jou nal o en i onmen al sciences 127 (2023) 222–233
A ailable online a www.sciencedi ec .com
w w w . e l s e i e . c o m / l o c a e / j e s
E alua ion o a c op o a ion wi h biological
inhibi ion po en ial o a oid N
2
O emissions in
compa ison wi h syn he ic ni i ica ion inhibi ion
Ad ián Bozal-Leo i
1 , ∗, Ma io Co ochano-Monsal e
1
, Luis M. A egui
2
,
Ped o M. Apa icio-Tejo
3
, Ca men González-Mu ua
1
1
Depa men o Plan Biology and Ecology, Uni e si y o he Basque Coun y (UPV/EHU), Apdo. 644, E-48080, Bilbao
48940, Spain
2
Ins i u e o Inno a ion and Sus ainable De elopmen in Food Chain (ISFOOD), Public Uni e si y o Na a e,
Pamplona 31006, Spain
3
Ins i u e o Mul idisciplina y Resea ch in Applied Biology (IMAB), Public Uni e si y o Na a e, Pamplona 31006,
Spain
a i c l e i n o
A icle his o y:
Recei ed 17 Janua y 2022
Re ised 22 Ap il 2022
Accep ed 25 Ap il 2022
A ailable online 4 May 2022
Keywo ds:
Fallow
So ghum
C op o a ion
Ni i ica ion inhibi o
N-cycling genes
Soil mine al ni ogen
a b s a c
Ag icul u e has inc eased he elease o eac i e ni ogen o he en i onmen due o
c ops’ low ni ogen-use e iciency (NUE) a e he applica ion o ni ogen- e ilise s. P ac-
ices like he use o s abilized- e ilise s wi h ni i ica ion inhibi o s such as DMPP (3,4-
dime hylpy azole phospha e) ha e been adop ed o educe ni ogen losses. O he wise, co e
c ops can be used in c op- o a ion-s a egies o educe soil ni ogen pollu ion and bene i
he ollowing cul u e. So ghum ( So ghum bicolo ) could be a good candida e as i is d ough
ole an and i s cul u e can educe ni ogen losses de i ed om ni i ica ion because i ex-
uda es biological ni i ica ion inhibi o s (BNIs). This wo k aimed o e alua e he e ec o
allow-whea and so ghum co e c op-whea o a ions on N
2
O emissions and he g ain
yield o win e whea c op. In addi ion, he sui abili y o DMPP addi ion was also analyzed.
The use o so ghum as a co e c op migh no be a sui able op ion o mi iga e ni ogen
losses in he subsequen c op. Al hough so ghum–whea o a ion was able o educe 22%
he abundance o amoA , i p esen ed an inc emen o 77% in cumula i e N
2
O emissions
compa ed o allow–whea o a ion, which was p obably ela ed o a g ea e abundance o
he e o ophic-deni i ica ion genes. On he o he hand, he applica ion o DMPP a oided he
g ow h o ammonia-oxidizing bac e ia and main ained he N
2
O emissions a he le els o
un e ilized-soils in bo h o a ions. As a conclusion, he use o DMPP would be ecommend-
able ega dless o he o a ion since i main ains NH
4
+
in he soil o longe and mi iga es
he impac o he c op esidues on ni ogen soil dynamics.
© 2022 The Resea ch Cen e o Eco-En i onmen al Sciences, Chinese Academy o
Sciences. Published by Else ie B.V.
This is an open access a icle unde he CC BY license
( h p://c ea i ecommons.o g/licenses/by/4.0/ )
∗Co esponding au ho .
E-mail: [email p o ec ed] (A. Bozal-Leo i).
h ps://doi.o g/10.1016/j.jes.2022.04.035
1001-0742/© 2022 The Resea ch Cen e o Eco-En i onmen al Sciences, Chinese Academy o Sciences. Published by Else ie B.V. This is
an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ )
jou nal o en i onmen al sciences 127 (2023) 222–233 223
In oduc ion
Since he beginning o he g een e olu ion, he applica-
ion o ni ogen (N) e ilise s o ag icul u al c ops has in-
c eased he le el o eac i e ni ogen p esen in he biosphe e
( Subba ao e al., 2017 ). Ammonium (NH
4
+
) can be p esen in
he soil a e being applied wi h e ilise s, bu also be e-
leased om o ganic ma e hanks o mic obial ac i i y in a
p ocess called “mine alisa ion”( Coskun e al., 2017 ). In ae obic
soils, NH
4
+ is oxidised by chemoli hoau o ophic ammonia-
oxidising bac e ia (AOB) and a chaea (AOA) in wha is known
as ni i ica ion. None heless, ni i ica ion is mainly d i en by
AOB a he han AOA in soils ecei ing ni ogen- e ilise s
( Di e al., 2009 , 2010 ). Fi s , ni i ie s oxidise NH
4
+
o hyd ox-
ylamine (NH
2
OH) h ough he enzyme ammonium monooxy-
genase (AMO) which is encoded by he amoA gene ( A p and
S ein, 2003 ). NH
2
OH is hen con e ed o ni i e (NO
2
−) and
inally ni i e-oxidising bac e ia (NOB) oxidise i o ni a e
(NO
3
−) ( Könneke e al., 2005 ). As an anion, NO
3
−is suscep-
ible o be los h ough leaching because i s nega i e cha ge
is epelled by nega i ely cha ged soil colloids ( Fiencke e al.,
2005 ). In anoxic condi ions, NO
3
−is he subs a e o he deni-
i ica ion p ocess. Du ing deni i ica ion, NO
3
−is sequen ially
educed o NO
2
−, ni ic oxide (NO) and ni ous oxide (N
2
O)
by enzymes encoded by he genes na G, ni K, ni S and no B
( Hochs ein and Tomlinson, 1988 ). In his p ocess, he emission
o N
2
O is a ha m ul h ea o he en i onmen since N
2
O is a
gas wi h a global wa ming po en ial (GWP) ha is 265 ime
g ea e han ha o CO
2
in a 100-yea pe iod ( IPCC, 2014 ). Fi-
nally, bac e ia ha bou ing nosZI o nosZII genes can ca y ou
a comple e educ ion o N
2
O o N
2
. None heless, 40% o deni-
i ie s lack o he genes o pe o m his las s ep ( Hallin e al.,
2018 ).
