Rangelo I (2025) Ba ley
Rhizosphe e Bac e iome Dynamics
unde O ganic and Mine al Inpu s:
The impo ance o in e c opping
p edecesso . Me aba coding and
Me agenomics 9: e167231. h ps://
doi.o g/10.3897/mbmg.9.167231
Me aba coding and Me agenomics 9: 595–620 (2025)
DOI: 10.3897/mbmg.9.167231
596
Me aba coding and Me agenomics 9: 595–620 (2025), DOI: 10.3897/mbmg.9.167231
S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
In oduc ion
Mic oo ganisms in he soil a e key d i e s o ag oecosys em unc ionali y, con ib-
u ing o essen ial p ocesses such as nu ien mine alisa ion, main enance o soil
s uc u e, and p omo ion o plan de elopmen . The concep o he mic obiome
has e ol ed om a pu ely axonomic desc ip ion o a unc ional, sys ems-le el un-
de s anding o mic obial in e ac ions in speci ic habi a s (Be g e al. 2020). These
mic obial popula ions a e in ol ed in undamen al p ocesses such as nu ien cy-
cling, o ganic ma e decomposi ion, and he egula ion o plan heal h, di ec ly in-
luencing soil e ili y and c op yields (Fie e e al. 2007; Van de Heijden e al. 2008).
The s uc u e and unc ion o hese mic obial assemblages a e closely linked o
land managemen decisions, including e ilisa ion me hods, c opping sys ems,
and illage in ensi y (La kin 2015). As conce ns abou en i onmen al sus ainabili y
g ow, he e is inc easing a en ion on how ag icul u al in e en ions can main ain
o imp o e mic obial biodi e si y, which is c i ical o he long- e m p oduc i i y and
s abili y o ag oecosys ems (Escude o-Ma inez e al. 2022).
One s a egy gaining ac ion is he in eg a ion o legumes in o c opping sys-
ems h ough in e c opping o g een manu ing. Legumes such as Vicia sa i a
L. can ix a mosphe ic ni ogen h ough symbiosis wi h hizobia, en iching soil
nu ien pools wi hou he need o syn he ic e ilise s (Peoples e al. 2009).
The exuda ion o oo me aboli es om legumes can s imula e mic obial ac i -
i y and inc ease mic obial ichness in he hizosphe e. When inco po a ed in o
soil as g een manu e, legume biomass con ibu es o ganic inpu s ha enhance
soil s uc u e and biochemical unc ion, c ea ing a a ou able en i onmen o
mic obial g ow h and ac i i y (Lazcano e al. 2013; Xiao e al. 2023). These
e ec s o en ansla e in o imp o ed pe o mance o subsequen ce eal c ops,
such as ba ley, h ough enhanced nu ien a ailabili y and disease supp ession.
O ganic inpu s, including compos , e micompos , and biocha , a e also wide-
ly used o s imula e mic obial p ocesses and es o e soil heal h. Compos and
e micompos p o ide easily accessible ca bon and mic obial inoculan s ha
suppo enzyma ic ac i i y and mic obial biomass de elopmen (A ancon e al.
2006; Lazcano e al. 2013). Biocha , a ca bon- ich ma e ial de i ed om he py-
olysis o biomass, is alued o i s po ous s uc u e and high su ace a ea, which
o e physical niches o mic obial colonisa ion and enhance nu ien and wa e
e en ion in soil (Lehmann and Joseph 2015). These amendmen s can con ibu e
o building esilien soil mic obial ne wo ks and imp o ing o e all soil unc ion-
ali y, pa icula ly unde o ganic o educed-inpu sys ems (Lehmann e al. 2011).
Recen ad ances in sequencing echnologies, including amplicon-based and
me agenomic app oaches, now enable esea che s o dissec mic obial commu-
ni y dynamics a high esolu ion. By a ge ing genes such as he 16S RNA o ap-
plying sho gun me agenomics, scien is s can simul aneously assess axonomic
di e si y and he me abolic po en ial o soil mic obial communi ies (Nissimo
2017; P osse 2020). Despi e hese de elopmen s, ield-based s udies ha in-
eg a e legume in e c opping, o ganic amendmen s, and mic obial communi y
p o iling emain limi ed, especially unde empe a e Eu opean condi ions. This
s udy in es iga es he impac o inco po a ing a legume-ce eal mix u e (A ena
sa i a × Vicia sa i a) as g een manu e, ollowed by sp ing ba ley cul i a ion, on
he composi ion and unc ion o hizosphe e bac e ial communi ies unde a y-
ing e ilisa ion egimes. Mic obial communi y s uc u e and unc ional po en ial
597
Me aba coding and Me agenomics 9: 595–620 (2025), DOI: 10.3897/mbmg.9.167231
S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
we e assessed using 16S RNA gene sequencing and me agenomic analysis.
The p esen s udy aims o e eal how e ilise ype and he legacy e ec s o
c opping sys em in luence soil bac e iome di e si y in he hizosphe e o odde
win e ba ley. This s udy ocuses on he bac e ial componen o he soil mic obi-
ome and ecognises ha nu ien cycling and soil heal h a e shaped by he com-
bined ac i i ies o bac e ia, ungi, and p o ozoa wi hin he b oade soil ood web.
Ma e ials and me hods
Field desc ip ion and clima ic condi ions
The ield ial was ca ied ou in 2023 a he T aining and Expe imen al S a ion
o he Ag icul u al Uni e si y o Plo di , Bulga ia (42°08'15.4"N, 24°48'16.4"E).
The soil a he si e is classi ied as Mollic Flu isol, acco ding o he FAO sys-
em, o med on allu ial deposi s wi h a humus- ich uppe ho izon and a an-
si ion in o a ca bona e laye . The egion alls wi hin he empe a e con inen al
clima e zone o Sou he n Bulga ia, sui able o long- e m ag oecological ex-
pe imen a ion (Shile e al. 2024). The en i onmen al condi ions du ing he
s udy could be cha ac e ized as a e age, aking in o accoun he a e age al-
ues o mul i-yea da a. The empe a u es did no show signi ican a ia ions
om he op imal o he ba ley de elopmen . The a e age amoun o p ecip-
i a ion a he end o 2022 was 75 mm, while a he beginning o 2023, i was
138 mm, wi h a lowe amoun in Ma ch.
