A icle h ps://doi.o g/10.1038/s41467-023-43926-4
C op o a ion and na i e mic obiome
inocula ion es o e soil capaci y o supp ess
a oo disease
Yanyan Zhou
1,12
,ZhenYang
2,3,12
,JinguangLiu
2
,XudongLi
1
, Xingxiang Wang
2,4
,
Chuanchao Dai
3
, Taolin Zhang
2,4
,Víc o J.Ca ión
5,6,7,8
, Zhong Wei
9
,
Fuliang Cao
10
, Manuel Delgado-Baque izo
11
&XiaogangLi
1,2,10
I is widely known ha some soils ha e s ong le els o disease supp ession
and p e en he es ablishmen o pa hogens in he hizosphe e o plan s.
Howe e , wha soils a e be e supp essing disease, and how managemen can
help us o boos disease supp ession emainunclea .He e,weusedfield,
g eenhouse and labo a o y expe imen s o in es iga e he e ec o manage-
men (monoc opping and o a ion) on he capaci y o hizosphe e mic o-
biomes in supp essing peanu oo o disease. Compa ed wi h c op o a ions,
monoc opping esul ed in mic obial assemblies ha we e less e ec i e in
supp essing oo o diseases. Fu he , he deple ion o key hizosphe e axa in
monoc opping, which we e a a disad an age in he compe i ion o limi ed
exuda es esou ces, educed capaci y o p o ec plan s agains pa hogen
in asion. Howe e , he supplemen a ion o deple ed s ains es o ed hizo-
sphe e esis ance o pa hogen. Taken oge he , ou findings highligh he ole
o na i e soil mic obes in figh ing disease and suppo ing plan heal h, and
indica e he po en ial o using mic obial inocula o egene a e he na u al
capaci y o soil o figh disease.
Some soils ha e a la ge capaci y o suppo disease supp ession
han o he s helping o p e en he es ablishmen o pa hogens in
he hizosphe e o plan s1,2. Howe e , he cha ac e is ics defining
hese soils a e la gely unknown. The hizosphe e e e s o he soil
di ec ly associa ed wi h he oo s. This complex en i onmen ,
en iched wi h ca bon and nu ien s, is home o a di e se mic obial
communi y ha plays an essen ial ole in p omo ing plan g ow h
and heal h3,4. This hizosphe e mic obiome se es as he fi s line o
de ense agains plan pa hogens, wi h di ec consequences o plan
disease ou comes5–7. Because o his, ad ancing ou mechanis ic
unde s anding on how a ia ions in he assembly o hizosphe e
mic obiomes influence plan disease is o pa amoun impo an o
p o ide inno a i e s a egies o imp o ing plan heal h and
p oduc i i y.
Recei ed: 2 No embe 2022
Accep ed: 24 No embe 2023
Check o upda es
1
S a e Key Labo a o y o T ee Gene ics and B eeding, College o Ecology and En i onmen , Nanjing Fo es y Uni e si y, Nanjing 210037, China.
2
S a e Key
Labo a o y o Soil & Sus ainable Ag icul u e, Ins i u e o Soil Science, Chinese Academy o Sciences, Nanjing 210008, China.
3
Jiangsu Key Labo a o y o
Mic obes and Func ional Genomics, College o Li e Sciences, Nanjing No mal Uni e si y, Nanjing 210023 Jiangsu, China.
4
Ecological Expe imen al S a ion o
Red Soil, Chinese Academy o Sciences, Ying an 335211, China.
5
Depa amen o de Mic obiología, Facul ad de Ciencias, Campus Uni e si a io de Tea inos s/n,
Uni e sidad de Málaga, 29010 Málaga, Spain.
6
Ins i u o de Ho o u icul u a Sub opical y Medi e ánea La Mayo a (IHSM) UMA-CSIC, 29010 Málaga, Spain.
7
Ins i u e o Biology, Leiden Uni e si y, Syl iusweg 72, 2333 BE Leiden, The Ne he lands.
8
Depa men o Mic obial Ecology, Ne he lands Ins i u e o Ecology
(NIOO-KNAW), D oe endaalses eeg 10, 6708 PB Wageningen, The Ne he lands.
9
College o Resou ces and En i onmen al Science, Nanjing Ag icul u al
Uni e si y, Nanjing 210095, China.
10
Co-Inno a ion Cen e o Sus ainable Fo es y in Sou he n China, Nanjing Fo es y Uni e si y, Nanjing 210037, China.
11
Labo a o iode Biodi e sidad y Funcionamien o Ecosis émico, Ins i u o de Recu sos Na u ales y Ag obiología de Se illa (IRNAS), CSIC, Se illa, Spain.
12
These
au ho s con ibu ed equally: Yanyan Zhou, Zhen Yang. e-mail: xgli@nj u.edu.cn
Na u e Communica ions | (2023) 14:8126 1
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C op managemen is known o ha e a c i ical ole in egula ing
hizosphe e mic obiomes. In China, peanu ep esen s one o he mos
p ofi able c ops. Howe e , he yield and quali y o his p oduc ha e
been epo ed o be g ea ly comp omised by soil-bo ne diseases,
especially unde in ensi e monoc opping managemen s. In sub-
opical China, 10–40% o c op yield is los due o inc easing disease
p essu e, wi h peanu oo o caused by ungal pa hogens being a
majo cons ain 8. Un o una ely, ew e ec i e con ol measu es a e
a ailable, and he use o chemical pes icides is limi ed by en i on-
men al conce ns9. C op o a ions, including di e en c op a ie ies,
ha e been p oposed o mi iga e he nega i e impac s o pa hogens on
c op p oduc ion, by b eaking he link be ween plan hos and pa ho-
gens, becoming a non-expensi e al e na i e o disease con ol. Ye ,
how managemen by egula ing he impac o hizosphe e mic obial
communi ies on peanu oo o diseases emains la gely unknown.
Mic obial and plan -based ools, such as syn he ic mic obial
communi ies (SynComs) and oo -de i ed me aboli es, a e p omising
s a egies o figh agains soil-bo ne pa hogens and plan disease, and
can help o p omo e plan heal h. Howe e , wha s a egies wo k bes
unde con as ing managemen emains poo ly unde s ood. Recen
s udies ha e shown ha hizosphe e mic obiome assembly is sig-
nifican ly influenced by he selec ion e ec s o plan oo me abolisms.
As a esul , di e en ou comes o plan heal h can be os e ed
depending on which mic obial popula ions a e able o ake ad an age
o he oo me aboli es10,11. Thus, ag icul u al p ac ices could po en-
ially exploi he di e ences in oo me abolisms o a ious c ops o
dis up he di ec ional selec ion o main aining he s abili y o ag i-
cul u al ecosys em12,13. Simila ly, SynComs a e cu en ly being de el-
oped as bio-p oduc s o con ol soil-bo ne disease. Ye , he e a e
needs o unde s anding he impac s o hese SynComs on c op hi-
zosphe e and as managemen ools o plan heal h.
He e, we conduc ed mul iple expe imen s o un a el he influ-
ence o c op managemen on he capaci y o he hizosphe e mic o-
biome o suppo disease supp ession. The findings e ealed ha
unde peanu monoc opping, he e was a selec i e impo e ishmen o
key bac e ia, wi h weake esponses o oo exuda es, bu ha we e
associa ed wi h he capaci y o hizosphe e o supp ess pa hogen
in asion. The es o a ion o hese bac e ia b ough back he capaci y
o soils o figh disease supp ession. Ou s udy ad ances ou knowl-
edge on how o de elop inno a i e s a egies o p o ide a o able
suppo o sus ainable ag icul u e.