Because o hese ni ogen leaks and ans o ma ions, ag i-
cul u e p esen s a low ni ogen-use-e iciency (NUE), since
c ops only assimila e an a e age o 30%–50% o he ni o-
gen applied wi h e ilise s ( Wendebo n, 2020 ). This leads
ield-c op ag icul u e, such as whea , o be esponsible o
mo e han 61% o o al global an h opogenic N
2
O emissions
( Mon zka e al., 2011 ). The e o e, we mus guide ag icul u al
sys ems owa ds sus ainabili y in o de o main ain adequa e
p oduc ion le els while educing he amoun o eac i e ni o-
gen los o he en i onmen . Some o he op ions o achie e
his goal and educe ni ogen-oxide emissions a e he op i-
misa ion o ni ogen supply and synch onisa ion wi h c op
demand o he applica ion o s abilised ni ogen e ilise s
wi h syn he ic ni i ica ion inhibi o s (SNIs) ( Thapa e al.,
2016 ). SNIs inhibi he AMO enzyme delaying he oxida ion
o NH
4
+ o NO
2
−, gi ing plan s mo e ime o abso b he
NH
4
+ ( Keeney, 1983 ; Ruse and Schulz, 2015 ). Se e al chemi-
cal compounds wi h ni i ica ion inhibi ion ac i i y ha e been
de eloped ( Subba ao e al., 2006 ). The 3,4-dime hylpy azole
phospha e (DMPP) is one o he mos wo ldwide used SNIs
( Gilsanz e al., 2016 ). In a 10 imes lowe applica ion a e,
DMPP p esen s simila e iciency o ano he wo ldwide used
SNI, dicyandiamide (DCD) ( Ruse and Schulz, 2015 ). In mi-
c ocosm expe imen s, DMPP abili y o dec ease N
2
O emis-
sions inc ease up o 90% ( Bozal-Leo i e al., 2021 ; Co ochano-
Monsal e e al., 2021a ). In ield condi ions, educ ions be ween
35% and 50% in N
2
O emissions a e epo ed wi h a highe
main enance o soil NH
4
+
con en o longe pe iod, wi hou
any dele e ious e ec s on he yields o di e en c ops such as
whea ( Hué ano e al., 2015 ; Duncan e al., 2017 ), pas u e and
co n ( Hué ano e al., 2018 ; Nai e al., 2020 ).
Ne e heless, ecen s udies ha e shown an impac o
dime hylpy azole-based inhibi o s on non- a ge o ganisms
( Co ochano-Monsal e e al., 2020 , 2021a ). The e o e, he po-
en ial isks o soil heal h o using SNIs in he long e m
should be conside ed. As an al e na i e, he use o co e
c ops, in a c op o a ion, wi h he abili y o modi y he
soil ni ogen cycle h ough he elease o oo exuda es is
also conside ed a good s a egy o educe ni ogen pollu ion
( Subba ao e al., 2013 ). This allelopa hy, which is known as
biological ni i ica ion inhibi ion (BNI), is highligh ed in he
amewo k o sus ainable ag icul u e based on he use o en-
i onmen ally iendly ag onomic p ac ices ( Subba ao e al.,
2013 ; Zhang e al., 2015 ). Some o hese biological ni i ica ion
inhibi o s (BNIs) ha e he po en ial o inhibi no jus AOB bu
also AOA ( By nes e al., 2017 ), and hus imp o e he ni ogen
e en ion in soils, in luencing posi i ely c op NUE and mi i-
ga e GHG emissions. Among c ops, so ghum ( So ghum bicolo
L.) has he g ea es BNI- eleasing capaci y ( Subba ao e al.,
2017 ), which makes i ad isable o use i in c op o a ions as a
co e c op. Thus, because o a wakened ni i ica ion, N pol-
lu ion in he ollowing c op should be educed. So ghum is
d ough ole an ( Hadebe e al., 2017 ), so i can de elop despi e
he d y summe clima e o a eas wi h humid Medi e anean
condi ions. Mo eo e , i has a sho g ow h cycle, which p o-
ides win e c ops enough ime o se le. In addi ion, he
use o co e c ops also b ing mul iple en i onmen al ben-
e i s such as an imp o ed soil s uc u e and e ili y, weed
con ol and a educ ion in nu ien leaching and soil e osion
( Muhammad e al., 2019 ; Ga land e al., 2021 ). Fu he mo e,
he use o a co e c op is a e y e icien ool in educing
he amoun o leachable NO
3
−in soil ( Cons an in e al., 2010 ;
Plaza-Bonilla e al., 2015 ).
The p esen wo k aimed o e alua e he e ec o wo di -
e en no- ill c op o a ions: (1) a so ghum-whea o a ion, in
which he possible bene i s o a co e c op wi h BNI po en ial
will be analysed in e ms o soil mine al ni ogen con en , N
2
O
emissions and g ain yield o win e whea ; in compa ison o
(2) a con en ional allow-whea o a ion. In his s udy, he ap-
plica ion o a syn he ic ni i ica ion inhibi o (DMPP) will be
also conside ed as (1) a con ol o ni i ica ion inhibi ion o
compa e wi h he po en ial BNI ac i i y o so ghum, and (2) a
complemen o inc easing he sus ainabili y o hese whea
o a ions in e ms o N
2
O emissions.
1. Ma e ials and me hods
1.1. Expe imen al design
This wo k was conduc ed in Pamplona, no he n Spain
(42 °47’N, 1 °37’W, 450 m abo e sea le el) du ing he 2019/2020
g owing season. The soil cha ac e is ics o he uppe ho i-
zon be o e he s a o he expe imen a e gi en in Table 1 ,
while daily p ecipi a ion and mean empe a u es a e shown
224 jou nal o en i onmen al sciences 127 (2023) 222–233
Table 1 –Physical and chemical p ope ies o he soil col-
lec ed in 0 –30 cm dep h laye in Pamplona be o e he
s a o he expe imen (42 °47
N, 1 °37
W, 450 m abo e sea
le el, Na a e, Spain).
