Ci a ion: Flo es-Dua e, N.J.;
Na a o-To e, S.; Ma eos-Na anjo,
E.; Redondo-Gómez, S.; Pajuelo, E.;
Rod íguez-Llo en e, I.D. Nodule
Syn he ic Bac e ial Communi y as
Legume Bio e ilize unde Abio ic
S ess in Es ua ine Soils. Plan s 2023,
12, 2083. h ps://doi.o g/10.3390/
plan s12112083
Academic Edi o s: Olga A. Aleyno a
and Kons an in V. Kisele
Recei ed: 21 Ap il 2023
Re ised: 17 May 2023
Accep ed: 22 May 2023
Published: 24 May 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
plan s
A icle
Nodule Syn he ic Bac e ial Communi y as Legume Bio e ilize
unde Abio ic S ess in Es ua ine Soils
No is J. Flo es-Dua e 1, Sal ado a Na a o-To e 1,* , En ique Ma eos-Na anjo 2, Susana Redondo-Gómez 2,
Eloísa Pajuelo 1and Ignacio D. Rod íguez-Llo en e 1,*
1Depa amen o de Mic obiología y Pa asi ología, Uni e sidad de Se illa, 41012 Se ille, Spain;
[email p o ec ed] (N.J.F.-D.); [email p o ec ed] (E.P.)
2Depa amen o de Biología Vege al y Ecología, Uni e sidad de Se illa, 41012 Se ille, Spain;
[email p o ec ed] (E.M.-N.); [email p o ec ed] (S.R.-G.)
*Co espondence: [email p o ec ed] (S.N.-T.); [email p o ec ed] (I.D.R.-L.)
Abs ac :
Es ua ies a e ecologically impo an ecosys ems pa icula ly a ec ed by clima e change
and human ac i i ies. Ou in e es is ocused on he use o legumes o igh agains he deg ada ion
o es ua ine soils and loss o e ili y unde ad e se condi ions. This wo k was aimed o de e mine
he po en ial o a nodule syn he ic bac e ial communi y (SynCom), including wo Ensi e sp. and wo
Pseudomonas sp. s ains isola ed om Medicago spp. nodules, o p omo e M. sa i a g ow h and nodu-
la ion in deg aded es ua ine soils unde se e al abio ic s esses, including high me al con amina ion,
salini y, d ough and high empe a u e. These plan g ow h p omo ing (PGP) endophy es we e able
o main ain and e en inc ease hei PGP p ope ies in he p esence o me als. Inocula ion wi h he
SynCom in po s con aining soil enhanced plan g ow h pa ame e s ( om 3- o 12- old inc ease in d y
weigh ), nodula ion ( om 1.5- o 3- old inc ease in nodules numbe ), pho osyn hesis and ni ogen
con en (up o 4- old unde me al s ess) unde all he con olled condi ions es ed. The inc ease in
plan an ioxidan enzyma ic ac i i ies seems o be a common and impo an mechanism o plan
p o ec ion induced by he SynCom unde abio ic s ess condi ions. The SynCom inc eased M. sa i a
me als accumula ion in oo s, wi h low le els o me als ansloca ion o shoo s. Resul s indica ed ha
he SynCom used in his wo k is an app op ia e ecological and sa e ool o imp o e Medicago g ow h
and adap a ion o deg aded es ua ine soils unde clima e change condi ions.
Keywo ds:
abio ic s ess; legumes nodula ion; plan g ow h p omo ing endophy es; SynCom;
deg aded es ua ine soils
1. In oduc ion
Es ua ies a e complex ecosys ems ha p o ide ma e ial and nonma e ial bene i s
o humans [
1
,
2
]; howe e , es ua ies also su e human dis up ion and o e exploi a ion,
esul ing in hei impo e ishing [
3
]. Clima e change a ec s hese coas al ecosys ems by
inc easing sea su ace empe a u e, hea (o cold) wa es and equency o ex eme e en s,
such as s ong educ ion in ain all (and consequen ly, d ough e en s) [
4
]. Modi ica ion o
wa e quali y, mig a ion pa e ns and plan g ow h a e also among he e ec s o clima e
change on es ua ies [
5
]. In ha way, he combined e ec s o human p ac ices, pa icula ly
indus ial and ag icul u al ac i i ies, and clima e change ha e con ibu ed o inc eased
es ua ine soils deg ada ion, limi ing plan de elopmen and di e si y. Vege al co e plays
a c ucial ole in es ua ine ecosys ems conse a ion, since plan s educe soil compac ion
and e osion, inc ease he con en o o ganic ma e and mic oo ganisms’ biodi e si y and
imp o e soil hyd aulic and s uc u al p ope ies [
6
]. Fu he mo e, eco e y o deg aded
es ua ine soils implies e ege a ion, p e e ably using au och honous plan species ha
should include legumes. Legumes can ix a mosphe ic ni ogen h ough hei in e ac ion
wi h soil bac e ia commonly named hizobia [
7
], imp o ing soil heal h and e ili y and
p o iding a la ge p opo ion o he ni ogen equi emen s o plan s [8].