Resul s
Monoc opping agg a a es peanu oo o se e i y
To de e mine he e ec s o di e en ag icul u al egimes on peanu
heal h, we conduc ed a field expe imen om 2012 o 2016 aiming o
quan i y he occu ence o peanu (A achis hypogaea L.) oo o unde
monoc opping and o a ion egimes (Fig. 1a). Resul s indica ed ha
disease index (DI) apidly inc eased om 2012 o 2016 in monoc op-
ping peanu s ha we e examined a flowe ing s age, compa ed wi h
c ops subjec ed o o a ions (DI
2012
= 2.1, DI
2016
=8.0,Fig.2a, b). In ac ,
we showed ha he DI o peanu s in o a ion sys ems emained s able
(DI
2012
=1.6, DI
2016
= 3.1, Fig. 2b). We u he showed ha he DI o
monoc opping and o a ion began o di e ge in 2014, wi h a sig-
nifican ly highe DI in monoc opping compa ed wi h c ops subjec ed
o o a ion (S uden ’s - es , =8.276,d =16,P< 0.001, Fig. 2b). These
esul s indica ed ha long- e m monoc opping can agg a a e peanu
oo o disease.
Based on he di e ence o DI be ween monoc opped and o a ion
peanu s a flowe ing s age, we specifically acked he changes in DI a
di e en peanu g ow h s ages in 2018. A peanu seedling s age, he
DI o peanu s g own on monoc opped and o a ion plo s we e e y
low (DI
o a ion
=3.87, DI
monoc opping
=4.38), and we did no find sig-
nifican di e ences in DI o he wo egimes a his pa icula s age
(S uden ’s - es , =−0.660, d =16,P=0.519,Fig.2c). Howe e , om
flowe ing s age, he DI in peanu s unde monoc opping egime d a-
ma ically inc eased, and was 2.6 imes highe han ha o peanu s
sown unde he o a ion egime a pod-bea ing s age (S uden ’s - es ,
=−7.901, d =16,P< 0.001, Fig. 2c), indica ing he amplified se e i y
o oo o du ing de elopmen al s age.
To iden i y he pa hogen associa ed wi h his oo o disease, we
cha ac e ized he communi y composi ion o ungi in heal hy and
diseased peanu oo s using Illumina sequencing (Fig. 1b). A o al o
347,214 in e nal ansc ibed space 1 (ITS1) eads we e ob ained
( ange, 31,995–44,592 eads pe sample), clus e ing in o 183 ungal
OTUs a ≥97% sequence iden i y. Se e al axa om Fusa ium sp. ha e
been epo ed wi h abili y o cause wil ing symp oms in peanu s, bu
specific pa hogen lying behind peanu oo o s emains o be
disco e ed14,15. Resul s indica ed OTU177 ( axonomically assigned o F.
oxyspo um)andOTU90( axonomicallyassigned oF. solani)we e
significan ly en iched in diseased peanu oo . The ela i e abundance
o hese wo ungal species was 34.1 and 2712.7 imes highe in diseased
han in heal hy peanu oo s espec i ely (Fig. 2d). In o de o ob ain
cul u es associa ed wi h he po en ial pa hogens, we isola ed 38 ungi
om he diseased peanu oo s. Based on g ow h mo phology, 20
isola es we e selec ed o 18S RNA sequencing and subsequen ly we e
iden ified as F. oxyspo um (5), Penicillium sp. 196F (1), F. solani (3),
Tala omyces pinophilus (2), Tala omyces e uculosus (4), and Neo-
cosmospo a s ia a (5) (Supplemen a y Fig. 1). Nex , we confi med he
pa hogenici y o he wo isola es linked wi h F. oxyspo um and F. solani.
F. oxyspo um was highly pa hogenic (51 ± 11%), esul ing in significan ly
highe disease incidence han ha obse ed upon inocula ion wi h F.
solani (35 ± 9%) (S uden ’s - es , = 5.580, d =58,P< 0.001) (Fig. 2e).
Since o he Fusa ium we e no en iched and cul i able, we iden ified
ou isola ed s ain (F. oxyspo um) as he mos likely o ganism behind
he oo o disease obse ed in he s udied peanu fields.
We conduc ed u he quan i a i e eal- ime PCR (qPCR) analyses
o gain deepe insigh s in o he abundance and dynamics o F. oxy-
spo um in he peanu hizosphe e a di e en g ow h s ages. Con-
sis en wi h he esul s o he disease index, he e was no significan
di e ences be ween monoc opping and o a ion egimes in F. oxy-
spo um abundance a he peanu seedling s age (S uden ’s - es ,
=−1.525, d =10,P= 0.158, Fig. 2 ). Howe e , F. oxyspo um abundance
in he hizosphe e o monoc opped peanu was significan ly highe
han ha o o a ion du ing he nex de elopmen al s ages (S uden ’s
- es , =2.980,d =10,P<0.05,Fig.2 ). Dynamics in pa hogen accu-
mula ion a e seedling s age in monoc opped peanu showed a pa -
allel pa e n o ha in peanu oo o disease, significan ly a ec ing
plan g ow h and educing plan yield (S uden ’s - es , P<0.05,Sup-
plemen a y Fig. 2). Taken oge he , ou esul s sugges ed ha he
e ec i e coloniza ion o F. oxyspo um in he peanu hizosphe e has
c i ical consequences o plan heal h in c ops subjec ed o
monoc opping.
C op managemen s ha e significan e ec s on he peanu hi-
zosphe e mic obiome
The bac e ial mic obiome plays a c ucial ole in influencing he
se e i y o soil-bo ne diseases compa ed o ungi. Plan oo s can
e ec i ely ec ui a highe di e si y and ichness o he hizosphe e
bac e ial communi y o supp ess he in asion o pa hogens, pa icu-
la ly hose o igina ing om ungal sou ces16–18. The e o e, we in es i-
ga ed he impac s o c op managemen s on he bac e ial mic obiome
o peanu hizosphe e a di e en plan g ow h s ages and bulk soil in
ou 2018 field expe imen . In o al, we ob ained 1,369,637 sequences
om 48 soil samples. Nonme ic mul idimensional scaling (NMDS)
based on he B ay–Cu is dissimila i y ma ix e ealed significan di -
e ences in bac e ial communi y o monoc opping and o a ion a all
de elopmen s ages (ANOSIM, P< 0.001, Fig. 3a). Sequen ially, we
compa ed he a ia ion o hizosphe e bac e ial communi y be ween
monoc opping and o a ion a seedling s age. Resul s indica ed ha
A icle h ps://doi.o g/10.1038/s41467-023-43926-4
Na u e Communica ions | (2023) 14:8126 2
183 OTUs we e en iched and 156 OTUs we e deple ed by monoc op-
ping (P
adjus ed
<0.05,Fig.3b). Disc imina ing OTUs mainly clus e ed in
P o eobac e ia and Ac inobac e io a (Fig. 3c). Fu he , he a e age
ela i e abundance o 122 deple ed OTUs (accoun o 82.4% o he
disc imina ing OTUs), and 148 en iched OTUs (accoun o 83.1% o he
disc imina ing OTUs) in monoc opping was <0.1% (Fig. 3c, d).