Expe imen al design and soil amendmen s
The s udy was s uc u ed wi hin a wo-yea c op o a ion scheme, aimed a as-
sessing he legacy e ec s o legume-based g een manu e and di e en e ili-
sa ion p ac ices on soil mic obial di e si y. In sp ing o he i s yea (2022), wo
di e en c opping s a egies we e implemen ed using a andomized block lay-
ou wi h h ee eplica es pe ea men : (i) monocul u e oa s (A ena sa i a L., c .
Max), and (ii) in e c opped oa s wi h e ch (Vicia sa i a L., c . Ob aze s 666) a a
3:1 seeding a io ( e ch: oa s), o aling 220 kg/ha. Each plo measu ed 5 × 7 m
(35 m2). Sowing was done in Ma ch 2022. The monoc op oa s we e ha es ed
a he end o June, while he in e c opped biomass was ploughed in o he soil
as g een manu e a a dep h o 30 cm. In he same yea (2022), a beginning o
No embe , all plo s we e cul i a ed wi h win e ba ley (Zemela cul i a ) o e al-
ua e esidual and in e ac i e e ec s o p io c opping and e ilisa ion schemes.
Two main ield se ups we e es ablished: (1) plo s p e iously en iched wi h g een
manu e, and (2) con en ional plo s wi hou g een manu e. Fi e e ilisa ion ea -
men s we e applied o bo h ields: no e ilisa ion (Con ol1, Con ol2); mine al
e ilise (MF1, MF2), NPK 15:15:15, 100 kg/ha, e micompos (VC1, VC2), 12 /
ha; combined mine al e ilise and e micompos (MF1.VC1, MF2.VC2) a hal
concen a ions; and biocha (Bch1, Bch2), 10 /ha. In his s udy, di e en o ganic
amendmen s we e applied and a e de ined as ollows: Compos (Com) e e s
o ma u e plan -based compos applied alone; Ve micompos (VC) is compos
p ocessed h ough he ac i i y o ea hwo ms; and Biocha compos (BC o
BC+Com) deno es a mix u e o biocha blended wi h compos p io o soil appli-
ca ion. Fo cla i y, he e m o ganic inpu s is used h oughou he manusc ip as
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Me aba coding and Me agenomics 9: 595–620 (2025), DOI: 10.3897/mbmg.9.167231
S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
a gene al desc ip o o all o ganic ea men s (Com, VC, and BC+Com), whe eas
indi idual abb e ia ions a e employed when speci ic ea men s a e discussed.
All ea men s we e a anged in a andomised comple e block design wi h h ee
eplica es pe ea men , esul ing in a o al o 30 ba ley plo s. A he ipening
s age o ba ley, hizosphe e soil samples we e collec ed om each plo by an-
domly selec ed plan s. This layou enabled he s udy o e ilisa ion and g een
manu ing e ec s on hizosphe e mic obial s uc u e unde ield condi ions
(Shile e al. 2024). Samples we e kep on ice du ing anspo , sie ed o 2 mm,
and s o ed a –80 °C un il DNA ex ac ion (Kulma iski 2020; Na alio e al. 2024).
DNA was ex ac ed om 0.5 g o soil using he DNeasy Powe Soil Ki (Qiagen,
Hilden, Ge many) ollowing s anda d p o ocols.
The analy ic da a showed su icien concen a ions o mos mac oelemen s
as o al con en in he biocha (ni ogen, po assium, calcium, and magnesium)
(Table 1). The phospho us and i on con en we e highe in he e micompos .
The analy ical p ocedu e is desc ibed p e iously by Shile e al. 2024.
The soil analyses a he beginning o he s udy ha e shown a ela i ely low
o al o ganic ca bon con en (TOC) – 1.14% (Table 2). Soil eac ion was sligh ly
alkaline wi h low elec ical conduc i i y (EC). The soil is poo ly supplied wi h
ni ogen and well s ocked wi h phospho us and po assium.
Me agenomic sequencing
DNA quali y con ol
Ex ac ed DNA quali y and quan i y we e assessed using a Qubi 2.0 Fluo ome e
(In i ogen, Ca lsbad, CA, USA) and 1% aga ose gel elec opho esis. All sam-
ples me he quali y s anda ds de ined by No ogene Co. L d., including pa ame-
e s o DNA in eg i y, pu i y, and concen a ion (Schloss e al. 2009).
Table 1. P ope ies o e micompos and biocha on a d y weigh basis. Da a show
mean and s anda d e o (n = 3).
Uni Ve micompos Biocha
TOC (%) 18.08 ± 1.48 65.00
pH 7.79 ± 0.01 –
EC (µS cm-1)1685 ± 10.0 –
To al N (%) 1.29 ± 0.17 1.31
P(%) 1.05 ± 0.08 0.64
K(%) 1.61 ± 0.21 7.95
Ca (%) 3.11 ± 0.32 4.28
Mg (%) 0.72 ± 0.07 3.06
Fe (%) 0.51 ± 0.05 0.13
Table 2. Physicochemical p ope ies o he soil be o e applica ion o amendmen s on a
d y weigh basis. Da a show mean and s anda d e o (n = 3).
pH EC (µS cm-1)
Accessible N
(N-NH4+N-NO3)
(mg 100 g-1)
P2O5
(mg 100 g-1)
K2O
(mg 100 g-1)TOC (%)
Soil 8.22 ± 0.1 121.0 ± 1.7 1.54 ± 0.03 3.58 ± 0.28 9.9 ± 0.1 1.14 ± 0.09
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Me aba coding and Me agenomics 9: 595–620 (2025), DOI: 10.3897/mbmg.9.167231
S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
Lib a y p epa a ion and sequencing
Me agenomic lib a ies we e cons uc ed h ough DNA agmen a ion, end- epai ,
A- ailing, and adap e liga ion, ollowing Illumina’s s anda d p o ocol. Whe e nec-
essa y, lib a ies unde wen PCR ampli ica ion. Following quan i ica ion and quali y
con ol, sequencing lib a ies we e dilu ed o 1.5 nM and subjec ed o pai ed-end se-
quencing (2 × 150 bp) on he Illumina No aSeq 6000 sys em (Quince e al. 2017).