Soil ex u e Soil chemical p ope ies
Sand 38.6% pH
a 8.3
Sil 31.8 C:N a io 8.9
Clay 29.6 N
b
(g/kg) 1.4
O ganic
ma e
c
(g/kg)
21.5
CaCO
3
d
(g/kg) 20.3
Mg
d
(mg/kg) 53.5
K
d
(mg/kg) 270.0
Ca
d
(mg/kg) 2735.7
P
e
(mg/kg) 11.5
a 1:2.5 ( m / V ) soil:wa e
b Kjeldahl diges ion ( Keeney, 1983 );
c Walkley and Black, 1934
d NH
4
AcO, MAPA, 1994 ;
e Wa anabe and Olsen, 1965 .
in Appendix A Fig. S1. A bi ac o ial expe imen al design
(c op o a ion and ype o whea e ilisa ion) was imple-
men ed. The c op o a ions we e: (1) allow–whea o a ion
( allow–whea ); and (2) so ghum co e c op wi hou ni ogen
e ilisa ion–whea o a ion (so ghum–whea ). The soil ea -
men s we e a anged in wo blocks. In he i s block, ad en i-
ious plan s we e desicca ed on May 20, 2019 using RoundUp
(a glyphosa e-based he bicide) (36% W / V , Fe ibe ia, Spain) a
a a e o 1.5 L/ha, dose ha is ou inely applied in no- ill sys-
ems om his egion. In he second one, so ghum ( So ghum
bicolo L. a . PR88P68 Pionee Co e a Ag iscience
R , USA) was
sown unde no- ill condi ions a a a e o 15 kg/ha on May 20,
2019. The so ghum co e c op was c ushed on Oc obe 14, 2019
and le on he soil su ace. One mon h be o e so ghum e mi-
na ion, soil NH
4
+
-N con en om allow and so ghum plo s
we e 2.0 and 1.7 kg N/ha, espec i ely, while he soil NO
3
−-
N con en was 43.7 and 7.0 kg N/ha o allow and so ghum
plo s. Win e whea ( T i icum aes i um L. c . Ma copolo RAGT
R ,
Spain) was sown unde no- ill condi ions and o e so ghum
s o e a a a e o 220 kg/ha on Oc obe 31, 2019. Wi hin each
block o c op o a ion ( allow–whea and so ghum–whea ),
h ee whea e ilise ea men s we e applied in ou andom
plo s eplica ions o indi idual size o 25 m
2
(5 m ×5 m): (1)
con ol wi hou e ilisa ion (Con ol); (2) e ilised wi h am-
monium sulpha e 21%-Ni ogen (AS); and (3) e ilised wi h
ammonium 21%-Ni ogen sulpha e combined wi h DMPP
(AS + DMPP). The e ilisa ion a e was 90 kg N/ha applied in
a single dose on Feb ua y 28, 2020 a he beginning o s em
elonga ion (Z30) acco ding o he Zadoks scale ( Zadoks e al.,
1974 ). The AS ea men used ammonium sulpha e (99%, Ag o
Ibe ia S.L., Spain) as e ilise , and AS + DMPP ea men used
ENTEC
R (Ag o Ibe ia S.L., Spain), which con ains ammonium
sulpha e (99%), and DMPP (99%) a a a e o 0.8% o he NH
4
+
-
N p esen in he ENTEC
R . The whea was ha es ed on July
24, 2020.
1.2. Whea soil geochemical analysis and wa e con en
Soil NH
4
+ and NO
3
−con en s we e i s de e mined he day
be o e applying he ea men s. Samples we e hen aken 10,
30 and 60 days pos - e ilisa ion. Th ee soil subsamples (3 cm
diame e ×0.3 m deep) we e aken om each plo , ocks
and s ones we e emo ed and inally hey we e homogenised.
Nex , 100 g esh weigh o he homogenised subsamples we e
mixed wi h 200 mL o 1 mol/L KCl (99%, PanReac Química,
Spain) and shaken o 1 h a 165 /min. The soil solu ion was
il e ed h ough Wha man No. 1 il e pape (GE Heal hca e,
Spain) o emo e pa icles and hen h ough Sep-Pak Classic
C18 125 ˚
A ca idges (Wa e s, USA) o elimina e o ganic ma -
e . The NH
4
+
con en o he il e ed solu ion was de e mined
using he Be helo me hod ( Pa on and C ouch, 1977 ) and he
NO
3
−con en as desc ibed by Cawse (1967) .
The soil wa e con en was also measu ed each ime he
soil and/o GHG we e sampled. Two subsamples (3 cm diam-
e e ×0.3 m deep) we e aken andomly om he ield. Rocks
we e emo ed and he soil subsamples we e d ied a 80 °C
in a ci cula ion o en o 72 h un il hey eached a cons an
d y weigh . Wa e con en was exp essed as he pe cen age o
wa e - illed po e space (WFPS, %) as pe Linn and Do an (1984) ,
Eq. (1) :
WFPS =
(
C ×D
b
)
×(
1 −(
D
b
/D
p
) )
−1 (1)
whe e, C (g) is he soil g a ime ic wa e con en , D
b
(Mg/m
3
)
is he bulk densi y; D
p
(Mg/m
3
) is he pa icle densi y.
D
p
was aken as 2.65 Mg/m
3
. D
b
was measu ed a he be-
ginning o he expe imen and was ound o be 1.0 Mg/m
3
.
1.3. Measu emen o N
2
O emissions om whea soils
N
2
O soil emissions we e measu ed using he closed cham-
be me hod ( Chadwick e al., 2014 ). Samples we e collec ed
3 imes/week o 2 weeks a e whea e ilisa ion, hen
2 imes/week o he nex 2 weeks and 1 imes/week up
o day 60. Conside ing he diu nal a ia ion o emissions
( Baggs and Blum, 2004 ), sampling was pe o med be ween
10:00 a.m. and 1:00 p.m. To accoun o soil he e ogenei y, ou
chambe s (20 cm diame e ×16 cm high once inse ed in he
soil) we e placed in each plo and wo we e sampled on al e -
na e days. Linea i y was checked and gas samples we e aken
jus a e closing he chambe s and hen 45 min la e . 20 mL o
gas was aken om each chambe and s o ed a o e p essu e
in p e-e acua ed 12 mL glass ials. Samples we e analysed
in a gas ch oma og aph (GC) (7890A, Agilen , USA) equipped
wi h an elec on cap u e de ec o o N
2
O de ec ion. A capil-
la y column (IA KRCIAES 6017: 240 °C, 30 m ×320 μm) was used
and he samples we e injec ed using a headspace au osam-
ple (HT3, Teledyne Tekma , USA). N
2
O s anda ds we e anal-
ysed a he same ime as he samples. Gas emission a es we e
calcula ed by conside ing he a ia ion in gas concen a ion
om he beginning o he end o he 45 min ( Menéndez e al.,
2008 ). Cumula i e N
2
O emissions du ing he sampling pe iod
we e es ima ed by a e aging he a e o loss be ween wo suc-
cessi e de e mina ions, mul iplying ha a e age a e by he
leng h o he pe iod be ween he measu emen s, and adding
ha amoun o he p e ious cumula i e o al ( Menéndez e al.,
2008 ). Soil empe a u e (a a dep h o 10 cm) was measu ed
jou nal o en i onmen al sciences 127 (2023) 222–233 225
be o e aking he gas samples. The ai empe a u e was mea-
su ed 3 imes du ing he 45 min gas sampling pe iod o ge he
a e age.