Plan s 2023,12, 2083. h ps://doi.o g/10.3390/plan s12112083 h ps://www.mdpi.com/jou nal/plan s
Plan s 2023,12, 2083 2 o 16
One example o deg aded es ua ine ecosys em is he join es ua y o he Odiel and
Tin o i e s in Huel a egion (SW Spain). Due o bo h na u al and an h opogenic ac i i-
ies, his es ua y became one o he mos pollu ed a eas in he wo ld [
9
]. Le els o oxic
me als—such as As, Cu, Pb o Zn—exceeding hose allowed by egional and na ional
legisla ion in na u al pa ks, ag icul u al and indus ial soils [
10
] has been eco ded in bo h
Odiel and Tin o i e ma shes in he las decade [
11
,
12
]. These le els o soil con amina ion
make hei eco e y manda o y. Se e al wo ks using au och honous halophy es and hei
associa ed hizosphe ic and endophy ic bac e ia aimed o alle ia e he ecological e ec s
o me al con amina ion in his es ua y ha e been epo ed. These publica ions e alua ed
he inc ease o he phy o emedia ion po en ial o Spa ina ma i ima [
13
,
14
], A h ocnemum
ma os achyum [
12
] o Salico nia amosissima [
15
] in es ua ine soils bioaugmen ed wi h bac-
e ia om hei mic obiomes. In gene al, he selec ed bac e ia enhanced plan g ow h in
p esence o me als and inc eased me al accumula ion in oo s, imp o ing he phy os a-
biliza ion po en ial o he halophy es. Bac e ia cha ac e ized and used in hese wo ks
showed se e al plan -g ow h p omo ing (PGP) p ope ies, such as phospha e solubilisa-
ion, ni ogen ixa ion o auxins and side opho es p oduc ion, among o he s, ha could be
esponsible o he e ec s obse ed in plan g ow h [16].
Mo e ecen ly, he po en ial use o legumes, pa icula ly Medicago sa i a, combined
wi h bo h izosphe ic and endophy ic bac e ia o imp o e es ua ine soils e ili y has been
in es iga ed [
17
,
18
]. In bo h wo ks, inocula ion wi h selec ed bac e ia esul ed in inc easing
M. sa i a g ow h and nodula ion, amelio a ing plan pho osyn hesis and physiological
pa ame e s. In his con ex , we ha e desc ibed ou endophy es syn he ic bac e ial com-
muni y (SynCom), isola ed om Medicago spp. nodules o plan s na u ally g owing in
Odiel i e high ma shes soils, capable o p omo ing M. sa i a g ow h, nodula ion and phy-
os abiliza ion abili y in me als con amina ed es ua ine soils [
19
]. This SynCom included
wo nodule inducing Ensi e sp. s ains and wo Pseudomonas sp. nodule endophy es wi h
mul iple PGP p ope ies.
The aims o his wo k we e o e alua e he abili y o he Pseudomonas-Ensi e SynCom
o enhance M. sa i a g ow h and nodula ion a es unde high me al, sal , high empe -
a u e o d ough s ess in es ua ine soils, in an a emp o igh agains he p esen and
u u e consequences o clima e change and human ac i i ies on he soil e ili y o Odiel
i e ma shes.
2. Resul s
2.1. SynCom Cha ac e iza ion unde Abio ic S ess
In a p e ious wo k, we isola ed and cha ac e ized endophy es inhabi ing inside
nodules o Medicago spp. g owing in deg aded es ua ine soils. A syn he ic bac e ial
communi y (SynCom) was designed based on bac e ial PGP p ope ies, ly ic enzyma ic
ac i i ies and ole ance o hea y me als (Supplemen a y Table S1) [
19
]. The SynCom was
made up o ou s ains: Pseudomonas sp. N4, Pseudomonas sp. N8, Ensi e sp. N10 and
Ensi e sp. N12 [
19
]. In his s udy, he PGP p ope ies and he ly ic enzyma ic ac i i ies o
hese s ains we e es ed in he p esence o hea y me als (Table 1). All s ains main ained he
PGP p ope ies in absence o me als, wi h he excep ion o s ains N10 and N12, which did
no g ow in a minimal medium wi hou a ni ogen sou ce in p esence o me als. I should
be no ed ha bio ilm capaci y was p esen in all s ains, bu he abso bance dec eased
deeply in p esence o hea y me als (Table 1). In e es ingly, he ACC deaminase ac i i y
o he N4 and N8 s ains inc eased in he p esence o hea y me als, while N10 and N12,
which did no show he ACC deaminase ac i i y in he absence o me al, showed ac i i y
in hei p esence (Table 1).