Bioin o ma ic analysis
Da a p ep ocessing and quali y con ol
The aw sequencing da a unde wen quali y con ol wi h as p 0.23.1 (Chen e
al. 2018a), which in ol ed emo ing adap e sequences, eads con aining ambig-
uous nucleo ides, and hose wi h quali y sco es below Q20. Ba code and p ime
egions we e immed be o e me ging he high-quali y pai ed-end eads using
FLASH 1.2.11 (Magoč and Salzbe g 2011). De ec ion and emo al o chime ic
sequences we e pe o med using sea ch 2.16.0 (Rognes e al. 2016), e e enc-
ing he SILVA 138 da abase (Quas e al. 2013) o imp o ed accu acy.
Fea u e able cons uc ion and axonomic assignmen
Amplicon Sequence Va ian s (ASVs) we e in e ed using he DADA2 plugin in
QIIME2 2022.2 (Callahan e al. 2016). Taxonomic classi ica ion was conduc -
ed using a Naï e Bayes classi ie ained on he 515F/806R egion om he
SILVA 138 e e ence se . ASV ables we e a e ied o he minimum ead dep h
o downs eam compa isons.
Di e si y and communi y composi ion
Alpha di e si y indices (Chao1, Shannon, Simpson, Pielou’s e enness, Good’s
co e age) and be a di e si y me ics (B ay-Cu is, weigh ed and unweigh ed
UniF ac) we e calcula ed using QIIME2. O dina ion analyses including P incipal
Coo dina es Analysis (PCoA), P incipal Componen Analysis (PCA), and Non-
me ic Mul idimensional Scaling (NMDS) we e conduc ed in R 4.2.0 (R Co e Team
2022) using he egan, ggplo 2, and ade4 packages. G oup-le el di e ences we e
assessed ia Analysis o Simila i ies (ANOSIM) and Pe mu a ional Mul i a ia e
Analysis o Va iance (PERMANOVA) using he egan package (Ande son 2001)
Phylogene ic and s a is ical analyses
Mul iple sequence alignmen s o ep esen a i e ASVs we e pe o med wi h
MAFFT (Ka oh and S andley 2013), and phylogene ic ees we e cons uc ed
using Fas T ee in QIIME2 (P ice e al. 2010). Visualisa ion o mic obial compo-
si ion was ca ied ou using s acked ba plo s, hea maps (hea map), and e na y
plo s, implemen ed in R so wa e 4.2.0 (R Co e Team 2022; h ps://www. -p oj-
ec .o g/ accessed on 06.07.2025). Venn and lowe diag ams ep esen ing
sha ed and unique ASVs we e gene a ed using he VennDiag am package and
cus om SVG-based sc ip s.
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Me aba coding and Me agenomics 9: 595–620 (2025), DOI: 10.3897/mbmg.9.167231
S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
Da a a ailabili y
The 16S RNA sequencing da a om examined soils we e submi ed o he
NCBI gene bank unde he BioP ojec accession numbe PRJNA1297989 wi h
he submission ID: SUB15499394 eleased on 29 July 2025. The BioSample
accessions o he analysed en samples a e SAMN50254222, SAMN50254223,
SAMN50254224, SAMN50254225, SAMN50254226, SAMN50254227,
SAMN50254228, SAMN50254229, SAMN50254230, and SAMN50254231. The
da a eco ds a e accessible using he ollowing link: h ps://www.ncbi.nlm.nih.
go /biop ojec /1297989, accessed on 29 July 2025.
Resul s
Sequencing da a p ocessing
Row PE (Pai ed-End Reads) alues ange om 62,941 (MF2) o 78,820 (VC1),
which is a easonable ange o 16S RNA gene amplicon sequencing (Table 3).
No sample shows an ex eme ou lie , meaning sequencing dep h was ai ly
consis en . The combined (Me ged Reads) s ep me ges he pai ed-end eads
in o longe sequences. The me ging a es a e high ac oss all samples (close
o 100%), indica ing good sequencing quali y and p ope o e lapping ead e-
gions. Success ul me ging sugges s accu a e ead alignmen wi hou majo is-
sues in adap e imming o sequence quali y. Quali ied (Clean Tags in Table 3).
The pe cen ages emain high ac oss all samples, showing da a unde wen
minimal loss due o low-quali y eads. This e lec s ha sequencing un had
high quali y, and da a loss a his s age was minimal. Nochime (E ec i e Tags)
indica es he p esence o chimae as. Con ol1 d opped om 63,766 o 54,656
(~14.3% educ ion), and VC1 d opped om 77,093 o 70,624 (~8.4% educ ion).
Chimae a a es a y sligh ly be ween samples. MF1.VC1 and Bch2 show low-
e chimae a a es, indica ing hese samples had ewe a e ac s. GC con en
anges om 56.03% o 57.02%, which is biologically consis en and wi hin he
expec ed ange o mos mic obial communi ies. All samples exhibi Q20 > 98%
and Q30 > 95%, demons a ing excellen sequencing accu acy. The high Q20/
Q30 sco es con i m minimal sequencing e o s and high da a eliabili y.
Analysis o ela i e axonomic abundance
This s acked ba plo ep esen s he ela i e abundance o bac e ial axa a he
phylum le el ac oss di e en samples (Fig. 1). The ba plo o ela i e abun-
dance a he phylum le el illus a es consis en dominance o P o eobac e ia,
Ac inobac e io a, and Acidobac e io a ac oss all ea men s, in line wi h hei
es ablished oles in nu ien cycling and soil heal h. The p opo ional dis ibu-
ion o hese and o he phyla a ied depending on he e ilisa ion egime and
he applica ion o g een manu e.