Rhizosphe e mic obiome o c ops unde o a ion supp ess he
in asion o F. oxyspo um
To examine he ole o hizosphe e communi y in he occu ence o
peanu oo o , we u he collec ed soils om field plo s o bo h
monoc opping and o a ion o g ow peanu a a g eenhouse expe i-
men (Fig. 1c). The hizosphe e mic obiome o peanu s a seedlings
was ha es ed o de e mine he abili y o F. oxyspo um inhibi ion.
An i ungal ac i i y agains F. oxyspo um was es ed in an agonism
assay o bo h ola ile o ganic compounds (VOCs)-media ed and
di ec ed mic ocosms. The esul s showed ha he hizosphe e
mic obiomes o peanu s g own unde o a ion egime we e able o
supp ess he g ow h o F. oxyspo um,by45–56% highe han mono-
c opped egime (Fig. 4a, b, S uden ’s - es , P< 0.01). This highligh ed
he significance o an agonism in hizosphe e communi y agains
pa hogen in asion.
We hen in es iga ed he po en ial ole o VOCs in media ing
hese esul s. We knew ha VOCs p oduced by he hizosphe e bac-
e ial communi y can play an impo an ole in p e en ing ungal
pa hogen in ec ion19,20. Thus, we used gas ch oma og aphy–mass
spec ome y (GC-MS) o de e mine he composi ion o VOCs p o-
duced by he hizosphe e mic obiome o monoc opped and o a ion
peanu . O he VOCs de ec ed, dime hyl sulfide, 2,5-dime hylcyclo-
hexanone, and 6-me hyl-3,5-pen adien-2-one p oduced by he
Monoc opping
Ro a ion
a
Field expe imen
×3 plo s
×3 plo s
Cul i able
po en ial pa hogens
Flowe ing
Isola ion and
ITS iden i ica ion Pa hogenici y su eillance
Communi y
analysis
Heal hy Diseased
DNA ex ac ion
Analysis o
pa hogenic ungal communi y
Sampling ime
●
●
●
●
●
●
●
●
●
●
●
●
−0.4
−0.2
0.0
0.2
−0.5 0.0 0.5
PCoA1 (53.43%)
PCoA2 (19.51%)
OTU90
OTU177
De elopmen al pe iod
Disease index
Disease dynamic cu e
Monoc opping oo s
b
Pa hogen de e mina ion
2012 2018
Peanu s
Maize Po a o Soybean
Peanu s Peanu s
Peanu s Peanu s
2014
Peanu s
Peanu s
2016
Peanu s
Peanu s
Peanu s Peanu s
Flowe ing
Seedling
Pod-bea ing
Monoc opping
Ro a ion
c
Po expe imen
Rhizosphe e soil
Cul i able
mic obiome
×10 po s
Communi y-pa hogen in e ac ion
An agonis ic
VOCs analysis
Isola ion and
16S RNA iden i ica ion
ATCG
+
Communi y
analysis
+
Func ional
analysis
Monoc opping Ro a ion
●
●
●
●
●
●
●
●
Quo um sensing
S aphylococcus au eus in ec ion
Two−componen sys em
ABC anspo e s
Phospho ans e ase sys em (PTS)
Fola e biosyn hesis
be a−Alanine me abolism
Tau ine and hypo au ine me abolism
Galac ose me abolism
Alanine, aspa a e and glu ama e me abolism
0.1 0.2 0.3 0.4
ko00250
ko00052
ko00430
ko00410
ko00790
ko02060
ko02010
ko02020
ko05150
ko02024
Di ec ed VOCs-media ed
2018
2012
2014
2016
Rhizosphe e
soil
Fig. 1 | Flow cha s o field expe imen s o peanu disease in es iga ion,
iden ifica ion o po en ial pa hogens and po expe imen . a F om 2012,
expe imen al plo s we e managed unde wo c opping egimes ( ea men s):
peanu monoc opping and o a ion. Fo monoc opping, peanu s we e con-
inuously plan ed om 2012 o 2018. Fo o a ion, peanu was g own fi s (2012),
and hen maize (Zea mays L.), po a o (Solanum ube osum), and soybean (Glycine
max) we e o dinally plan ed in e e y o he peanu plan ing yea . The disease index
o peanu was in es iga ed a he flowe ing s age in 2012, 2014, and 2016. A 2018,
peanu disease was in es iga ed a seedling, flowe ing, and pod-bea ing s age, and
he hizosphe e soil was sampled o communi y analysis. bRoo s om ma u e
monoc opped plan s wi h heal hy and diseased we e used o pa hogenic com-
muni y analysis and po en ial pa hogen iden ifica ion. cDu ing he end o 2018
plan ing season, soil samples we e collec ed om six plo s and used o po
expe imen s. The hizosphe e soil was collec ed a he seedling s age o cul u able
mic obiome–pa hogen in e ac ion, communi y analysis, and unc ional analysis.
A icle h ps://doi.o g/10.1038/s41467-023-43926-4
Na u e Communica ions | (2023) 14:8126 3
hizosphe e mic obiome om o a ion-g own peanu we e no
de ec ed in he hizosphe e mic obiome om monoc opped peanu
(Supplemen a y Fig. 3). Fu he , he ela i e con en s o α-aco enol,
dime hyl disulfide, and 1,3-xylene we e significan ly educed unde he
monoc opping egime compa ed wi h o a ion (S uden ’s - es ,
P< 0.05). Fu he in i o expe imen s o pa hogen supp ession using
s anda d VOCs (dime hyl sulfide, 2,5-dime hylcyclohexanone, 6-
me hyl-3,5-pen adien-2-one and 1,3-xylene), e ified ha hese specific
VOCs de ec ed in peanu hizosphe e o o a ion egime significan ly
inhibi ed pa hogen g ow h, e en a low concen a ions (0.5–5.0 μg/
mL; Supplemen a y Fig. 4).
In o de o p o ide a mo e de ailed unde s anding on he
mechanisms behind hese esul s, we u he pe o med an-
sc ip ome analysis o cul i able mic obiome om monoc opping
and o a ion hizosphe e o unde s and he associa ed unc ional
a ia ion o F. oxyspo um inhibi ion (Fig. 1c). We ex ac ed mRNA o
cul i able mic obiome om monoc opping and o a ion hizo-
sphe e a e he VOCs-media ed an agonism assay, and ansc ibed i
in o cDNA o sequencing. Pa hways en iched in di e en Kyo o
Encyclopedia o Genes and Genomes (KEGG) o hology unc ional
ca ego ies in cul i able hizosphe e mic obiomes om monoc op-
ping and o a ion we e analyzed. The me abolic pa hways ha we e
p ima ily esponsible o he di e ences in KO unc ional ca ego ies
in he monoc opping and o a ion included ABC anspo e s and
Two-componen sys em (Fig. 4c). To be specific, he exp ession o
genes associa ed wi h pa hogen inhibi ion, including Isopen enyl-
diphospha e Del a-isome ase (K01823), Glu ama e deca boxylase
(K01580), and 1-py oline-5-ca boxyla e dehyd ogenase (K00294)
we e significan ly highe in o a ion han ha in monoc opping
(Fig. 4d, S uden ’s - es , P< 0.05).