Rega ding he ly ic enzyma ic ac i i ies, all s ains showed pec inase ac i i y in he
p esence o hea y me als, while no in hei absence (Table 1). The N10 and N12 main ained
he DNAse ac i i y bu los he abili y o deg ade cellulose in he p esence o me als
(Table 1). On he o he hand, s ains N4 and N8 only showed pec inase ac i i y in he
p esence o me als, losing bo h p o einase ac i i y and N8 cellulase ac i i y (Table 1).
Plan s 2023,12, 2083 3 o 16
Table 1.
PGP p ope ies and enzyma ic ac i i ies in p esence o a mix o hea y me als, he max-
imum ole able concen a ion o NaCl and PEG, and he maximum ole able empe a u e o he
bac e ial s ains.
PGP P ope ies N4 N8 N10 N12
Phospha e solubiliza ion 12 15 12 13
IAA p oduc ion 3.884 5.729 3.051 4.115
Bio ilm o ma ion 0.132 0.104 0.102 0.149
ACC deaminase ac i i y 29.634 19.117 15.388 16.571
N ixa ion + + – –
Enzyma ic ac i i ies
DNAse – – + +
Amylase – – – –
Cellulase – – – –
Lipase – – – –
Pec inase + + + +
P o ease – – – –
Chi inase – – – –
Maximum Tole able
Concen a ion
NaCl 1.5 1.5 2 2
PEG 30 25 30 30
Maximum empe a u e 40 ◦C 40 ◦C 45 ◦C 45 ◦C
“+”: p esence o he ac i i y; “–”: absence o he ac i i y. Values o phospha e solubiliza ion and side opho es
p oduc ion exp ess he diame e o he halo in mm. Values o IAA p oduc ion a e exp essed in mg
·
L
−1
. Values
o ACC deaminase ac i i y a e exp essed in
µ
moles
α
-ke obu y a e
·
mg p o ein
−1·
h
−1
). Values o maximum
ole able concen a ion o NaCl and PEG a e exp essed in M and pe cen age, espec i ely. N4: Pseudomonas sp.
N4, N8: Pseudomonas sp. N8, N10: Ensi e sp. N10, and N12: Ensi e sp. N12.
In addi ion o hese p ope ies, he abili y o hese bac e ia o g ow unde saline
condi ions was es ed. All s ains ole a ed high concen a ions o NaCl eaching alues o
1.5 M in he case o s ains N4 and N8, and 2 M o s ains N10 and N12 (Table 1).
Rega ding ole ance o d ough , bac e ial s ains we e exposed o di e en concen a-
ions o polye hylene glycol (PEG) since his polyme simula es he d ough condi ions.
S ains N4, N10, and N12 we e able o g ow a 30% o PEG, and s ain N8 a 25% PEG.
Finally, all s ains ole a ed empe a u es simila o o g ea e han 40 ◦C (Table 1).
2.2. Inocula ion wi h he SynCom Enhances M. sa i a G ow h unde S ess ul Condi ions
The u ili y o he designed SynCom o p omo e he g ow h o M. sa i a unde high
me al s ess was es ed using s e ilized soil om he low ma shes in he Odiel i e es ua y.
Me al concen a ions in his soil a e p esen ed in Table 2.
Table 2.
Physicochemical p ope ies and mic onu ien s concen a ions o soil om Rio Odiel es ua y.
Physicochemical p ope ies
Loca ion Tex u e (%) * O ganic
ma e ial (%)
Conduc i i y
(µS·cm−1)pH
High ma sh 72/13/15 0.90 ±0.01 13.1 ±0.3 6.8 ±0.2
Middle ma sh 61/21/18 1.59 ±0.05 13.7 ±0.2 6.8 ±0.1
Me al/loids concen a ion (mg·kg−1)
Loca ion As Cd Cu Zn
High ma sh 27.9 ±2.2 0.38 ±0.01 316.7 ±3.2 345.0 ±7.1
Middle ma sh 162.4 ±5.6 0.86 ±0.03 537.5 ±6.2 865.2 ±5.6
Values a e mean ±S.D. (n = 3). * Tex u e (sand/sli /clay pe cen age).
Plan s 2023,12, 2083 4 o 16
The inocula ed plan s we e signi ican ly alle and lea ie han he non-inocula ed ones,
showing ha he SynCom up o h ee- old he leng h and i e- old shoo d y weigh o he
shoo compa ed o non-inocula ed plan s (Figu e 1A,B). Simila esul s we e obse ed in
oo s compa ing non-inocula ed and inocula ed plan s, al hough hose plan s inocula ed
wi h he SynCom showed a simila leng h o oo s han hose inocula ed wi h Ensi e sp.