T ea men s VC, MF.VC, and o a lesse ex en Bch, showed ele a ed le els
o Ac inobac e io a and Bac e oido a, phyla known o hei sap ophy ic and
deg ada i e capabili ies. This shi sugges s inc eased a ailabili y o o ganic
subs a es ollowing he inco po a ion o legume biomass, which can s imula e
mic obial axa in ol ed in o ganic ma e decomposi ion and nu ien elease.
601
Me aba coding and Me agenomics 9: 595–620 (2025), DOI: 10.3897/mbmg.9.167231
S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
In con as , plo s ea ed wi h mine al e ilize o le as un ea ed con ols
exhibi ed ela i ely highe p opo ions o Acidobac e io a, o en associa ed
wi h oligo ophic condi ions and mo e s able, slowe nu ien cycling dynam-
ics (Fig. 1). These ea men s also showed educed abundance o phyla such
as Gemma imonado a and Myxococco a, indica ing a less di e se and less
unc ionally dynamic mic obial communi y. The biocha ea men p esen ed
a unique phylum dis ibu ion, wi h sligh ly ele a ed Ve ucomic obio a and
Chlo o lexi, consis en wi h p e ious indings ha biocha al e s mic ohabi a
condi ions such as ae a ion, pH, and mois u e con en , he eby selec ing o
specialized mic obial axa. The esul s unde sco e ha g een manu e inco po-
a ion, pa icula ly in combina ion wi h compos o mine al e ilize s, enhances
mic obial di e si y and shi s he axonomic s uc u e owa d copio ophic, unc-
ionally ac i e axa. These mic obial changes e lec imp o ed soil e ili y and
biological unc ioning unde in e c opping-based, o ganic-amended sys ems.
Table 3. Sequencing da a analysis.
Sample RawPE Combined Quali ied Nochime Base(n ) A glen(n ) GC, % Q20,% Q30,%
Con ol1 65518 64580 63766 54656 22898990 418.97 56.65 98.71 95.43
MF1 66110 65551 64810 59174 24743668 418.15 56.10 98.87 95.80
VC1 78820 78024 77093 70624 29567160 418.66 56.03 98.86 95.81
MF1.VC1 68808 68308 67339 59460 24800258 417.09 56.27 98.74 95.35
Bch1 66344 65837 64947 56883 23728533 417.15 57.02 98.77 95.48
Con ol2 70347 69768 68827 64043 26761127 417.86 56.74 98.81 95.70
MF2 62941 62431 61527 55209 23012585 416.83 56.74 98.70 95.21
VC2 70455 69639 68724 62112 25927482 417.43 56.97 98.75 95.58
MF2.VC2 63834 63011 62111 54694 22854201 417.86 56.47 98.74 95.42
Bch2 64928 64102 63302 54227 22642699 417.55 56.51 98.75 95.54
Legend – Abb e ia ions o sample codes: Con ol1-un e ilized con ol a e g een manu e, Con ol2 – Ba ley g own wi hou e ilisa ion
; MF1-Ba ley g own a e oa - e ch g een manu e and e ilized wi h mine al e ilize , MF2 – e ilized wi h mine al e ilize , VC1 - Ba ley
g own a e oa - e ch g een manu e and e ilized wi h e micompos , VC2 – e ilized wi h e micompos , MF1.VC1 – Ba ley g own
a e oa - e ch g een manu e, e ilized wi h compos + mine al e ilize , MF2.VC2 – e ilized wi h compos + mine al e ilize , Bch1 -
Ba ley g own a e oa - e ch g een manu e, e ilized wi h biocha , Bch2 – e ilized wi h biocha only.
Figu e 1. Rela i e axonomy abundance o each sample in di e en axonomic le el. a
phylum le el.
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Analysis o ela i e axonomic abundance a he genus le el
The genus-le el ba plo (Fig. 2) e eals a consis en dominance o Sphingomonas
ac oss all ea men s, highligh ing i s ubiqui y and po en ial ecological ele-
ance in he ba ley hizosphe e. This genus, known o i s capaci y o deg ade
complex o ganic compounds and p omo e plan heal h, was mos abundan in
compos - and biocha -amended soils, pa icula ly in MF.VC and VC ea men s.
This sugges s ha g een manu e inco po a ion en iched copio ophic gene a
linked o nu ien ans o ma ion and o ganic ma e decomposi ion.
O he no able gene a include Lysobac e , No ihe spi illum, and Gemma imonas,
which oge he con ibu ed o a mode a e p opo ion o he communi y in Fig. 2.
These gene a a e o en associa ed wi h ca bon and ni ogen cycling and showed
sligh inc eases in compos and biocha ea men s, possibly e lec ing he en-
hanced a ailabili y o oo exuda es and o ganic subs a es. In con as , con ol
and mine al-only (MF) ea men s exhibi ed lowe ichness and di e si y a he
genus le el, wi h g ea e p opo ions classi ied unde “O he s,” indica ing ei he
lowe axonomic esolu ion o dominance o a e/uncul u ed axa. Massilia and
A enimonas we e mo e p ominen in biocha -amended soils (Bch1, Bch2), sug-
ges ing hei po en ial p e e ence o he al e ed physicochemical p ope ies
(e.g., pH, po osi y) associa ed wi h biocha . These esul s con i m ha g een
manu e, pa icula ly when combined wi h o ganic amendmen s, en iches bene-
icial mic obial axa a he genus le el, con ibu ing o imp o ed soil heal h and
po en ial plan g ow h-p omo ion unde in e c opping sys ems.