Cul i able hizosphe e mic obiome unde monoc opping and
o a ion
To unde s and whe he imma u e mic oflo a was associa ed wi h
dec eased hizosphe e esis ance in monoc opping seedlings, we fi s
collec ed cul i able hizosphe e mic obiome om he abo e an ag-
onis ic expe imen s. Conside ing he di e ence in inhibi ion abili y o
he hizosphe e mic obiome agains F. oxyspo um on aga pla es
be ween monoc opping and o a ion, we fi s compa ed he cha ac e -
is ics o he whole cul i able mic obiome by Illumina sequencing. The
numbe o sequences o he cul i able mic obiome anged om 14,804
o 22,748, wi h clus e ing as 714 OTUs a 97% simila i y. Since he cul i-
able mic obiome sequence came om all bac e ial colonies g owing on
aga pla es, we hen de e mined OTUs deple ed om monoc opping o
o a ion hizosphe e cul u es. Resul s indica ed 362 OTUs, mos
belonging o Bacillus,En e obac e ,Esche ichia−Shigella,Pan oea,
En e ococcus,Pseudomonas,Lysinibacillus,Paenibacillus,Kluy e a,and
Jeo galibacillus, we e no de ec ed in he hizosphe e o monoc opped
peanu (Supplemen a y Fig. 5). By con as , 257 OTUs, mos belonging o
Bacillus,Pseudomonas,Vi gibacillus,Bu kholde ia−Pa abu kholde ia,
Rals onia,Esche ichia−Shigella,Klebsiella,Gemma imonas,Acine o-
bac e ,Paenibacillus,andLysinibacillus, we e no de ec ed in he hizo-
sphe e o o a ion- egime peanu (Supplemen a y Fig. 5).
In o de o ob ain he deple ed and en iched s ains in mono-
c opping hizosphe e, we isola ed 173 bac e ial s ains om aga
pla es o hizosphe e cul u es o 16S RNA sequencing. These
Fig. 2 | In es iga ion o peanu disease and iden ifica ion o pa hogen in long-
e m field expe imen . a Symp oms o heal hy and diseased peanu s in he field.
bDisease index o peanu oo o om 2012 o 2016 a he flowe ing pe iod
(P<0.001,n= 9 biologically independen samples). cDisease index o peanu oo
o in monoc opping and o a ion egimes a 2018 ac oss di e en g owing pe i-
ods (P<0.001,n= 9 biologically independen samples). dSignifican ly inc eased
OTUs (Fusa ium sp.) in diseased oo s compa ed wi h heal hy oo s. −1.5 o 1.5
ep esen s he ela i e abundance o no malized OTUs. eTes o pa hogenici y o
he po en ial pa hogens Fusa ium spp. (P< 0.001, n= 30 biologically independen
samples). Ho izon al ba s wi hin boxes ep esen he median. The ops and bo -
oms o boxes ep esen 75 h and 25 h qua iles, espec i ely. The uppe and lowe
whiske s ep esen he ange o non-ou lie da a alues. The abundance o F.
oxyspo um in peanu hizosphe e o monoc opping and o a ion egimes in 2018
field expe imen (P<0.05,n= 6 biologically independen samples). As e isks
abo e he ba s indica e s a is ically significan di e ences be ween he ea men s
based on wo-sided es s by S uden ’s - es (*P< 0.05, **P< 0.01, ***P<0.001).Each
ba s ep esen s he mean ± SD.
A icle h ps://doi.o g/10.1038/s41467-023-43926-4
Na u e Communica ions | (2023) 14:8126 4
sequenced s ains we e mainly belonged o Paenibacillus sp., Pan oea
sp., Bacillus sp., Fic ibacillus sp., Lysinibacillus sp., En e obac e sp.,
Spo osa cina sp., Pseudomonas sp., Bu kholde ia sp., S eno-
ophomonas sp., Se a ia sp., A h obac e sp., Bu iauxella sp., and
B e undimons sp. (Supplemen a y Fig. 6). Fu he , in o de o de e -
mine he consis ency o deple ed o en iched OTUs wi h he abo e
s ains, we compa ed he simila i y be ween he 16 S RNA sequences
o he isola ed s ains and he OTUs ha we e deple ed o en iched in
he cul i able mic obiome o monoc opping hizosphe e (Fig. 4e).
When he sequence simila i y be ween isola ed s ains and deple ed o
en iched OTUs exceeded 97%, he s ain was conside ed ep esen a-
i e o he co esponding OTU. We excluded he 16S RNA sequence o
Bacillus sp., a highly abundan genus widely p esen in he peanu
hizosphe e, om he analysis. Among hem, s ains isola ed om he
hizosphe e o o a ion-g own peanu s, i.e., Paenibacillus sp. (R60),
Pan oea sp. (R05), Lysinibacillus sp. (R06), En e obac e sp. (R09),
Spo osa cina sp. (R07), Fic ibacillus sp. (R37), and Pseudomonas sp.
(R26), we e 97% simila o OTUs deple ed om he hizosphe e o
monoc opped peanu s, while s ains belonging o S eno ophomonas
sp. (C20) and Bu kholde ia sp. (C63) we e 97% simila o OTUs en i-
ched in he hizosphe e o monoc opped peanu s (Fig. 4e). Indeed, he
abundance o he OTUs associa ed wi h he deple ed s ains was e y
low in he o a ion plan hizosphe e (Fig. 4e).
Cha ac e is ics o deple ed s ains esponding o peanu oo
exuda es
Plan oo exuda es a e one o he sou ces ha egula e hizosphe e
mic obial assembly21. To unde s and whe he he deple ion o low
abundance o axa was associa ed wi h esponsi eness o plan oo
exuda es, we collec ed oo exuda es o peanu s g own on mono-
c opping and o a ion soil, and e alua ed he e ec s o oo exuda es
on bac e ial g ow h in i o. In his ega d, all deple ed bac e ial
s ains had less sensi i e esponse o oo exuda es o monoc opped
peanu as compa ed o ha om o a ion peanu (S uden ’s - es ,
P< 0.001, Fig. 4 ). Howe e , monoc opping oo exuda es p omo ed
he g ow h o Bu kholde ia and S eno ophomonas ha we e en i-
ched in monoc opping hizosphe e (S uden ’s - es , P< 0.001,
Fig. 4 ). Compa ed o he en iched s ains, he g ow h o deple ed
s ains inc eased less in esponding o oo exuda es om mono-
c opping peanu .
Nex , we de e mined po en ial plan g ow h-p omo ing p op-
e ies o he abo e s ains in i o. In o al, deple ed s ains o
monoc opping had po en ial g ow h-p omo ing p ope ies. O
hese, En e obac e and Pan oea p oduced side opho es, and
Bacillus and Spo osa cina solubilized o ganic phospho us (Supple-
men a y Table 1). Acco dingly, Paenibacillus and Lysinibacillus p o-
duced mo e han 25 mg indoleace ic acid (IAA) pe L. Excep Pan oea
and Lysinibacillus, all he o he deple ed s ains inhibi ed he g ow h
o F. oxyspo um (Supplemen a y Table 1). Ne e heless, Bu -
kholde ia, a monoc opping en iched s ain, solubilized phospho us
and p oduced side opho es and IAA, bu did no inhibi he ungal
g ow h.
Finally, we de e mined he in e ac ion be ween any wo deple ed
bac e ia. No significan enhancemen o inhibi ion was obse ed
be ween any wo s ains (Supplemen a y Fig. 7), indica ing indepen-
den g ow h and s able coexis ence among deple ed s ains.