N10 alone (Figu e 1B). Howe e , he oo s o he plan s inocula ed wi h SynCom we e
hicke , wi h mo e seconda y oo s and oo hai s han he plan s inocula ed wi h s ain
N10 (Figu e 1A). The numbe o nodules was signi ican ly highe in plan s inocula ed wi h
SynCom (Figu e 1C) and he o al N con en was also highe —close o 300% mo e N han
in non-inocula ed plan s (Figu e 1D).
Plan s 2023, 10, x FOR PEER REVIEW 5 o 17
Figu e 1. G ow h pa ame e s and nodula ion. (A) D y weigh o shoo ( op g aphs) and oo s
(bo om g aphs), (B) leng h o shoo and oo s, (C) numbe o nodules, and (D) ni ogen con en in
non-inocula ed and inocula ed M. sa i a plan s a e 60 days in po s unde g eenhouse condi ions
using di e en subs a es. Values a e means ± S.D. (n = 16). Di e en le e s and symbols indica e
means ha a e signi ican ly di e en om each o he (One-way ANOVA, LSD es , p < 0.0001). C-:
non inocula ion; N10: inocula ion wi h Ensi e sp. N10; CSN: inocula ion wi h he SynCom o med
by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensi e sp. N10, and Ensi e sp. N12; hea y me al
ea men : soil om he middle ma shes o he Odiel i e as subs a e; sal ea men : s e ilized soil
om he uppe ma shes o he Odiel i e supplemen ed wi h 60 mM o NaCl as subs a e; d ough
ea men : s e ilized soil om he uppe ma shes o he Odiel i e as subs a e and plan s exposed
o d ough s ess; high empe a u e ea men : s e ilized soil om he uppe ma shes o he Odiel
i e as subs a e and plan s exposed o 40 °C.
2.3. Inocula ion wi h he SynCom Enhances M. sa i a Pho osyn he ic Pa ame e s unde
S ess ul Condi ions
In addi ion o he imp o emen in plan g ow h and nodula ion, he bac e ial
SynCom also imp o ed he pho osyn he ic pa ame e s in a s a is ically signi ican way,
compa ed wi h plan s no inocula ed o inocula ed wi h Ensi e sp. N10 in he p esence o
all he abio ic s ess condi ions (Figu e 2). Unde high concen a ions o hea y me als, he
ne pho osyn he ic a e showed he g ea es imp o emen a e inocula ion wi h SynCom
(Figu e 2B).
Figu e 1.
G ow h pa ame e s and nodula ion. (
A
) D y weigh o shoo (
op
g aphs) and oo s
(
bo om
g aphs), (
B
) leng h o shoo and oo s, (
C
) numbe o nodules, and (
D
) ni ogen con en in
non-inocula ed and inocula ed M. sa i a plan s a e 60 days in po s unde g eenhouse condi ions
using di e en subs a es. Values a e means
±
S.D. (n= 16). Di e en le e s and symbols indica e
means ha a e signi ican ly di e en om each o he (One-way ANOVA, LSD es , p< 0.0001).
C-: non inocula ion; N10: inocula ion wi h Ensi e sp. N10; CSN: inocula ion wi h he SynCom
o med by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensi e sp. N10, and Ensi e sp. N12; hea y me al
ea men : soil om he middle ma shes o he Odiel i e as subs a e; sal ea men : s e ilized soil
om he uppe ma shes o he Odiel i e supplemen ed wi h 60 mM o NaCl as subs a e; d ough
ea men : s e ilized soil om he uppe ma shes o he Odiel i e as subs a e and plan s exposed
o d ough s ess; high empe a u e ea men : s e ilized soil om he uppe ma shes o he Odiel
i e as subs a e and plan s exposed o 40 ◦C.
To assess he e ec o SynCom inocula ion unde salini y s ess, s e ilized soil om
he uppe ma shes o he Odiel i e was supplemen ed wi h s e ile NaCl solu ion o each
a inal NaCl concen a ion o 60 mM in he soil, as desc ibed in Sec ion 4.
Plan s inocula ed wi h Ensi e sp. N10 signi ican ly imp o ed hei biomass and leng h
compa ed o non-inocula ed plan s (Figu e 1A,B). A highe imp o emen was measu ed in
plan s inocula ed wi h SynCom, inc easing he shoo biomass by 2300% compa ed o non-
Plan s 2023,12, 2083 5 o 16
inocula ed plan s (Figu e 1A). Howe e , he SynCom-inocula ed plan s showed he lowes
alues o oo leng h, al hough he oo s we e hicke and p esen ed mo e la e al oo s
han in he o he wo condi ions, as e lec ed in oo biomass (Figu e 1A,B). Fu he mo e,
SynCom amelio a ed plan nodula ion, showing almos h ee imes he numbe o nodules
coun ed in plan s inocula ed wi h N10 (Figu e 1C). The o al ni ogen con en in plan s also
inc eased in p esence o bac e ia and he highes amoun was obse ed in plan s inocula ed
wi h SynCom (Figu e 1D).