Hea map o genus le els
The hea map highligh s he di e en ial abundance o dominan bac e ial gen-
e a ac oss ea men s, clus e ed bo h by axonomic a ilia ion and ea men
ype (Fig. 3). Samples ea ed wi h g een manu e, especially MF.VC, and VC,
exhibi ed clea shi s in mic obial composi ion compa ed o mine al e ilisa-
ion (MF) and un ea ed con ols. Gene a such as Noca dioides, Ag omyces,
Rub obac e , and Soli ub obac e (phylum: Ac inobac e io a) we e no ably
Figu e 2. Rela i e axonomy abundance o each sample in di e en axonomic le el a
genus le el.
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S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
en iched in g een manu e and biocha (Bch) ea men s, e lec ing inc eased
mic obial ac i i y ela ed o o ganic ma e decomposi ion. These gene a a e
o en associa ed wi h cellulose deg ada ion, plan -g ow h p omo ion, and esil-
ience o en i onmen al s ess.
Con e sely, RB41, Lysobac e , and Sphingomonas we e mo e abundan in
con ol and mine al e ilize ea men s. Thei p e alence in nu ien -limi ed
en i onmen s may indica e a compe i i e ad an age in lowe -ca bon soils. The
ela i ely lowe p esence o copio ophic gene a in MF and Con ol samples
sugges s educed mic obial ichness and unc ional di e si y. Dis inc pa -
e ns we e also e iden o No ihe spi illum (P o eobac e ia), which showed
Figu e 3. Hea map showing he ela i e abundance o he 35 dominan gene a ac oss all samples. The X-axis ep esen s
sample codes, and he Y-axis ep esen s gene a. Hie a chical clus e ing is shown on he le . The Z-sco e indica es
de ia ion om he mean abundance, and he colou g adien e lec s abundance le els: ed/o ange = highe , yellow =
in e media e, blue = lowe .
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ela i e o Con ol1 (0.132; 0.451) and MF1 (0.114; 0.451), sugges ing ha his
ea men al e s mic obial communi ies subs an ially. In con as , he lowes
dissimila i y was obse ed be ween Con ol1 and MF1 (0.098; 0.417), ollowed
by MF1 and VC1 (0.143; 0.434), implying ha hese indi idual soil ea men s
showed limi ed e ec s on o e all mic obial composi ion, main aining simila i y
wi h he con ol g oup. The combina ion MF1.VC1 esul ed in a mode a e shi
om Con ol1 (0.182; 0.446), highligh ing a possible in e ac ion e ec . While
axonomically close o he con ol han Bch2, i s ill demons a ed mode a e o
high dissimila i y when compa ed wi h o he ea men s such as MF2 (0.146;
0.455) and MF1.VC1 (0.160; 0.424). This sugges s ha he speci ic Bacillus
s ain o o mula ion used in Bch1 induced a unique mic obial esponse.
Con ol1 and Con ol2 (0.132; 0.451) exhibi ed measu able dissimila i y due
o mic o-en i onmen al a iabili y o he e ogenei y in mic obial baseline con-
di ions. Be a di e si y ma ix con i ms ha ea men s con aining biocha in-
oculan s and MF2.VC2 d i e he g ea es di e gence in mic obial communi-
y s uc u e, while MF1, VC1, and hei indi idual applica ions show minimal
de ia ion om he con ol. These indings a e consis en wi h PCA and PCoA
esul s, ein o cing he conclusion ha combined biological amendmen s can
eshape hizosphe e mic obial communi ies, likely h ough syne gis ic nu ien
e ec s, mic obial in e ac ions, and hos plan esponses. These esul s align
wi h p e ious s udies epo ing ha in eg a ed o ganic and mic obial inpu s
can signi ican ly modi y soil mic obial assemblages and enhance unc ional
di e si y (Ha mann e al. 2015; Singh e al. 2020). Biocha amendmen s ha e
been shown o enhance mic obial di e si y and al e communi y assembly by
p o iding addi ional habi a and in luencing soil chemis y (Lehmann e al.
2011). Ve micompos applica ions a e consis en ly associa ed wi h imp o ed
soil s uc u e and inc eased abundance o bene icial ungal axa (Domínguez
and Edwa ds 2011; Pa hma and Sak hi el 2012). Likewise, compos inpu s p o-
mo e s able mic obial ne wo ks and s imula e unc ional guilds in ol ed in nu-
ien cycling (Bonanomi e al. 2016). Ou esul s a e in line wi h hese s udies,
sugges ing ha he combined use o o ganic inpu s and g een manu ing can
os e esilien mic obial communi ies ha suppo bo h soil heal h and c op
p oduc i i y.
Discussion
This s udy demons a es ha bo h e ilisa ion s a egy and c opping sys em
signi ican ly shape he s uc u e and unc ional po en ial o hizosphe e mic o-
bial communi ies in ba ley ag oecosys ems. Using 16S RNA gene amplicon se-
quencing, we cha ac e ised he mic obial axonomic composi ion and me abolic
capabili ies ac oss soils amended wi h mine al e ilise , compos , compos -min-
e al blends, and biocha , unde con en ional monocul u e and oa - e ch in e c op-
ping sys ems. Ac inobac e io a and P o eobac e ia domina ed ac oss all ea -
men s, highligh ing hei cen al oles in soil ecosys em unc ioning (Ha mann e
al. 2015; Fie e 2017) (Figs 1–4). The high abundance o Ac inomyce o a—pa ic-
ula ly gene a such as Noca dioides, Soli ub obac e , and Rub obac e sugges s a
s able and esilien mic obial co e capable o o ganic ma e decomposi ion and
an ibio ic p oduc ion, aligning wi h obse a ions in o ganically managed soils
(Lehmann e al. 2011; Zhang e al. 2023) (Fig. 4). Biocha - ea ed soils displayed
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he g ea es communi y di e gence, likely due o biocha ’s capaci y o modi y
soil s uc u e, pH, and mois u e e en ion, os e ing unique mic obial niches. The
MF.VC and Bch ea men s en iched a b oade ange o axa, including bo h dom-
inan phyla (e.g., Ac inobac e io a, Bac e oido a) and less abundan gene a wi h
key unc ional oles in ca bon and ni ogen cycling.