1.4. Abundance o N-cycle ela ed mic oo ganisms in
whea soils
Quan i a i e polyme ase chain eac ion (qPCR) was used o
quan i y he abundance o ni i ying and deni i ying genes.
Soil DNA was isola ed om 0 o 30 cm soil samples ( h ee sub-
samples pe plo ) collec ed a 10 days pos - e ilisa ion. DNA
was ex ac ed om 0.25 g o d y soil using he Powe Soil
R
DNA Isola ion Ki (Qiagen, Ge many) wi h he modi ica ions
desc ibed in Ha e e al. (2014) . The concen a ion and qual-
i y o DNA ex ac s we e de e mined by means o spec opho-
ome y (NanoD op
R 1000, The mo Scien i ic, USA).
The 16S RNA gene ( o quan i ica ion o o al bac e ial
abundance) and unc ional ma ke genes in ol ed in bac e-
ial ni i ica ion ( amoA ) and deni i ica ion ( ni K, ni S, nosZI and
nosZII ) we e ampli ied by means o qPCR using SYBR
R P e-
mix Ex TaqTM II (Taka a-Bio Inc., Japan) and gene-speci ic
p ime s (Appendix A Table S1) in he S epOnePlus
TM Real-
Time PCR Sys em. Each qPCR eac ion was pe o med in ip-
lica e o each sample. Da a analysis was ca ied ou wi h
S epOnePlus
TM So wa e 2.3 (The mo Scien i ic, USA). S an-
da d cu es we e p epa ed om se ial dilu ions o linea ised
plasmids wi h inse ions o he a ge gene anging om 10
7
o
10
3
gene copies/μL. The numbe o copies o a ge gene/g am
o d y soil ( N ) was calcula ed acco ding o a modi ied equa ion
( Eq. (2) ) de ailed in Beh ens e al. (2008) :
N = (N
pe eac ion
×V
DNA ex ac ed
/V
DNA used
×W) / [ DN A
ex ac ed
]
(2)
whe e, N
pe eac ion is he numbe o a ge gene
copies/ eac ion; V
DNA ex ac ed is he olume o DNA ex-
ac ed; V
DNA used is he olume o DNA used pe eac ion;
W (g) is he weigh o d y soil ex ac ed. [DNA
ex ac ed
] is he
ex ac ed DNA concen a ion.
1.5. Whea c op yield pa ame e s
G ain yields we e calcula ed based on a ha es ed a ea o
7.5 m
2
(1.5 m ×5 m) pe plo and adjus ed o a mois u e con-
en o 12%. An a ea o 0.36 m
2
pe plo was measu ed o cal-
cula e he numbe o spikes/m
2
and d y weigh o 1000 g ains.
The o al g ain ni ogen con en was analysed by applying
he Kjeldhal p ocedu e (AOAC, 1980) wi h a Kjel ec Au osam-
ple Sys em 1035 (Teca o , Spain) a e g inding he g ain and
passing i h ough a 1 mm sc een. G ain p o ein con en was
aken as 5.7 imes he o al ni ogen con en ( Telle , 1932 ). The
yield-scaled N
2
O emissions (YSNE) we e exp essed as he a-
io be ween he amoun o N emi ed as N
2
O and he abo e-
g ound ni ogen up ake ( an G oenigen e al., 2010 ). The ni o-
gen use e iciency (NUE) (kg d y ma e /kg N) was de e mined
as, Eq. (3) :
NUE =
(
W
N x
−W
N0
)
/W
ni ogen (3)
whe e, W
N
x
(kg) is he d y ma e ob ained when 90 kg N/ha
we e added; W
N0 (kg) is he d y ma e ob ained wi h no
e ilise applica ion. W
ni ogen (kg) is he weigh o applied
ni ogen.
1.6. S a is ical analysis
The esul s om soil mine al ni ogen con en de e mina ions
we e subjec o a wo-way (c op o a ion, “R”; and e ilise
ea men , “F”) analysis o a iance s a is ical analysis. The
esul s o N
2
O measu emen s, mic obial quan i ica ion and
g ain yield pa ame e s we e analysed by one-way ANOVA us-
ing Duncan’s mul iple ange es o sepa a ion o means be-
ween ea men s and he Mann–Whi ney U es was used o
compa e he wo ea men s wi h he SPSS s a is ical so wa e
package (2016, IBM SPSS S a is ics o Windows, Ve sion 24.0.
A monk, NY, IBM Co p, USA). p -Values < 0.05 we e conside ed
o be s a is ically signi ican di e ences.