●
●
●
●
●
●
●
●
Bulk soil
Pod-bea ing
Seedling
Flowe ing
●Monoc opping
Ro a ion
●●●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
−0.4
−0.2
0.0
0.2
0.4
−0.4 0.0 0.4
NMDS1
NMDS2
R=0.9499
P<0.001
S ess=0.10
0
10
20
30
40
−10 0 10
log2(FC)
−log10(pVal)
Down(156)
Up (183)
Monoc opping en iched(183) Monoc opping deple ed(156)
Acidobac e io a
Ac inobac e io a
Unassigned
Bac e oido a
Chlo o lexi
Fi micu es
P o eobac e ia
0
0 - 0.1%
0.1% - 1%
> 1%
OTU abundance
Monoc opping Ro a ion Monoc opping Ro a ion
d
ab
c
Fig. 3 | Changes o hizosphe e mic obial communi y in 2018 peanu season.
aNon-me ic mul idimensional scaling (NMDS) o mic obial communi ies in hi-
zosphe e o monoc opping and o a ion a seedling, flowe ing and pod-bea ing
s ages. bCompa ed wi h o a ion, en iched o deple ed OTUs in monoc opping
seedlings hizosphe e (DESeq2, P< 0.05, FDR adjus men ). c,dPhylogene ic ees
cons uc ed by en iched/deple ed OTUs in monoc opping seedlings hizosphe e
and he ela i e abundance.
A icle h ps://doi.o g/10.1038/s41467-023-43926-4
Na u e Communica ions | (2023) 14:8126 5

Deple ed s ains associa ed wi h pa hogen supp ession by hi-
zosphe e mic obiome
We nex asked why he deple ion o specifics ainsdep essed he
abili y o monoc opped peanu hizosphe e o supp ess F. oxyspo um.
We fi s analyzed he abili y o di e en SynComs con aining 7, 4, and 2
deple ed s ains o impac F. oxyspo um pa hogenici y (Fig. 5aand
Supplemen a y Table 2). O e all, SynCom con aining he 7, 4, o 2
deple ed s ains significan ly supp essed mycelial de elopmen o F.
oxyspo um (ANOVA, F= 38.483, P< 0.001, Fig. 5b). When he s ain was
inac i a ed, he inhibi ion disappea ed. Mo eo e , he inhibi ion e ec
was posi i ely co ela ed wi h he di e si y o he deple ed s ains.
When he e we e 7 deple ed s ains, SynCom showed he bes inhibi-
ion o F. oxyspo um. This indica ed ha all deple ed s ains pa icipa e
in he supp ession o ungal pa hogens ia syne gis ic in e ac ions22.
Nex , we supplemen ed suspension o SynComs wi h di e en
deple ed s ains o hizosphe e mic obiome om monoc opped
ab
R07 Spo osa cina
R06 Lysinibacillus
R37 Fic ibacillus
R60 Paenibacillus
R09 En e obac e
R05 Pan oea
R26 Pseudomonas
C20 S eno ophomonas
C63 Bu kholde ia
0
5
10
15
Rela i e abundance (× 10
-4
%)
OTU6966
OTU12223
OTU9456
OTU8675
OTU9041
OTU9439
OTU8770
OTU9294
OTU10342
cd
e
Monoc opping
Ro a ion
●
●
●
●
●
●
●
be a−Alanine me abolism
Two−componen sys em
Quo um sensing
Galac ose me abolism
ABC anspo e s
Tau ine and hypo au ine me abolism
Phospho ans e ase sys em (PTS)
Alanine, aspa a e and glu ama e me abolism
0.00 0.04 0.08 0.12 0.16
Rich Fac o
Pa hway Name
Gene Numbe
●
5
10
●
●
●
●
●
●
Monoc opping
Ro a ion
Rhizosphe e
bac e ial
communi y
F. oxyspo um
Isopen enyl-
diphospha e
Del a-isome ase
K01823
0.0
0.1
0.2
0.3
0.4
0.5
TPM
K01580 K00294
Glu ama e
deca boxylase
1-py oline-
5-ca boxyla e
dehyd ogenase
Ro a ion
Monoc opping
Pseudomonas
En e obac e
Spo osa cina
Paenibacillus
Lysinibacillus
Pan oea
Fic ibacillus
Bu kholde ia
S eno ophomonas
0.0
0.1
0.2
0.3
0.4
0.5
OD 600nm
***
***
***
*** ***
*** ***
***
***
0
20
40
60
80
100
Inhibi ion (%)
Di ec ed
Ro a ionMonoc opping Ro a ionMonoc opping
0
20
40
60
80
100
Inhibi ion (%)
VOCs-media ed
Ro a ion
Monoc opping
Ro a ion
Monoc opping
***
*
*** *** ***
Fig. 4 | Inhibi ion o pa hogens by hizosphe e mic oo ganisms in mono-
c opping and o a ion and de e mina ion o deple ed bac e ia in monoc op-
ping hizosphe e. a Di ec ed inhibi o y e ec o hizosphe e mic obial
communi y on F. oxyspo um (P=0.006,n= 3 biologically independen samples).
bVOCs-media ed inhibi o y e ec o hizosphe e mic obial com=muni y on F.
oxyspo um (P=0.006,n= 3 biologically independen samples). cEn ichmen ana-
lysis o di e en ial genes o cul u able mic oo ganisms in hizosphe e o mono-
c opping and o a ion. Rich Fac o , he a io o he numbe o di e en ial genes in
he me abolic pa hway o he numbe o all genes anno a ed o he pa hway (n=3
biologically independen samples). dAbundance o ep esen a i e genes asso-
cia ed wi h pa hogen inhibi ion in cul i able mic oo ganisms o monoc opping
and o a ion. TPM, ansc ip s Pe million (P< 0.001, n= 3 biologically independen
samples). Each ba s ep esen s he mean ± SD. eThe ela i e abundances o OTUs
in he monoc opping and o a ion samples. The blue ba s show he ela i e
abundance o monoc opping-en iched OTUs in monoc opping samples (<0.002%
in o a ion samples). The ed ba s show he ela i e abundance o monoc opping-
deple ed OTUs in o a ion samples (<0.002% in monoc opping samples). The nine
deple ed o en iched OTUs wi h >97% simila i y o isola es a e shown (n= 3 bio-
logically independen samples). Each ba s ep esen s he mean ± SEM. E ec s o
peanu oo exuda es om monoc opping and o a ion on g ow h o
monoc opping-deple ed and en iched bac e ia (P<0.001,n= 12 biologically
independen samples). OD, op ical densi y. As e isks abo e he ba s indica e s a-
is ically significan di e ences be ween ea men s based on wo-sided es s by
S uden ’s - es (*P< 0.05, **P< 0.01, ***P< 0.001). Each ba s ep esen s he
mean ± SD.
A icle h ps://doi.o g/10.1038/s41467-023-43926-4
Na u e Communica ions | (2023) 14:8126 6
peanu s and co-cul u ed on a pla e wi h F. oxyspo um (Fig. 5c). When
he monoc opping hizosphe e suspensions we e supplemen ed
wi h SynComs, he pa hogen g ow h was inhibi ed by 64.4%, wi h
he inhibi ion 4- old highe han ha o he con ol wi hou hizo-
sphe e suspension (ANOVA, F= 33.501, P< 0.001, Fig. 5d). By con-
a y, an inac i a ed SynCom did no make up o he supp ession
abili y o monoc opping hizosphe e mic obiome (Fig. 5d). The
supp ession abili y on pa hogen inc eased wi h he supplemen a-
ion o SynCom di e si y, indica ing deple ed s ains compensa e
he abili y o monoc opping hizosphe e mic obiome o esis
pa hogen in asion.