Despi e he p esence o a sha ed co e mic obiome ac oss ea men s, mi-
c obial ichness and e enness we e signi ican ly enhanced in o ganic and
g een manu e-amended plo s compa ed o mine al e ilised and con ol ea -
men s (Fig. 5). This sugges s ha in eg a ed nu ien managemen enhances
ecological s abili y and unc ional edundancy, which a e c ucial o esilien
soil sys ems. The phylogene ic and composi ional di e ences obse ed u -
he suppo he hypo hesis ha o ganic inpu s in oduce a g ea e a ie y o
subs a es and mic ohabi a s, d i ing mic obial di e si ica ion. The abundance
o copio ophic and unc ionally e sa ile gene a, pa icula ly in g een manu e
plo s, con i ms he ecological bene i o hese p ac ices o sus aining soil bi-
ological heal h and c op p oduc i i y. The in eg a ion o leguminous g een ma-
nu e (oa - e ch) no ably inc eased alpha di e si y, pa icula ly in in e c opped
and compos - ea ed plo s, suppo ing p e ious e idence ha legumes s imu-
la e hizosphe e mic obial ichness h ough ni ogen ixa ion and exuda e-me-
dia ed ec ui men (Peoples e al. 2009; Shakya e al. 2013; Philippo e al. 2013;
B ooke e al. 2015; Pe ko a e al. 2025)
The ele a ed Shannon and Chao1 indices in VC1 and VC2 plo s sugges
ha g een manu e imp o es niche a ailabili y and subs a e he e ogenei y,
os e ing mo e di e se and e en mic obial assemblages (Lazcano e al. 2013)
(Table 4). The de ec ion o a co e mic obiome ac oss ea men s—comp ising
o e 560 sha ed species—sugges s ha ba ley main ains a s able mic obial
conso ium i espec i e o soil amendmen . The p esence o o e 1,000 unique
species in VC1 highligh s he signi ican ecological impac o in e c opping and
g een manu e inco po a ion. This en ichmen may e lec mic obial adap a ion
o ele a ed nu ien a ailabili y and complex oo a chi ec u es cha ac e is ic
o mixed legume-ce eal sys ems (Cong e es and Van Ee d 2015; Finney e al.
2016). Impo an ly, while mine al e ilise s suppo ed high mic obial abun-
dance, hei sole applica ion was associa ed wi h lowe e enness and di e si y
compa ed wi h o ganic o mixed ea men s, echoing conce ns o e long- e m
mic obial homogeniza ion unde in ensi e inpu egimes (Chen e al. 2018b).
Cu en esul s sugges ha mine al inpu s, when used in isola ion, may a ou
copio ophic axa and educe unc ional edundancy. Toge he , hese indings
unde sco e he syne gis ic bene i s o in e c opping and o ganic e ilisa ion
o main aining ich, di e se, and unc ionally obus mic obial communi ies. By
in eg a ing oa - e ch g een manu e wi h compos -based amendmen s, a m-
e s can os e a hizosphe e mic obiome conduci e o nu ien cycling, plan
heal h, and ag oecosys em sus ainabili y.
Be a di e si y analysis in Fig. 8 e ealed dis inc communi y shi s d i en by
e ilisa ion ype. Bch1 and Bch2 exhibi ed he mos p onounced di e gence
om o he ea men s, consis en wi h biocha ’s known capaci y o al e soil
physicochemical p ope ies and p o ide mic ohabi a s o specialised axa
(Lehmann e al. 2011; Liu e al. 2010).
The clus e ing ees based on Unweigh ed and Weigh ed UniF ac dis anc-
es e eal dis inc pa e ns in bac e ial communi y s uc u e ac oss ea men s
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(Fig. 9). Unweigh ed UniF ac (Fig. 9A) conside s only he p esence o absence
o axa and shows clea sepa a ion be ween ea men g oups, especially be-
ween Con ol, MF, and MF.VC samples. This indica es ha ea men s in lu-
ence he axonomic composi ion, e en among low-abundance axa (Lozupone
e al. 2005). In con as , he Weigh ed UniF ac ee (Fig. 9B), which accoun s o
axon abundances, highligh s di e ences in dominan mic obial lineages. MF1.
VC1 and MF2.VC2 clus e s closely, sugges ing a sha ed en ichmen o abundan
phyla such as P o eobac e ia, Ac inobac e io a, and Acidobac e io a, as seen
in he ela i e abundance ba plo s (Zhang e al. 2023). The dis inc clus e ing
o Bch1 and Bch2 in bo h ees suppo s he idea ha his ea men p omo es
a unique mic obial assemblage, po en ially d i en by speci ic soil o plan -as-
socia ed ac o s. These esul s emphasise ha bo h communi y composi ion
and abundance p o iles a e a ec ed by managemen p ac ices, and combining
Unweigh ed and Weigh ed UniF ac analyses p o ides a mo e comp ehensi e
iew o mic obial communi y dynamics (Lozupone and Knigh 2011).
Figu e 9. Clus e ing ee based on B ay-Cu is dis ance in species abundance (A) unweigh ed and (B) weigh ed Uni ac
dis ance. The dend og am (le ) coupled wi h a s acked ba plo o ela i e species abundance ( igh ), o e ing insigh
in o communi y composi ion and simila i y among ea men s.