Rega ding M. sa i a g ow h and nodula ion unde d ough s ess, his abio ic s ess
pa icula ly a ec ed he de elopmen o plan shoo s (Figu e 1A). Again, plan s inocula ed
wi h he SynCom showed he highes d y weigh and leng h alues o he oo s and shoo s,
while plan s inocula ed wi h he Ensi e s ain epo ed highe alues han non-inocula ed
plan s (Figu e 1A,B). Al hough SynCom signi ican ly enhanced M. sa i a nodula ion and
plan ni ogen con en (Figu e 1C,D), ni ogen con en was pa icula ly a ec ed by his
s ess condi ion.
Finally, he esponse o M. sa i a g ow h a e a hea shock and he e ec o bac e-
ial inocula ion in plan p o ec ion we e e alua ed. The high empe a u e a ec ed he
de elopmen o he shoo in longe ex ension han he de elopmen o oo s (Figu e 1A,B).
Inocula ion wi h he SynCom signi ican ly imp o ed oo and shoo de elopmen . Plan s
inocula ed wi h SynCom unde hea s ess showed he longes oo s and he highes d y
weigh compa ed wi h he o he s ess condi ions. Conce ning nodula ion, al hough in-
ocula ion wi h SynCom inc eased nodule numbe , he empe a u e pa icula ly a ec ed
nodula ion, showing hese plan s he lowes numbe o nodules among plan s unde he
di e en s ess condi ions (Figu e 1C). Howe e , he ni ogen con en was simila o hose
eco ded in plan s unde sal o hea y me al s ess (Figu e 1D).
2.3. Inocula ion wi h he SynCom Enhances M. sa i a Pho osyn he ic Pa ame e s unde
S ess ul Condi ions
In addi ion o he imp o emen in plan g ow h and nodula ion, he bac e ial SynCom
also imp o ed he pho osyn he ic pa ame e s in a s a is ically signi ican way, compa ed
wi h plan s no inocula ed o inocula ed wi h Ensi e sp. N10 in he p esence o all he
abio ic s ess condi ions (Figu e 2). Unde high concen a ions o hea y me als, he ne
pho osyn he ic a e showed he g ea es imp o emen a e inocula ion wi h SynCom
(Figu e 2B).
Plan s 2023, 10, x FOR PEER REVIEW 6 o 17
Figu e 2. Pho osyn he ic pa ame e s a e he exposi ion o di e en s ess ul condi ions. (A) To al
chlo ophyll con en , (B) ne pho osyn he ic a e (AN), (C) maximum quan um e iciency o PSII
pho ochemis y (F /Fm), (D) quan um e iciency o PSII (ΦPSII), (E) in insic wa e use e iciency
(iWUE), and (F) elec on anspo e iciency (ETR) in non-inocula ed and inocula ed M. sa i a
plan s a e 60 days in po s unde g eenhouse condi ions in he case o hea y me al and sal
ea men s, and du ing he s ess in he case o d ough and high empe a u e ea men s. Values
a e means ± S.D. (n = 16). Di e en le e s and symbols indica e means ha a e signi ican ly di e en
om each o he . (One-way ANOVA, LSD es , p < 0.0001). C-: non inocula ion; N10: inocula ion
wi h Ensi e sp. N10; CSN: inocula ion wi h he SynCom o med by Pseudomonas sp. N4, Pseudomonas
sp. N8, Ensi e sp. N10, and Ensi e sp. N12; hea y me al ea men : soil om he middle ma shes o
he Odiel i e as subs a e; sal ea men : s e ilized soil om he uppe ma shes o he Odiel i e
supplemen ed wi h 60 mM o NaCl as subs a e; d ough ea men : s e ilized soil om he uppe
ma shes o he Odiel i e as subs a e and plan s exposed o d ough s ess; high empe a u e
subs a es: s e ilized soil om he uppe ma shes o he Odiel i e as subs a e and plan s exposed
o 40 °C.
Plan s ea ed wi h 60 mM o NaCl and inocula ed wi h SynCom showed inc eases
o e 100% in o al chlo ophyll con en , PSII e iciency, iWUE and elec on anspo a e
compa ed wi h single inocula ed o non-inocula ed plan s (Figu e 2A,D–F).
Unde d ough s ess, he o al chlo ophyll con en and iWUE signi ican ly inc eased
hei alues in SynCom-inocula ed plan s (Figu e 1A,E).
On he o he hand, high empe a u es pa icula ly a ec ed plan pho osyn hesis
(Figu e 2). Inocula ion wi h he SynCom inc eased all he pa ame e s measu ed, bu he
alues o hese pa ame e s we e, in gene al, clea ly lowe han he alues obse ed in
plan s unde he o he s ess condi ions. In pa icula , he ne pho osyn he ic a e (AN),
he quan um e iciency o PSII (ΦPSII) and he elec on anspo e iciency (ETR) showed
e y low alues unde his condi ion (Figu e 2B,D,F).