Fu he , we es ed he compensa o y e ec o he deple ed s ains
in expe imen s wi h s e ile peanu seedlings (Fig. 5e). Compa ed wi h
seedling inocula ion wi h he monoc opping hizosphe e suspension,
supplemen a ion wi h he deple ed s ains significan ly imp o ed
hizosphe e esis ance o F. oxyspo um in asion (ANOVA, F=71.755,
P< 0.001, Fig. 5 ). Consis en wi h in i o an agonism, he bes inhi-
bi o y e ec was ob ained when he SynCom composed o 7 s ains
was supplemen ed. In he ea men wi h no SynCom inocula ion, he
oo o p o ec ion collapsed. Howe e , compa ed wi h he ea men
wi h hizosphe e communi y, supplemen a ion o he deple ed s ains
did no a ec plan g ow h (ANOVA, P> 0.05, Supplemen a y Fig. 8).
h
7 s ains
4 s ains
2 s ains
d
7 s ains4 s ains2 s ains
An agonism
ab
e
g
SynCom
0/2/4/7 s ains
RhiCom
+
An agonism
F. oxyspo um RhiCom + SynCom
RhiCom
Con ol
0/2/4/7 s ains
Replenishmen expe imen in i o
Field expe imen
c
An agonism o SynCom in i o
Replenishmen expe imen in i o
Con ol
Con ol RhiCom 0 2 4 7
0
1
2
3
4
Mycelial diame e (cm)
(s ains)
abbccdde
Con ol RhiCom 0 2 4 7
0
20
40
60
80
100
120
Incidence (%)
(s ains)
abb c cd
abbc
247
0
20
40
60
80
100
120
Incidence (%)
Con ol
(s ains)
inac i a ed
7 s ains
21 35
combina ions
0 s ain
Mycelial diame e (cm)
(s ains)
Con ol 0 2 4 7
0
1
2
3
4
5
aa b b c
1 combina ion, 4 plo s
35 combina ion, 35 plo s
21 combina ion, 21 plo s
1 combina ion, 4 plo s
andomized block
Fig. 5 | Assessmen o supp ession abili y o monoc opping-deple ed s ains on
pa hogen de elopmen and peanu oo o . a O e iew o expe imen s pe -
o med o de e mine he supplemen al e ec o monoc opping-deple ed s ains
on pa hogen in asion. bE ec s o e-inocula ion o di e en s ain combina ions
on he g ow h o F. oxyspo um (P<0.001,n
con ol
=8,n
7
=8,n
4
=35×8,n
2
=21×8,
n
0
= 8; biologically independen samples). cO e iew o a eplenishmen expe i-
men pe o med o de e mine he inhibi o y e ec o he addi ion o deple ed
s ains on F. oxyspo um in i o. dInhibi ion o F. oxyspo um by monoc opping
hizosphe e communi y supplemen ed wi h di e en s ain combina ions
(P<0.001,
con ol
=6,n
7
=6,n
4
=35×6,n
2
=21×6,n
0
=6).eO e iew o a eplen-
ishmen expe imen pe o med o de e mine he disease index o peanu s in i o.
E ec o monoc opping hizosphe e communi y supplemen ed wi h di e en
s ain combina ions on oo o occu ence (P<0.001,
con ol
=5,n
7
=5,n
4
=35×5,
n
2
=21×5,n
0
=5).gRandom block design o a field ial plo . hE ec s o e-
inocula ion o di e en s ain combina ions on oo o occu ence in he field
(P<0.001,n
con ol
=4,n
7
=4,n
4
=35×4,n
2
= 21 × 4). In figu es b, d, and h, di e en
le e s abo e he ba s indica e s a is ically significan di e ences be ween ea -
men s. P alues we e calcula ed using ANOVA’s es based on wo-sided. RhiCom,
ea men wi h monoc opping hizosphe e communi y; SynCom, supplemen ed
syn he ic communi y composed o monoc opping-deple ed s ains o mono-
c opping hizosphe e communi y; 0, a SynCom composed o 7 inac i a ed deple-
ed s ains;2, SynComs composed o 2 deple ed s ains; 4, SynComs composed o 4
deple ed s ains; 7, a SynCom composed o 7 deple ed s ains. Eachba s ep esen s
he mean ± SD. Ho izon al ba s wi hin boxes ep esen he median. The ops and
bo oms o boxes ep esen 75 h and 25 h qua iles, espec i ely. The uppe and
lowe whiske s ep esen he ange o non-ou lie da a alues.
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Na u e Communica ions | (2023) 14:8126 7
Finally, we conduc ed field expe imen s o e i y he compensa-
o y e ec o he deple ed s ains on he occu ence o oo o in
monoc opped peanu s (Fig. 5g). Peanu seeds we e inocula ed wi h
SynComs composed o 7, 4, o 2 s ains by soaking. Resul s indica ed
compa ed wi h he con ol wi hou hei inocula ion, SynComs sig-
nifican ly educed he incidence o peanu oo o (ANOVA, F= 16.169,
P< 0.001, Fig. 5h). SynComs composed o 7 bac e ial s ains we e mos
e ec i e (Fig. 5h). Simila ly, no SynCom es ed significan ly p omo ed
peanu g ow h compa ed o he con ol (ANOVA, P> 0.05), indica ing
hei biocon ol e ec in he plan hizosphe e a he han a di ec
p omo ion o plan esis ance (Supplemen a y Fig. 9).
Discussion
In he cu en s udy, we in es iga ed he influence o ag icul u al
p ac ices on soil disease supp ession. We u he manipula ed he
mic obiome o ou soils aiming o b ing back he capaci y o hizo-
sphe e o figh pa hogens and disease. We ound ha he deple ion o
hizosphe e specific bac e ia in esponse o monocul u es o peanu
and i s associa ed oo exuda es can educe he capaci y o soils o
supp ess disease and u he de e mined he u u e esponses o plan
o disease ou comes. On he con a y, c op o a ions suppo ed soils
wi h a g ea e capaci y o supp ess disease, and we we e able o es o e
hizosphe e communi ies o p o ec soils om pa hogens using
mic obial innocula. Ou s udy adds o he consensus ha mono-
cul u es ha e a educed soildisease supp ession capaci y compa ed o
c op o a ions.
Ou s udy e ealed ha monocul u es had a educed soil disease
supp ession capaci y. In pa icula , ou fi e-yea field peanu expe i-
men , monoc opping significan ly inc eased he incidence o oo o
disease, compa ed wi h fields subjec ed o o a ion-g own peanu .
Thus, hese con as ing ag icul u al managemen s suppo ed opposi e
capaci y o con ol disease occu ence by p e en ing soil-bo ne
pa hogen in asion. We hen acked oo o occu ence o e an
en i e peanu g owing pe iod in 2018. The esul s showed ha disease
indices and pa hogen abundances we e no significan ly di e en
unde monoc opping and o a ion egimes a he seedling s age.
Howe e , he e was a d ama ic inc ease in disease incidence a la e
g ow h s ages in monoc opped peanu compa ed wi h o a ion-g own
peanu . In e es ingly, we ound significan di e ences in he hizo-
sphe e mic obiome o peanu s in monocul u e and c op o a ion om
seedling s age o he en i e plan de elopmen . These di e ences we e
associa ed wi h di e en disease ou comes, indica ing he impo ance
o hizosphe e mic obiome o he de elopmen o hese plan s and
hei disease. Recen s udies ha e shown ha di e ences in hizo-
sphe e mic obial composi ion o seedlings lead o changes in mic o-
bial unc ion, and e en ually di e en ia e in o wo di e en esul s
(diseased o heal hy)23. The e o e, ou wo k sugges s ha s eng h-
ening seedling managemen by ocusing on hizosphe e mic obiome
could con ibu e o main aining plan heal h.