A
B
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The PCA plo in Fig. 10 p o ides a wo-dimensional isualiza ion o he a i-
a ion in communi y s uc u e among he ea men g oups, based on p incipal
componen analysis. The i s p incipal componen (PC1) accoun s o 14.53%
o he o al a iance, while he second p incipal componen (PC2) explains
12.15%, oge he cap u ing 26.68% o he obse ed a ia ion. Dis inc clus e -
ing pa e ns we e obse ed among he ea men s. Samples om Con ol1,
MF1, and VC1 clus e ed closely in he uppe le quad an , indica ing ha hese
g oups sha ed simila mic obial o biochemical p o iles and esponded sim-
ila ly o he es ed condi ions. In con as , Con ol2 and MF2 we e loca ed in
he lowe igh quad an , clea ly sepa a ed om Con ol1 and MF1, sugges ing
di e gen p o iles po en ially associa ed wi h he e ec s o he ea men s o
en i onmen al di e ences be ween eplica es. MF2.VC2 and Bch2, g ouped
nea he cen e o he plo , implying mode a e o mixed esponses ha do no
s ongly d i e a ia ion along ei he PC1 o PC2. VC2 appea ed as a clea ou -
lie in he op igh quad an , e lec ing a p onounced de ia ion in communi-
y s uc u e, especially along PC2. Simila ly, Bch1 was loca ed dis an ly om
o he g oups in he lowe quad an , sugges ing a unique ea men e ec o
s ain-speci ic esponse. MF1.VC1, posi ioned be ween he clus e s o MF1
and VC1, displayed an in e media e p o ile, indica i e o a pa ial in e ac ion
e ec . Likewise, he combined ea men MF2.VC2 demons a ed a dis inc bu
cen ally loca ed p o ile, sugges ing a complex o balanced in e ac ion be ween
he indi idual componen s. PCA highligh s he ea men -d i en di e en ia ion
o he mic obial o biochemical communi y s uc u e. The close associa ion o
ce ain ea men s (Con ol1, MF1, and VC1) and he di e gence o o he s (MF2,
VC2, Bch1) unde sco e he a ied e ec s o expe imen al condi ions. Bch2 also
sugges s po en ial in a-g oup a iabili y o sensi i i y o en i onmen al o p o-
cedu al ac o s. These indings emphasize he ele ance o ea men -speci ic
and in e ac ion-dependen in luences on he unde lying biological sys em.
P incipal Coo dina es Analysis (PCoA) in Fig. 11 was pe o med o assess
be a-di e si y and isualize di e ences in mic obial communi y composi ion
Figu e 10. P incipal Componen Analysis (PCA) o Soil Mic obial Communi ies unde
Di e en Fe ilisa ion T ea men s.
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ac oss he expe imen al ea men s. This me hod allows o dimensionali y e-
duc ion based on pai wise dissimila i y me ics and is commonly used o e al-
ua e composi ional shi s in mic obial communi ies (Lozupone e al. 2005). In
he le panel o Fig. 11, he i s wo p incipal coo dina es explain 49.16% and
14.56% o he a iance, espec i ely. A clea clus e ing o MF1, VC1, and Con ol1
is obse ed in he uppe le quad an , sugges ing composi ional simila i y unde
hese ea men s. In con as , MF2.VC2 and Bch2 a e dis inc ly sepa a ed in he
igh hal o he plo , e lec ing a ma ked di e gence in mic obial communi y
s uc u e. Bch1 and MF1.VC1 show isola ed posi ions, indica ing unique mic o-
bial signa u es ha di e om bo h con ol and o he ea men g oups.
The igh panel o he PCoA plo , based on a di e en dis ance me ic, e-
eals simila bu mo e dispe sed clus e ing. He e, PC1 and PC2 explain 21.07%
and 12.73% o he o al a ia ion, espec i ely. Bch2 and MF2.VC2 clus e igh -
ly in he uppe igh quad an , indica ing a s ong ea men e ec om he
combined applica ion o Bacillus-based bios imulan s and e micompos . VC2
appea s as an ou lie in he lowe igh quad an , con i ming a dis inc mic o-
bial communi y composi ion. Meanwhile, MF1, Con ol1, and MF1.VC1 clus e
in he uppe le quad an , ein o cing hei composi ional simila i y. PCoA e-
sul s con i m ha speci ic ea men s—pa icula ly MF2, VC2, and Bch-based
amendmen s—induce signi ican shi s in mic obial communi y s uc u e com-
pa ed o he con ol. The clus e ing pa e ns indica e ha combina ions o mi-
c obial e ilize s and o ganic inpu s can lead o syne gis ic o addi i e e ec s,
eshaping he hizosphe e mic obiome. These indings a e consis en wi h p e-
ious s udies showing ha o ganic and mic obial amendmen s d i e dis inc
mic obial assemblages and in luence soil ecological unc ion (Ha mann e al.
2015; Ai e al. 2015; Singh e al. 2020).
The in luence o a ious e ilisa ion s a egies on soil mic obial communi-
ies was assessed unde wo con as ing c opping sys ems: a con en ional
sys em wi h di ec ba ley sowing (no p io co e c op), and an in e c opping
sys em u ilising oa - e ch g een manu e be o e ba ley cul i a ion. In he ab-
sence o e ilisa ion (Con ol), mic obial communi ies emained s able unde
Figu e 11. P incipal Coo dina es Analysis (PCoA) o Soil Mic obial Communi ies Based on UniF ac Dis ances.
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con en ional managemen , domina ed by Ac inomyce o a. Тhe in e c opped
con ol plo s suppo ed sligh ly highe mic obial ichness and unc ional di e -
si y, consis en wi h indings ha legume-based g een manu es enhance soil
mic obio a e en wi hou nu ien amendmen s (Zhang e al. 2018; Zhou e al.
2021). Mine al e ilisa ion induced a selec i e inc ease in s ess- ole an axa
such as Noca dioides and Rub obac e aceae, leading o educed di e si y in
con en ionally managed soils. In con as , he g een manu e in e c opping sys-
em p ese ed a mo e balanced mic obial s uc u e and acili a ed he appea -
ance o Acidobac e io a and Chlo o lexo a, phyla known o hei oles in o -
ganic ma e u no e and en i onmen al esilience (Janssen 2006; Kielak e al.
2016). Compos applica ion en iched decompose bac e ia unde con en ional
ba ley, especially Noca dioides, bu had a s onge e ec unde in e c opping
by p omo ing Soli ub obac e aceae and less abundan phyla. The inclusion
o compos signi ican ly enhanced mic obial di e si y and unc ionali y, pa -
icula ly when combined wi h g een manu e p ac ices, aligning wi h p e ious
epo s ha o ganic amendmen s s imula e mic obial ichness and p omo e
bene icial axa (Lazcano e al. 2013; Po ilai is e al. 2020).