2. Resul s and discussion
2.1. Ni i ying mic oo ganisms a e educed in
so ghum-whea c op o a ion
Fe ilisa ion had a s ong e ec on soil mine al ni ogen con-
en ( p < 0.01). Soil om bo h c op o a ions s a ed wi h a
simila soil NH
4
+ con en be o e e ilise applica ion o he
whea c op ( Fig. 1 a). A e he addi ion o he ni ogen e -
ilise , soil NH
4
+ con en inc eased o he same le el in he
AS and AS + DMPP ea men s o bo h c op o a ions, bu he e
was a signi ican dec ease in he AS ea men a 30 days pos -
e ilisa ion (DPF). Howe e , a he same ime, he use o DMPP
mean he soil e ained wice he amoun o NH
4
+
compa ed
o he AS ea men . Al hough he NH
4
+ con en o he AS
and AS + DMPP ea men s d opped o he le el o he Con ol
ea men a 60 DPF, DMPP was able o p olong he a ailabil-
i y o NH
4
+
a leas un il 30 DPF. These esul s a e compa able
o o he s udies ha applied DMPP as a SNI ( Hué ano e al.,
2015 , 2016 ; Liu e al., 2020 ). On he con a y, AS + DMPP e-
duced NO
3
−con en o 40% a 10 DPF and o 30% a 30 DPF
compa ed o he AS ea men ( Fig. 1 b). The Con ol ea -
men also main ained low soil NO
3
− alues h oughou he
expe imen . As NH
4
+
con en emained high and NO
3
−con-
en was low due o he delay in NH
4
+ oxida ion ( Ruse and
Schulz, 2015 ), AS + DMPP p esen ed he highes NH
4
+
/NO
3
−
a io o all he e ilised ea men s ( Fig. 1 c). AS ea men
showed a highe NH
4
+
/NO
3
− a io han Con ol ea men bu
i was only able o main ain 53% and 22% o AS + DMPP a io
a 10 and 30 DPF, espec i ely. Ne e heless, al hough he use
o DMPP ea men was able o main ain mo e NH
4
+ in he
soil, i can p esen some undesi ed e ec s. A highe e en-
ion o soil NH
4
+ con en could lead o an inc ease on NH
3
ola iliza ion. E en hough we did no measu e NH
3
ola iliza-
ion in ou expe imen s, me a-analysis es ima e ha he use
o SNIs can inc ease NH
3
ola iliza ion a ound 20% ( Qiao e al.,
2015 ). The e o e, despi e he ac ha SNIs applica ion alle i-
a es global wa ming po en ial de i ed om di ec N
2
O emis-
sions, hei po en ial nega i e side e ec s should be ully con-
side ed. On he o he hand, we did no ind any di e ences
be ween he wo c op o a ions in he main enance o soil
mine al ni ogen, in e ms o soil NH
4
+ and soil NO
3
−con-
en , du ing whea de elopmen ( Fig. 1 a and b). This may in-
dica e ha , e en hough co e c ops ca y bene i s o he ol-
lowing cul u e such as imp o ed soil physicochemical p op-
226 jou nal o en i onmen al sciences 127 (2023) 222–233
0
10
20
30
40
Soil NO3--N
(kg N/ha)
0
3
6
9
12
15
0204060
NH4+-N/NO3--N a io
Days pos - e iliza ion
0
40
80
120
Soil NH4+-N
(kg N/ha)
a
b
c
10 30 60
R*NSNS
F** ** **
R×F*NSNS
10 30 60
R*NSNS
F** ** NS
R×FNS NS NS
10 30 60
RNS NS NS
F** ** NS
R×FNS NS NS
Fig. 1 –Whea c op soil mine al ni ogen (0 –30 cm) e olu ion du ing 60 days pos - e ilisa ion in o m o NH
4
+
(a), NO
3
−(b)
and he a io o NH
4
+
-N/NO
3
−-N (c). Con ol: con ol wi hou e iliza ion; AS: e ilised wi h ammonium sulpha e;
AS + DMPP: e ilised wi h ammonium sulpha e + 3,4-dime hylpy azole phospha e. S a is ical analysis was made h ough
analysis o a iance ( wo-way ANOVA) showing he e ec o c op o a ion (R), e ilize ea men (F) and hei in e ac ion
(R ×F). Signi ican di e ences a e ma ked wi h an as e isk (
∗) when p < 0.05 and double as e isk (
∗∗) when p < 0.01.
jou nal o en i onmen al sciences 127 (2023) 222–233 227
0.0E+00
4.0E+06
8.0E+06
1.2E+07
1.6E+07
C
AS
AS+DMPP
C
AS
AS+DMPP
Fallow So ghum
ecnadn
uba BOA
(amoA g/seipoc )lios y d
0.0E+00
5.0E+08
1.0E+09
1.5E+09
2.0E+09
2.5E+09
lai e caB ecnadnuba
(16S RNA g/seipoc)l
ios y
d
Con ol
Con ol
Fallow-whea So ghum-whea
Fig. 2 –Abundance o o al bac e ia (a) and ammonia
oxidizing bac e ia (AOB) (b) a 10 days pos - e ilisa ion
(DPF) on soil o whea c op. Signi ican di e ences be ween
ea men s o “Fallow-whea ” o a ion a e ma ked wi h
lowe case le e s. Signi ican di e ences be ween
ea men s o “So ghum-whea ” o a ion a e ma ked wi h
capi al le e s. Fo bo h ANOVA, he Duncan Tes was used
( p < 0.05; n = 4). The Mann-Whi ney U es was used o
he compa ison be ween c op o a ions wi hin he same
e iliza ion ea men . Signi ican di e ences a p < 0.05
a e ma ked wi h an as e isk (
∗).
e ies (such as wa e holding capaci y, agg ega e s abili y and
C s ock) ( Buye e al., 2010 ; Lal, 2015 ; Poeplau and Don, 2015 )
o educed ni ogen losses ( Kaye and Quemada, 2017 ), in his
case, he use o so ghum as a summe co e plan a ion did no
a ec he e olu ion o soil mine al ni ogen du ing he ollow-
ing c op. None heless, he analysis o soil mine al ni ogen in
he subsequen cul u e may no be su icien ly sensi i e o de-
ec he e ec s o using so ghum as a co e c op.