2.4. Inocula ion wi h he SynCom Enhances M. sa i a S ess S a us unde S ess ul Condi ions
In his wo k, he s ess le el in plan s was also checked by measu ing an ioxidan
enzymes ac i i ies. Inocula ion wi h he SynCom inc eased an ioxidan enzyme ac i i ies
o e he alues eco ded in non-inocula ed o single-inocula ed plan s (Figu e 3). Unde
hea y me al s ess, his enzyma ic induc ion was also obse ed in plan s inocula ed wi h
s ain N10 compa ed o non-inocula ed plan s, bu wi h lowe di e ences (Figu e 3).
Enzyma ic ac i i ies we e highe in oo s han in shoo s (Figu e 3). In pa icula , ca alase
Figu e 2. Pho osyn he ic pa ame e s a e he exposi ion o di e en s ess ul condi ions. (A) To al

Plan s 2023,12, 2083 6 o 16
chlo ophyll con en , (
B
) ne pho osyn he ic a e (A
N
), (
C
) maximum quan um e iciency o PSII
pho ochemis y (F /Fm), (
D
) quan um e iciency o PSII (
ΦPSII
), (
E
) in insic wa e use e iciency
(iWUE), and (
F
) elec on anspo e iciency (ETR) in non-inocula ed and inocula ed M. sa i a plan s
a e 60 days in po s unde g eenhouse condi ions in he case o hea y me al and sal ea men s, and
du ing he s ess in he case o d ough and high empe a u e ea men s. Values a e means
±
S.D.
(n= 16
). Di e en le e s and symbols indica e means ha a e signi ican ly di e en om each o he .
(One-way ANOVA, LSD es , p< 0.0001). C-: non inocula ion; N10: inocula ion wi h Ensi e sp. N10;
CSN: inocula ion wi h he SynCom o med by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensi e sp.
N10, and Ensi e sp. N12; hea y me al ea men : soil om he middle ma shes o he Odiel i e as
subs a e; sal ea men : s e ilized soil om he uppe ma shes o he Odiel i e supplemen ed wi h
60 mM o NaCl as subs a e; d ough ea men : s e ilized soil om he uppe ma shes o he Odiel
i e as subs a e and plan s exposed o d ough s ess; high empe a u e subs a es: s e ilized soil
om he uppe ma shes o he Odiel i e as subs a e and plan s exposed o 40 ◦C.
Plan s ea ed wi h 60 mM o NaCl and inocula ed wi h SynCom showed inc eases
o e 100% in o al chlo ophyll con en , PSII e iciency, iWUE and elec on anspo a e
compa ed wi h single inocula ed o non-inocula ed plan s (Figu e 2A,D–F).
Unde d ough s ess, he o al chlo ophyll con en and iWUE signi ican ly inc eased
hei alues in SynCom-inocula ed plan s (Figu e 1A,E).
On he o he hand, high empe a u es pa icula ly a ec ed plan pho osyn hesis
(Figu e 2). Inocula ion wi h he SynCom inc eased all he pa ame e s measu ed, bu he
alues o hese pa ame e s we e, in gene al, clea ly lowe han he alues obse ed in
plan s unde he o he s ess condi ions. In pa icula , he ne pho osyn he ic a e (A
N
), he
quan um e iciency o PSII (
ΦPSII
) and he elec on anspo e iciency (ETR) showed e y
low alues unde his condi ion (Figu e 2B,D,F).
2.4. Inocula ion wi h he SynCom Enhances M. sa i a S ess S a us unde S ess ul Condi ions
In his wo k, he s ess le el in plan s was also checked by measu ing an ioxidan
enzymes ac i i ies. Inocula ion wi h he SynCom inc eased an ioxidan enzyme ac i i ies
o e he alues eco ded in non-inocula ed o single-inocula ed plan s (Figu e 3). Unde
hea y me al s ess, his enzyma ic induc ion was also obse ed in plan s inocula ed wi h
s ain N10 compa ed o non-inocula ed plan s, bu wi h lowe di e ences (Figu e 3).
Enzyma ic ac i i ies we e highe in oo s han in shoo s (Figu e 3). In pa icula , ca alase and
asco ba e pe oxidase ac i i ies we e no signi ican ly di e en in shoo s o non-inocula ed
and single-inocula ed plan s (N10) (Figu e 3C,D).
Conce ning salini y s ess, hese enzyma ic ac i i ies we e mo e p onounced in oo s
han in shoo s, whe e an ioxidan enzymes showed highe ac i i ies (Figu e 3). In gene al,
inocula ion wi h bac e ia induced he ac i i y o hese plan enzymes, and he highes
alues we e again eco ded in plan s inocula ed wi h SynCom (Figu e 3).