The combina ion o compos and mine al e ilisa ion led o s ong domi-
nance by Noca dioides and F ankiales unde con en ional managemen . Unde
in e c opping, his combina ion p omo ed g ea e mic obial e enness and en-
iched a e axa such as Gaiella occul a. This sugges s a syne gis ic e ec be-
ween o ganic and ino ganic inpu s when used wi h g een manu e, enhancing
mic obial unc ional edundancy—an impo an indica o o soil ecosys em e-
silience (Bane jee 2021). Biocha applica ion suppo ed dis inc mic obial as-
semblages domina ed by ca bon-deg ading bac e ia unde con en ional ba ley
cul i a ion. The in e c opping-biocha combina ion esul ed in he b oades mi-
c obial spec um, including en ichmen o niche-speci ic and a e axa as Gaiella.
These esul s co obo a e he hypo hesis ha biocha in combina ion wi h
legume-based g een manu es enhances mic obial niche complexi y and sup-
po s specialised unc ional g oups (Quilliam e al. 2013; Lehmann e al. 2011).
The oa - e ch g een manu e sys em imp o ed mic obial ichness, e enness,
and unc ional di e si y ac oss all e ilisa ion ea men s. In e c opping no
only mi iga ed he po en ial nega i e e ec s o mine al e ilisa ion bu also en-
hanced he pe o mance o o ganic amendmen s. These indings suppo he
in eg a ion o g een manu es wi h compos o biocha as e ec i e s a egies o
p omo e di e se and ecologically obus soil mic obiomes in ba ley-based c op-
ping sys ems. F om an ecological s andpoin , he applica ion o legume-based
g een manu e os e ed mic obial esilience and ecosys em complexi y, sup-
po ing a b oade ange o axa and po en ially inc easing unc ional edundan-
cy. Func ionally, hese ea men s a e likely o enhance nu ien cycling, o ganic
ma e u no e , and plan -mic obe in e ac ions, which a e c i ical o long- e m
soil e ili y and c op p oduc i i y. These indings demons a e ha in eg a ed
nu ien managemen combining g een manu e wi h o ganic amendmen s is a
iable s a egy o p omo e di e se, ecologically s able, and unc ionally obus
mic obial communi ies in ce eal c opping sys ems. Such p ac ices a e key o
ad ancing soil heal h, sus ainabili y, and ag oecological esilience unde mod-
e n ag icul u al p essu es. While he p esen esea ch concen a es on bac e i-
al communi ies, we acknowledge ha ungi and p o ozoa in e ac closely wi h
bac e ia in d i ing decomposi ion and nu ien mine alisa ion. Fu u e s udies
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S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
in eg a ing bac e ial, ungal, and p o ozoan analyses will p o ide a mo e com-
p ehensi e iew o soil unc ional ecology unde hese ag oecological p ac ices.
Conclusions
G owing in e c opped oa - e ch and inco po a ing hem a he ipening s age as
g een manu e, and applying e micompos , os e s mo e di e se, unc ionally
e sa ile, and ecologically esilien hizosphe e mic obiomes compa ed o soils
wi hou g een manu e o mine al inpu s.. The bac e ial biome ep esen s only a
pa o he complex soil mic obiome, and ha u u e me agenomic wo k should
encompass ungal and p o ozoan con ibu ions o ully elucida e soil ecosys-
em unc ioning. These indings ad oca e o he adop ion o in e c opping wi h
legume g een manu e and ailo ed o ganic amendmen s o suppo sus ainable
ce eal p oduc ion. Ecological and unc ional bene i s o he cu en s udy a e
he imp o ed di e si y unde g een manu e and combined ea men , which like-
ly ansla es in o mo e obus ni ogen and ca bon u no e , d i en by a b oade
sui e o deg ada i e and biosyn he ic pa hways. Communi ies in e micompos
and combined e ilisa ion plo s exhibi ed g ea e phylogene ic dispe sion, sug-
ges ing imp o ed capaci y o wi hs and en i onmen al pe u ba ions. Biocha ’s
abili y o s abilise soil pH and mois u e appea s o os e specialised conso ia,
po en ially imp o ing long- e m soil s uc u e and plan -mic obe in e ac ions.
Addi ional in o ma ion
Con lic o in e es
The au ho s ha e decla ed ha no compe ing in e es s exis .
E hical s a emen
No e hical s a emen was epo ed.
Use o AI
No use o AI was epo ed.
Funding
This esea ch was unded by he Bulga ian Na ional “Science Fund”, g an numbe KP-
06-DO 02/5. The au ho s acknowledge he inancial suppo h ough he pa ne s o he
Join Call o he Co und ERA-Ne s SusC op (g an No. 771134), FACCE ERA-GAS (g an
No. 696356), ICT-AGRI-FOOD (g an No. 862665) and SusAn (g an No. 696231).
Au ho con ibu ions
Concep ualiza ion, S.S.; me hodology, S.S. and M.P.; o mal analysis, M.P., V.P., N.M. and
I.N.; in es iga ion, M.P., V.P., I.N. and S.S.; esou ces, S.S.; w i ing – o iginal d a p epa a-
ion, M.P. and S.S.; w i ing – e iew and edi ing, M.P., S.S., I.R.; supe ision, S.S.; unding ac-
quisi ion, S.S. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Au ho ORCIDs
S e an Shile h ps://o cid.o g/0000-0002-1172-881X
Ma iana Pe ko a h ps://o cid.o g/0000-0001-5122-9575
617
Me aba coding and Me agenomics 9: 595–620 (2025), DOI: 10.3897/mbmg.9.167231
S e an Shile e al.: Ba ley hizosphe e bac e iome dynamics
I elina Neyko a h ps://o cid.o g/0009-0005-5163-3128
Da a a ailabili y
All o he da a ha suppo he indings o his s udy a e a ailable in he main ex .
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