Fe ilise ea men did no a ec he o al bac e ial abun-
dance (measu ed as 16S RNA gene abundance) ( Fig. 2 a). How-
e e , he AS ea men g ea ly enhanced ni i ica ion (in e ms
o bac e ial amoA gene abundance), especially in allow–whea
o a ion ( Fig. 2 b). E en hough ad en i ious plan s we e desic-
ca ed wi h glyphosa e-based he bicide o c ea e a allow plo ,
he g ea AOB g ow h in AS ea men in allow–whea o-
a ion indica ed he lack o dele e ious e ec s o glyphosa e
on ni i ying mic oo ganisms. Ou esul s ag ee wi h p e i-
ous s udies whe e was demons a ed ha highe doses o
glyphosa e o epea ed exposu e did no a ec ni i ying pop-
ula ions ( Alleg ini e al., 2017 ; Zabaloy e al., 2017 ). The appli-
ca ion o he DMPP was e y e ec i e a educing AOB abun-
dance in soils om bo h c op o a ions, e en down o he le -
els o he un e ilised Con ol, wi h educ ions o 56% and 40%
compa ed o AS in allow–whea and so ghum–whea o a-
ions, espec i ely. No wi hs anding ha some s udies in mi-
c ocosms ha e eached an AOB inhibi ion o o e 85% wi h
he use o DMP-based inhibi o s ( To albo e al., 2017 ; Bozal-
Leo i e al., 2021 ; Co ochano-Monsal e e al., 2021b ), ou
esul s a e simila o hose ob ained in ield s udies whe e
he AOB inhibi ion was e icien bu a lowe pe cen ages
( Kleineidam e al., 2011 ; Duncan e al., 2017 ). Al hough he
di e en c op o a ions did no a ec he soil ni ogen con-
en , i did in luence soil mic obial popula ions. We obse ed
a signi ican inc ease in he o al bac e ial abundance in soil
o so ghum–whea o a ion, as i was 24% and 34% highe
compa ed o allow–whea o he Con ol and AS ea men s,
espec i ely ( Fig. 2 a). Fu he mo e, he ype o c op o a ion
also a ec ed AOB abundance, as he le els o he Con ol and
AS ea men s o he so ghum–whea o a ion we e 35 and
22% lowe han he allow–whea plo s ( Fig. 2 b). This educ-
ion indica es ha , du ing i s de elopmen , so ghum migh
ha e exuded BNIs ha can keep ni i ie s inhibi ed un il he
nex c op. Dayan e al. (2010) indica ed ha he inhibi o y e -
ec o so ghum could pe sis o a leas 60 days a e he ha -
es was emo ed. In ou case, he BNIs may had a mo e en-
du ing e ec (140 days) because he so ghum was no elimi-
na ed om he cul i a ion soil and he expe imen was ca -
ied ou unde no- illage condi ions, which e a ds BNI deg a-
da ion ( Ro h e al., 2000 ). Thus, lea ing he so ghum s o e in
he soil unde no- illage condi ions ensu es slowe oo deg a-
da ion and he consequen elease o exuda es wi h a BNI ca-
paci y because such compounds a e p oduced exclusi ely in
he oo s ( Bae son e al., 2008 ).
2.2. N
2
O emissions a e a ec ed by he ype o o a ion
Daily N
2
O emissions anged om 0.89 o 13.74 g N
2
O-
N/(ha • day) in allow–whea o a ion and om 0.38 o 24.58 g
N
2
O-N/(ha • day) in so ghum–whea o a ion ( Fig. 3 a). The cu-
mula i e N
2
O emissions om he AS ea men s we e he
highes o all he e ilise ea men s wi h 382.9 and 678.3 g
N
2
O-N/ha in soil om he allow–whea and so ghum–whea
o a ions, espec i ely ( Fig. 3 b). As is well suppo ed elsewhe e
( Ruse and Schulz, 2015 ), DMPP educed cumula i e N
2
O emis-
sions o alues akin o he Con ol ea men , co espond-
ing o educ ions o 79% and 86% compa ed o he AS ea -
men o he allow–whea and so ghum–whea o a ions, e-
spec i ely. Since AOB popula ions we e educed in he whea
c op (see Sec ion 2.1 ), p obably because o he BNIs eleased
om so ghum, we also expec ed a dec ease in N
2
O emis-
sions (ei he due o a educ ion in N
2
O emi ed by ni i ie s
228 jou nal o en i onmen al sciences 127 (2023) 222–233
Fig. 3 – Daily (a) and cumula i e (b) N
2
O emission du ing 56 days pos - e ilisa ion on soil o whea c op. Signi ican
di e ences be ween ea men s o “Fallow-whea ” o a ion a e ma ked wi h lowe case le e s. Signi ican di e ences
be ween ea men s o “So ghum-whea ” o a ion a e ma ked wi h capi al le e s. Fo bo h ANOVA, he Duncan Tes was
used ( p < 0.05; n = 4). The Mann-Whi ney U es was used o he compa ison be ween c op o a ions wi hin he same
e iliza ion ea men . Signi ican di e ences a p < 0.05 a e ma ked wi h an as e isk (
∗).
o a dec ease in deni i ying ac i i y because o a delay in
he ans o ma ion o NH
4
+ in o NO
3
−). Howe e , as shown
in Fig. 3 a, N
2
O emissions om he AS ea men o so ghum–
whea o a ion we e highe han hose o he allow–whea
o a ion h oughou he en i e expe imen , esul ing in a 77%
inc ease in cumula i e N
2
O emissions ( Fig. 3 b). I should be
ema ked ha so ghum s o e is an ex a ca bon sou ce in
he soil, and he main mechanism connec ing ca bon cy-
cling wi h ni ogen gas emissions is he ca bon a ailabili y
in he soil ha enhances he e o ophic deni i ica ion, which
is one o he main p ocesses esponsible o N
2
O p oduc ion
( Da idson e al., 2000 ). Fu he mo e, N
2
O emissions a e ela ed
o soil wa e con en ( Da idson, 1991 ), wi h a h eshold o 60%
WFPS be ween wa e -limi ed and ae a ion-limi ed mic obial
p ocesses. In he p esen wo k, du ing N
2
O measu emen s,
he soil WFPS emained be ween 45% and 60% (Appendix A
Fig. S2), a ange in which deni i ying mic oo ganisms become
mo e ele an o N
2
O elease. The e o e, we a gue ha he in-
c emen in N
2
O emissions om soils in he so ghum–whea
o a ion is due o an enhanced he e o ophic deni i ica ion
because o a g ea e ca bon a ailabili y, as la ge po ions
o labile ca bon subs a es p omo e deni i ica ion eac ions
( Su ey e al., 2020 ). This g ea e abundance o he e o ophic
deni i ie s in so ghum–whea o a ions was e idenced by he
inc eased abundance o ni i e educ ase (NIR) enzyme con-
aining deni i ying bac e ia ( Fig. 4 a and b). The Con ol and AS
ea men s om he so ghum–whea o a ion showed a 56%
and 73% inc ease in ni K abundance compa ed o he equi a-
len ea men s on he allow–whea o a ion ( Fig. 4 a). On he
o he hand, he abundance o ni K was no a ec ed by any o
he ea men s (wi h o wi hou ni ogen) on he allow–whea
o a ion, bu he AS + DMPP ea men on he so ghum–whea
o a ion had a lowe ni K abundance han he Con ol and
AS ea men s. Compa ing c op o a ions in he case o ni S
abundance, an inc ease o 30% was only signi ican o he AS
ea men ( Fig. 4 b). Simila ly o ni K, ni S abundance was no
a ec ed by e ilise ea men in he allow–whea o a ion,
bu he AS + DMPP ea men p esen ed a lowe le el han he
Con ol and AS ea men s o he so ghum–whea o a ion.