A simila pa e n o ha desc ibed in plan s unde me al s ess was obse ed in plan s
exposed o a d ough and a high empe a u e pe iod. The ac i i ies o an ioxidan enzymes
inc eased in plan s inocula ed wi h he bac e ial communi y, al hough highe alues o
ac i i y we e obse ed in oo s han in shoo s in he case o d ough (Figu e 3).
2.5. Inocula ion wi h he SynCom Modi ies M. sa i a Me als/loids and Mic onu ien s
Accumula ion unde Hea y Me al and Salini y S ess
Finally, he concen a ion o he mos abundan and oxic me al/loids was measu ed in
oo s and shoo s in o de o de e mine hei accumula ion in plan issues when plan s we e
g own in soils wi h high concen a ions o hea y me als. As shown in Figu e 4, inocula ion
wi h SynCom p omo ed As, Cd, Cu and Zn, wi h signi ican di e ences compa ed o
he me als accumula ed in non-inocula ed o single-inocula ed plan s. These di e ences
in me al/loids accumula ion we e g ea e in oo s han in shoo s and, in all plan s, he
accumula ion was much highe in oo s han in shoo s.
Plan s 2023,12, 2083 7 o 16
In plan s ea ed wi h 60 mM NaCl, he concen a ion o elemen s such as K, Mn, Na
and P was highe in plan s inocula ed wi h he SynCom han in con ol plan s (Supplemen-
a y Table S2).
Plan s 2023, 10, x FOR PEER REVIEW 7 o 17
and asco ba e pe oxidase ac i i ies we e no signi ican ly di e en in shoo s o non-
inocula ed and single-inocula ed plan s (N10) (Figu e 3C,D).
Conce ning salini y s ess, hese enzyma ic ac i i ies we e mo e p onounced in oo s
han in shoo s, whe e an ioxidan enzymes showed highe ac i i ies (Figu e 3). In gene al,
inocula ion wi h bac e ia induced he ac i i y o hese plan enzymes, and he highes
alues we e again eco ded in plan s inocula ed wi h SynCom (Figu e 3).
A simila pa e n o ha desc ibed in plan s unde me al s ess was obse ed in
plan s exposed o a d ough and a high empe a u e pe iod. The ac i i ies o an ioxidan
enzymes inc eased in plan s inocula ed wi h he bac e ial communi y, al hough highe
alues o ac i i y we e obse ed in oo s han in shoo s in he case o d ough (Figu e 3).
Figu e 3. An ioxidan enzymes ac i i ies a e he exposi ion o di e en s ess ul condi ions. (A)
Guaiacol pe oxidase (GPx), (B) supe oxide dismu ase (SOD), (C) ca alase (CAT), and (D) asco ba e
pe oxidase (GPx) in shoo s ( op g aphs) and oo s (bo om g aphs) o non-inocula ed and inocula ed
M. sa i a plan s a e 60 days in po s unde g eenhouse condi ions in he case o hea y me al and
sal ea men s, and du ing he s ess in he case o d ough and high empe a u e ea men s.
Values a e means ± S.D. (n = 16). Di e en le e s and symbols indica e means ha a e signi ican ly
di e en om each o he . (One-way ANOVA, LSD es , p < 0.0001). C-: non inocula ion; N10:
inocula ion wi h Ensi e sp. N10; CSN: inocula ion wi h he SynCom o med by Pseudomonas sp. N4,
Pseudomonas sp. N8, Ensi e sp. N10, and Ensi e sp. N12; hea y me al ea men : soil om he middle
ma shes o he Odiel i e as subs a e; sal ea men : s e ilized soil om he uppe ma shes o he
Odiel i e supplemen ed wi h 60 mM o NaCl as subs a e; d ough ea men : s e ilized soil om
he uppe ma shes o he Odiel i e as subs a e and plan s exposed o d ough s ess; high
empe a u e subs a e: s e ilized soil om he uppe ma shes o he Odiel i e as subs a e and
plan s exposed o 40 °C.
2.5. Inocula ion wi h he SynCom Modi ies M. sa i a Me als/loids and Mic onu ien s
Accumula ion unde Hea y Me al and Salini y S ess
Finally, he concen a ion o he mos abundan and oxic me al/loids was measu ed
in oo s and shoo s in o de o de e mine hei accumula ion in plan issues when plan s
we e g own in soils wi h high concen a ions o hea y me als. As shown in Figu e 4,
Figu e 3.