We hen sough o unde s and he mechanisms behind he abili y
o he hizosphe e mic obiome o suppo pa hogen supp ession
du ing he seedling s age. As explained abo e, he abili y o he hi-
zosphe e mic obiome om monoc opped peanu s o inhibi pa ho-
gen was significan ly lowe han ha om o a ion-g own peanu s.
These esul s indica ed ha he mic oflo a a seedling s age could
de e mine he supp ession abili y o plan hizosphe e o pa hogen
in asion. The unde lying mechanisms behind inhibi ion would
associa e wi h he p oduc ion o ola ile o ganic compounds and
an ibio ics24,25. Fo ins ance, dime hyl sulfide, 2,5-dime hylcyclohex-
anone, and 6-me hyl-3,5-pen adien-2-one ha we e no de ec ed in he
hizosphe e mic obiome o monoc opping had s ong inhibi ion on
pa hogen g ow h e en a low concen a ions. In e ms o hizosphe e
mic obiome unc ion, he exp ession o genes associa ed wi h pa ho-
gen inhibi ion, including Isopen enyl-diphospha e Del a-isome ase,
Glu ama e deca boxylase, and 1-py oline-5-ca boxyla e
dehyd ogenase we e significan ly highe in o a ion compa ed wi h
monoc opping26. Ou esul s e ealed he po en ial mechanisms
behind he g ea e disease supp ession capaci y in c oplands unde
o a ion.
The nega i e influence o oo exuda es in monocul u e peanu
field on key bac e ia suppo ing supp ession may also explain he
educed capaci y o p o ec soils agains pa hogens and disease. This
highligh s he impo ance o low-abundance mic obial species eg-
ula ing plan hizosphe e esis ance, as he abili y o he communi y
assembly wi hou hem in he monoc opped peanu hizosphe e o
supp ess pa hogen g ow h was educed. In ou s udy, he g ow h-
p omo ing e ec o monoc opping oo exuda es on Bu kholde ia sp.
and S eno ophomonas sp. may lead o he occupa ion o ecological
niche, and hus a ec he hizosphe e deple ion o bac e ia wi h a
weak esponse o monoc opping oo exuda es such as Paenibacillus
sp., Pan oea sp., Lysinibacillus sp., En e obac e sp., Spo osa cina sp.,
Fic ibacillus sp., and Pseudomonas sp. Acco dingly, he en iched
s ains p e e en ially occupy oo esou ces, and he deple ed axa a e
he eby unable o e ec i ely pa icipa e in communi y assembly in he
monoc opped hizosphe e.
We hen in es iga ed whe he ein oducing he deple ed mic o-
bial axa could b ing back he capaci y o soils o esis pa hogen
in asions and a oid disease. Despi e he g owing in e es in SynComs,
he de ailed mechanisms by which hey ope a e emain la gely
unknown. We fi s de e mined he inhibi o y capaci y o mic obial
communi ies composed o andom combina ions o he dele ed
s ains in monocul u e compa ed wi h o a ion c oplands. Ou esul
e ealed ha mic obes ha we e los unde monocul u e, pa icipa ed
in he supp ession o ungal pa hogens ia syne gis ic in e ac ions. We
secondly ein oduced hese s ains in o he hizosphe e o mono-
c opped peanu s. The supplemen a ion significan ly alle ia ed
pa hogen a ack o peanu oo , s ongly suppo ing he no ion ha
he addi ion o low abundance mic obes du ing communi y succes-
sion can imp o e he o e all unc ion o he hizosphe e communi y27.
The inhibi o y capaci y o mic obial communi ies composed o an-
dom combina ions o s ains was consis en wi h an inc eased phylo-
gene ic di e si y, sugges ing ha all deple ed s ains pa icipa e in he
supp ession o ungal pa hogens ia syne gis ic in e ac ions22.Indeed,
ou mic obial inocula ion expe imen s e ealed ha he ein oduc-
ion o deple ed s ains s ongly p o ec ed he plan oo agains
pa hogen in ec ion, while ailing o p omo e plan g ow h. Da a
ob ained by di e en app oaches sugges s ha he subs an ial pa i-
cipa ion o he deple ed s ains in he esiden communi y assembly
could de e mine hei success in ulfilling unc ional se ices in he
hizosphe e4,28.
Applica ions o single- unc ion mic obes o SynComs composed
o mul i- unc ional mic obes gene ally ha e sa is ac o y ou comes in
he labo a o y se ing bu no in p ac ice. Indeed, lack o conside a ion
o he app op ia e ecological niches o unc ional s ains, ac o s
d i ing he a ia ion in esiden mic obial in e ac ions, and he impac
o en i onmen al ac o s on mic obial coloniza ion abili y can ha e
undesi able e ec s29.In hefield expe imen s p esen ed in he cu en
s udy, SynCom composed o 7 deple ed s ains showed an excellen
abili y o p e en oo o . SynComs composed o some deple ed
s ains also con olled oo o , sugges ing he s abili y o hei appli-
ca ion. Recen ly, a simplified SynCom, de eloped based on hos -
media ed mic obial communi y selec ion, had e ec i ely cap u ed he
dominan membe s o he maize oo mic obio a, inhibi ing pa hogen
coloniza ion30. Howe e , i is unlikely ha he such simplified SynComs
would ake o e all he unc ions o hizosphe e mic obio a31.Con-
e sely, specific mic obial axa ha d i e esiden mic obial commu-
ni y assembly can ampli y hizosphe e communi y unc ions. Fo
ins ance, he addi ion o biological agen s con aining Bacillus amylo-
lique aciens W19 p o ec ed banana om ungal pa hogen in ec ion by
ac i a ing specificplan -beneficial bac e ial gene a (e.g. Pseudomonas)
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o o m a plan -benefi ing conso ium27. Consequen ly, he cu en
s udy seeks o a ionally con ol plan disease om a holis ic mic obial
pe spec i e, in ha SymComs should swi ch co e om basic unc ions
o ac i a e communi y unc ionali y.
Taken oge he , ou esul s indica e ha managemen and hi-
zosphe e mic obiomes play a ole in suppo ing soil pa hogen sup-
p ession agains impo an soil disease. Ou wo k shows ha ,
compa ed wi h o a ion managemen s, in ensi e monoc opping s a-
egies weakened he capaci y o soils o p e en he en ance o
pa hogens o he hizosphe e, and p omo ed he de elopmen o soil-
bo ne diseases. Impo an ly, we p o ide expe imen al e idence ha
pa hogen esis ance in weakened hizosphe es may be es o ed by
applying a ge ed mic obial inocula. The e o e, ou s udy highligh s
he ole o managemen o figh agains soil c op disease and sugges s
he ac i e es o a ion o hizosphe e communi ies o p omo e soil
disease supp ession.
Me hods
Field si e desc ip ion and disease assessmen
F om Ap il 2012, we conduc ed a field expe imen a Yujiang Coun y,
Jiangxi P o ince, China. The soil a he s udy si e is classified as Udic
Fe osol (FAO classifica ion), co e ing an a ea o ~1 × 108ha in sou he n
China. Because o low soil o ganic ma e con en and e ili y, and
clima ic sui abili y, peanu (A achis hypogaea L.) is a pa icula ly
popula c op in he egion. The field expe imen included wo c op-
ping egimes ( ea men s): (1) monoc opping wi h peanu , he same
peanu cul i a (Ganhua-5) was consecu i ely plan ed o he g owing
season (Ap il–Augus ) o each yea ; and (2) o a ion, peanu was g own
fi s (2012), and hen maize (Zea mays L.), po a o (Solanum ube osum),
and soybean (Glycine max) we e o dinally plan ed in e e y o he
peanu plan ing yea (Fig. 1a). Th ee plo s o he wo c opping egimes
we e laid ou in a andomized block design, and lay allow a e ha es
un il he ollowing sowing pe iod. Commonly used managemen
p ac ices, including illage, e ilize applica ion, and weed con ol,
we e applied manually32.