In his case, ega ding N
2
O- educing bac e ia, nei he he c op
o a ions no he N ea men s a ec ed bo h nosZI and nosZII
genes abundances ( Fig. 4 c and d).
In con as o he soil mine al ni ogen, he changes in
he abundances o N- ela ed mic oo ganisms we e sensi-
i e enough o de ec he e ec s o using a co e c op.
In soil wi h allow–whea o a ion, AS ea men p esen ed
he highes amoA / ni K and amoA / ni K + ni S a ios o all
h ee ea men s ( Table 2 ). This means ha hose soils
we e mo e balanced owa ds ni i ica ion since he addi ion
o N- e ilisa ion inc eases he le el o ni i ica ion genes
( Ouyang e al., 2018 ). In he same manne , he e was a
highe a io be ween N
2
O p oduc ion in deni i ica ion and
N
2
O educ ion ( ni K + ni S )/( nosZI + nosZII ). On he o he hand,
he addi ion o DMPP dec eased amoA abundance, which
is why his ea men p esen ed he lowes amoA / ni K and
amoA / ni K + ni S a ios. Fe ilise ea men did no a ec he
ni i ying/deni i ying a ios in he so ghum–whea o a ion.
Ne e heless, he ype o c op o a ion in luenced hese a-
ios, as he e we e signi ican di e ences be ween hem. As
has been men ioned be o e, he highe a ailabili y o soil ca -
bon due o so ghum co e c op esidues migh be esponsi-
ble o an inc ease in deni i ica ion eac ions ( Palme and
Ho n, 2015 ; Su ey e al., 2020 ). Then, he alleged inc ease o
deni i ying mic oo ganisms con aining NIR enzyme because
o so ghum s o e ( Fig. 4 a and b), balanced he amoA / ni K
and amoA / ni K + ni S a ios owa ds deni i ica ion. This p o-
duced a 60% educ ion in he amoA / ni K a io and 55% in
he amoA / ni K + ni S a io o he Con ol ea men on he
jou nal o en i onmen al sciences 127 (2023) 222–233 229
Fig. 4 –Abundance o deni i ying bac e ia measu ed as he abundance o ni K (a), ni S (b), nosZI (c) and nosZII (d) genes a 10
days pos - e ilisa ion (DPF) on soil o whea c op. Signi ican di e ences be ween ea men s o “Fallow-whea ” o a ion a e
ma ked wi h lowe case le e s. Signi ican di e ences be ween ea men s o “So ghum-whea ” o a ion a e ma ked wi h
capi al le e s. Fo bo h ANOVA, he Duncan Tes was used ( p < 0.05; n = 4). The Mann-Whi ney U es was used o he
compa ison be ween c op o a ions wi hin he same e iliza ion ea men . Signi ican di e ences a p < 0.05 a e ma ked
wi h an as e isk (
∗).
Table 2 –The amoA/ni K, amoA /( ni K + ni S ) and (ni K + ni S)/(nosZI + nosZII) a io on soil o whea c op.
amoA/ni K amoA/(ni K + ni S) (ni K + ni S)/ ( nosZI + nosZII )
Fallow-
whea
Con ol 2.76 ±0.44 ab 1.77 ±0.24 ab 1.49 ±0.25 b
AS 3.37 ±0.45 a 2.79 ±0.61 a 2.27 ±0.15 a
AS + DMPP 1.86 ±0.29 b 1.25 ±0.19 b 2.36 ±0.18 a
So ghum-
whea
Con ol 1.11 ±0.14 A
∗0.78 ±0.14 A
∗2.45 ±0.14 B
∗
AS 1.64 ±0.09 A
∗1.26 ±0.05 A
∗3.00 ±0.24 A
∗
AS + DMPP 1.82 ±0.32 A 1.29 ±0.21 A 2.32 ±0.13 B
Signi ican di e ences be ween ea men s o “Fallow-whea ” o a ion a e ma ked wi h lowe case le e s. Signi ican di e ences be ween
ea men s o “So ghum-whea ” o a ion a e ma ked wi h capi al le e s. Fo bo h ANOVA, he Duncan Tes was used ( p < 0.05; n = 4). The
Mann-Whi ney U es was used o he compa ison be ween c op o a ions wi hin he same e iliza ion ea men . Signi ican di e ences a
p < 0.05 a e ma ked wi h an as e isk (
∗).
230 jou nal o en i onmen al sciences 127 (2023) 222–233
so ghum–whea o a ion compa ed o allow–whea . Fu he -
mo e, he educ ion o he AOB popula ion in he AS ea -
men ( Fig. 2 b) due o he po en ial elease o BNIs om
so ghum oo s also con ibu ed o he dec ease in amoA / ni K
and amoA / ni K + ni S a ios wi h a 51% and 54% educ ion, e-
spec i ely, compa ed o he allow–whea o a ion. Mo eo e ,
al hough he abundances o ni K and ni S migh inc ease due
o he ex a ca bon, no e ec s could be obse ed on he abun-
dance o nosZI and nosZII genes. We heo ize ha , somehow,
he use o so ghum as a co e c op a ou ed a scena io o in-
comple e deni i ica ion, which inc eased he emission o N
2
O
due o he lack o inc ease o he mic oo ganisms ha could
educe i comple ely o N
2
.