In he 2012, 2014, and 2016, all plo s we e plan ed wi h peanu ,
and he se e i y o oo o s was pe sis en ly e alua ed a flowe ing
s age. In 2018 peanu plan ing season, we u he examined peanu
oo o s a seedling, flowe ing and pod-bea ing s ages (Fig. 1a). Fo
each examina ion, 30 plan s om each plo we e ca e ully emo ed
om he soil, and peanu oo o was e alua ed using a fi e-class
a ing scale (0 = no lesions, 1 = small oo lesions, 2 = cen al oo
lesions, 3 = la ge oo lesions, 4 = dead plan )15. O e all, 540 plan s (6
plo s, 3 ime poin s) we e emo ed om he plo s o examina ion.
Following a gen le wash wi h ap wa e , he plan heigh , shoo and
oo esh weigh , oo leng h, and nodule numbe we e de e mined a
each sampling.
Sample collec ion and p ocessing
Bulk soil and hizosphe e soil samples we e collec ed be o e he pea-
nu s ha es in 2018. Be o e peanu plan ing, 10 soil co es wi h a dep h
o 20 cm we e andomly selec ed om each plo . E e y 5 soil co es
we e ully mixed in o a singlecomposi e soil sample as bulk soil. Excess
soil on he oo s was disca ded by gen ly shaking he plan s, and he
emaining soil pa icles a ached o he oo su ace we e collec ed as
hizosphe e soil. A seedling, flowe ing, and pod-bea ing s age, 10
peanu s we e andomly selec ed om each plo , and hizosphe e soil
o e e y 5 peanu plan s was ully mixed in o a single composi e soil
sample. In o al, 36 soil samples (6 plo s, 3 ime poin s) we e collec ed.
Soil DNA o hizosphe e soil collec ed om hizosphe e and bulk soil
wa e ex ac ed o mic obial communi y analysis. Roo s om heal hy
o se e e disease peanu s a pod-bea ing s ages we e su ace-s e ilized
in 3% hyd ogen pe oxide, and washed wi h s e ile wa e and 70%
e hanol. Excess fluid on he s e ilized oo su ace was wiped o using
s e ilized fil e pape s and oo samples we e cu in o pieces. All he
oo samples we e s o ed a −80 °C be o e DNA ex ac ion. In addi ion,
a po ion o diseased peanu oo s we e used o isola e po en ial
pa hogens.
Sequencing analysis o endophy ic ungal communi y
Roo samples we e g ound in liquid ni ogen and DNA was ex ac ion
using he MiniBEST Plan Genomic DNA Ex ac ion Ki (Taka a, Japan)
(Fig. 1b). Specific sequences we e amplified using he p ime pai s
ITS1F(5’-CTTGGTCATTTAGAGGAAGTAA-3’)/ITS2(5’-GCTGCGTTCTTC
ATCGATGC-3’). The samples we e ini ially dena u ed o 3 min a 95 °C;
his was ollowed by 27 cycles o dena u a ion (95 °C, 30 s), annealing
(55 °C, 30 s), and elonga ion (72 °C, 45 s). The PCR p og am ended wi h
a 10-min incuba ion a 72 °C. The PCR p oduc s we e sepa a ed by
elec opho esis on 1% aga ose gel. All samples we e pooled in equi-
mola concen a ions and hen sequenced wi h a pai ed-end p o ocol
a Majo bio Bio-Pha m Technology Co. L d. (Shanghai, China) using
he Illumina MiSeq pla o m, acco ding o he manu ac u e ’s
ins uc ions. Raw as q files we e quali y-fil e ed using QIIME33.Low-
quali y sequences (<150 bp long, wi h an a e age quali y sco e <25)
we e emo ed. The eads we e immed and assigned based on unique
7-base ba codes. The ba code and p ime sequences we e hen
emo ed. The o wa d and e e se eads we e inco po a ed in o ull-
leng h sequences based on he h esholds: o e lap leng h >10 bp and
misma ch a io <0.2. A e disca ding unqualified eads, he OTUs we e
assigned a 97% iden i y simila i y le el using UPARSE34. Chime ic
sequences we e iden ified and emo ed using UCHIME35. Taxonomic
assignmen was pe o med using UNITE da abase ( 7.0) o ungi36.
Isola ion and iden ifica ion o po en ial pa hogens
Po en ial ungal pa hogens we e isola ed om peanu oo s ha dis-
played disease symp om37.B iefly, a clean kni e was used o cu he
pa hogen-in ec ed oo s in o sec ions. The oo sec ions we e su ace-
s e ilized (subme ged in 4 / sodium hypochlo i e o 5 min), washed
( wo imes, in s e ile dis illed wa e ), and placed on a PDA pla e con-
aining s ep omycin and penicillin (20 μg/mL) o ob ain ungal
isola es38,39. DNA was ex ac ed om each ungal cul u e by using he
Fas DNA Spin Ki (MP Biomedicals, San a Ana, CA), acco ding o he
manu ac u e ’s ins uc ions. DNA sequences om he ungal 18S RNA
egion we e amplified using p ime s NS1(5′- GTAGTCATATGCT
TGTCTC-3′)andNS8(5′-TCCG-CAGGTTCACCTACGGA-3′)40.ThePCR
mix u e con ained (pe 50 μL) 1.5 U o Taq polyme ase (Red Taq, Sigma
Chemical Co.) and he ollowing eagen s: 1 × Sigma PCR bu e ,
0.20 mM PCR nucleo ide mix (P omega), 4.0mM MgCl
2
,6.25mg
bo ine se um albumin (Roche Diagnos ics), and 25 pmol o each p i-
me . Fo he amplifica ion eac ion, he DNA samples we e ini ially
dena u ed o 3 min a 95 °C. This was ollowed by 35 cycles o dena-
u a ion (94 °C, 30 s), annealing (57 °C, 30 s), and elonga ion (72 °C,
105 s).The PCR p og am ended wi h a 2-min incuba ion a 72 °C40.Each
agmen was compa ed phylogene ically o sequences o known
species in he GenBank da abase o he Na ional Cen e o Bio-
echnology In o ma ion (NCBI) by using BLAST. Phylogene ic ees
we e analyzed using MEGA 5. Phylogene ic ees we e cons uc ed by
using he neighbo -joining (NJ) me hod.
De e mina ion o pa hogenici y o po en ial pa hogens
Po en ial pa hogens we e highly en iched isola es om he diseased
oo s, including F. oxyspo um and F. solanum. A ungal plug (6-mm
diame e ) was ans e ed o PDA medium and cul u ed a 28 °C o
7 days. Ob ained e men a ion solu ion was fil e ed o emo e
mycelia, and cen i uged o 10 min (3000 × g) o e ain p ecipi a-
ion. The p ecipi a ed spo es we e e-suspended wi h s e ile wa e ,
and he concen a ion o spo e suspension was adjus ed o 109CFU/
mL. To confi m pa hogenici y o po en ial pa hogens, peanu s we e
plan ed in glasshouse (30 °C, 70% ela i e humidi y, ligh in ensi y
500 μMm
−2s−1). 10 mL spo e suspensions o F. oxyspo um o F.
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