Consequen ly, ocusing on he capaci y o modi y he ni-
i ying/deni i ying a io o he di e en c op o a ions, we
migh de elop a be e unde s anding o how soil N emis-
sions espond, such as he di e en N
2
O emissions o he
AS ea men . Howe e , since he AS + DMPP ea men signi -
ican ly inhibi ed AOB g ow h ( Fig. 2 b), soil NO
3
− o ma ion di-
minished compa ed o he AS ea men and he e was no
inc ease in ni K and ni S genes ( Fig. 4 a and b), ul ima ely e-
sul ing in simila ni i ying/deni i ying a ios be ween c op
o a ions. In addi ion, AS + DMPP ea men showed he lowe
( ni K + ni S )/( nosZI + nosZII ) a io, esul ing in a be e balance
be ween N
2
O p oduc ion/N
2
O educ ion. We he e o e sugges
he use o syn he ic NIs such as DMPP o educe he pollu-
ion de i ed om he use o so ghum as a co e c op. Mo e-
o e , Menéndez e al. (2012) epo ed ha he educ ion in N
2
O
emissions induced by DMPP is condi ioned by he magni ude
o he losses om he e ilise wi hou NIs. Thus, DMPP can
coun e ac highe N
2
O emissions wi h g ea e e iciency, as
can be obse ed in ou expe imen , wi h a 79% educ ion o
N
2
O emissions wi h espec o AS in he allow–whea o a-
ion e sus 86% in he so ghum–whea o a ion ( Fig. 3 b).
2.3. Yield pa ame e s a e no a ec ed by he ype o
o a ion
Plan ing win e whea a e a so ghum c op is a common
p ac ice, ye i can a ec he se lemen o whea seed.
Guenzi e al. (1967) demons a ed ha wa e ex ac s om
so ghum esidues could inhibi co n and whea seed ge -
mina ion. This e ec is due o he allelopa hic subs ances,
such as so goleone, ha so ghum eleases h ough i s oo s.
Ro h e al. (2000) sugges ed ha soil managemen is he key o
coun e ac ing hese e ec s. In soils wi h con en ional illage
managemen , so ghum exuda es a e quickly solubilised and
deg aded. In no- illage soils, by con as , so ghum emains e-
leasing hei deg ada ion compounds g adually and he e o e
a ec c op yield. E en so, he e ec s o so ghum esidues on
whea seed ge mina ion can be mi iga ed by inc easing he
seeding a e o delaying he plan ing o subsequen c ops un-
il he esidues ha e decomposed o wea he ed ( Wes on e al.,
2013 ). In ou expe imen , he whea sowing densi y was
220 kg/ha, which seems high enough o pallia e he e ec s o
he p e ious so ghum c op since he ype o c op o a ion did
no a ec he whea g ain yield o e ilised ea men s. As
expec ed, whea g ain yield was much highe o he AS and
AS + DMPP ea men s wi h 6616 and 6133 kg/ha, espec i ely,
o he allow–whea o a ion and 6230 and 6543 kg/ha o
he so ghum–whea o a ion (Appendix A Table S2), su pass-
ing he a e age o ha egion in 2019, which was 5000 kg/ha
( MAGRAMA, 2019 ). Simila ly, he use o SNIs did no a ec
g ain yield, which was in line wi h o he wo ks whe e DMPP
main ained he g ain yield o ain ed win e ce eals compa ed
o e ilise ea men s wi hou inhibi o ( A egui and Que-
mada, 2008 ; Hué ano e al., 2016 ). Fu he mo e, he numbe
o spikes/m
2
was also highe in he AS and AS + DMPP ea -
men s in bo h c op o a ions han in he Con ol ea men s.
Howe e , e en hough c op o a ions did no in luence he
g ain yield o e ilised ea men s, his was no he case o
he Con ol ea men , which p esen ed a dec ease in whea
g ain yield in he so ghum–whea o a ion (Appendix A Table
S2). This dec ease may be due o he compe i ion o nu ien s
be ween plan s and soil mic obes in soils wi h a low ni ogen
con en . I is assumed ha he e o ophic soil mic oo ganisms
a e s onge compe i o s o ino ganic N han plan s ( Kaye and
Ha , 1997 ). Mo eo e , he g ow h o hese mic oo ganisms is
ca bon-limi ed. Thus, when soil mine al ni ogen inc eased in
he Con ol ea men s, such as be ween 0 and 10 DPF when
mine alisa ion can be obse ed ( Fig. 1 a and b), he addi ional
C om so ghum s o e led o an inc ease in he o al bac e ial
abundance compa ed o he allow–whea o a ion ( Fig. 2 a),
and he e o e o a g ea e compe i ion agains he plan s o
soil ni ogen up ake. In addi ion, a hese s ages o whea
plan s g own in a so ghum–whea o a ion, he educ ion in
N up ake was e iden as he numbe o spikes/m
2
in he Con-
ol ea men o he so ghum–whea o a ion was lowe han
hose o he allow–whea o a ion. Despi e his, he e we e
no signi ican di e ences be ween e ilise ea men s o be-
ween c op o a ions on he numbe o g ains/spike and he
pe cen age o g ain p o ein (Appendix A Table S2). Fu he -
mo e, a ending o he ni ogen use e iciency (NUE), he e
we e also no di e ences be ween e ilisa ion ea men s o
c op o a ions. Finally, o e alua e he N
2
O e iciency o c op-
ping sys ems and de elop s a egies o op imal c op p oduc-
i i y, and hence minimise en i onmen al con amina ion, i
may be mo e in o ma i e o exp ess N
2
O emissions in ela-
ion o c op p oduc i i y (YSNE) ( an G oenigen e al., 2010 ;
Schwenke and Haigh, 2016 ). The AS ea men applied o
bo h c op o a ions p esen ed an inc eased YSNE compa ed
o he Con ol and AS + DMPP ea men s, which we e equally
low. These ea men s did no show any di e ences be ween
he wo c op o a ions, bu e ilising whea wi h AS in he
so ghum–whea o a ion had a 93% highe YSNE han whea
e ilised wi h AS in he allow–whea o a ion. Thus, when
so ghum was used as a co e c op, i p oduced wice he
N
2
O emissions/kg o ni ogen up ake because i inc eased he
gaseous ni ogen losses.
3. Conclusions
The use o so ghum in a c op o a ion migh no be a sui -
able op ion o mi iga e he ni ogen losses, such as N
2
O emis-
sions, de i ed om he ni ogen e ilise applica ion in he
subsequen cul u e. So ghum–whea o a ion did no p esen
any e ec on he main enance o soil NH
4
+ con en du ing
whea c op de elopmen , as he le els we e he same as he
allow–whea o a ion. Al hough he po en ial elease o BNIs