An ioxidan enzymes ac i i ies a e he exposi ion o di e en s ess ul condi ions. (
A
) Gua-
iacol pe oxidase (GPx), (
B
) supe oxide dismu ase (SOD), (
C
) ca alase (CAT), and (
D
) asco ba e pe ox-
idase (GPx) in shoo s ( op g aphs) and oo s (bo om g aphs) o non-inocula ed and inocula ed M.
sa i a plan s a e 60 days in po s unde g eenhouse condi ions in he case o hea y me al and sal
ea men s, and du ing he s ess in he case o d ough and high empe a u e ea men s. Values a e
means
±
S.D. (n= 16). Di e en le e s and symbols indica e means ha a e signi ican ly di e en
om each o he . (One-way ANOVA, LSD es , p< 0.0001). C-: non inocula ion; N10: inocula ion
wi h Ensi e sp. N10; CSN: inocula ion wi h he SynCom o med by Pseudomonas sp. N4, Pseudomonas
sp. N8, Ensi e sp. N10, and Ensi e sp. N12; hea y me al ea men : soil om he middle ma shes
o he Odiel i e as subs a e; sal ea men : s e ilized soil om he uppe ma shes o he Odiel
i e supplemen ed wi h 60 mM o NaCl as subs a e; d ough ea men : s e ilized soil om he
uppe ma shes o he Odiel i e as subs a e and plan s exposed o d ough s ess; high empe a u e
subs a e: s e ilized soil om he uppe ma shes o he Odiel i e as subs a e and plan s exposed
o 40 ◦C.
Plan s 2023,12, 2083 8 o 16
Plan s 2023, 10, x FOR PEER REVIEW 8 o 17
inocula ion wi h SynCom p omo ed As, Cd, Cu and Zn, wi h signi ican di e ences
compa ed o he me als accumula ed in non-inocula ed o single-inocula ed plan s. These
di e ences in me al/loids accumula ion we e g ea e in oo s han in shoo s and, in all
plan s, he accumula ion was much highe in oo s han in shoo s.
In plan s ea ed wi h 60 mM NaCl, he concen a ion o elemen s such as K, Mn, Na
and P was highe in plan s inocula ed wi h he SynCom han in con ol plan s
(supplemen a y Table S2).
Figu e 4. Me al/loids concen a ion a e he exposi ion o hea y me al s ess. (A) a senic (As), (B)
cadmium (Cd), (C) coppe (Cu), and (D) zinc (Zn) concen a ions in shoo s and oo s o M. sa i a
plan s inocula ed wi h di e en inoculums a e 60 days in po s unde g eenhouse condi ions using
soil om he middle ma shes o he Odiel i e as subs a e. Values a e means ± S.D. (n = 16).
Di e en le e s indica e means ha a e signi ican ly di e en om each o he (One-way ANOVA,
LSD es , p < 0.0001). C-: non inocula ion; N10: inocula ion wi h Ensi e sp. N10; CSN: inocula ion
wi h he SynCom o med by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensi e sp. N10, and Ensi e
sp. N12.
3. Discussion
In he las decade, ou e o s ha e been ocused on he eco e y o he deg aded
es ua ine soils o sou hwes Spain using au och honous halophy es and hei associa ed
bac e ia [11–15]. Recen ly, we ha e p oposed he combined use o hizobia and plan
g ow h p omo ing nodule endophy es o inocula e au och honous Medicago spp. plan s in
an a emp o eco e deg aded es ua ine soils by inc easing hei e ili y and educing
me als impac in plan s [19]. We selec ed an endophy ic bac e ial SynCom con aining wo
Ensi e sp. and wo Pseudomonas sp. s ains wi h se e al PGP p ope ies ha we e able o
p omo e M. sa i a g ow h and nodula ion on deg aded es ua ine soils. M. sa i a is a c op
o age plan widely used in Spain o cen u ies and, al hough i is no a na i e species, i
is a na u alized one. Ou pu pose in his wo k was o u he elucida e he u ili y o he
designed nodule SynCom o enhance M. sa i a adap a ion and g ow h in es ua ine soils
unde a ple ho a o s esses in a clima e change scena io. Odiel i e es ua ine (SW Spain)
is loca ed in a egion ha expe iences long pe iods o d ough , in which empe a u es can
each 40 °C a cen al hou s o he day du ing summe s, unde a hea y sun. The u u e
p ospec i e is e en wo se, wi h longe pe iods o d ough and high empe a u es.
M. sa i a plan s subjec ed o abio ic s essing si ua ions inc eased hei g ow h,
nodula ion a e compa ed wi h single inocula ed plan s and imp o ed hei
pho osyn he ic pa ame e s in esponse o inocula ion wi h ou nodule SynCom. These
Figu e 4.
Me al/loids concen a ion a e he exposi ion o hea y me al s ess. (
A
) a senic (As),
(
B
) cadmium (Cd), (
C
) coppe (Cu), and (
D
) zinc (Zn) concen a ions in shoo s and oo s o M. sa i a
plan s inocula ed wi h di e en inoculums a e 60 days in po s unde g eenhouse condi ions using
soil om he middle ma shes o he Odiel i e as subs a e. Values a e means
±
S.D.
(n= 16)
.
Di e en le e s indica e means ha a e signi ican ly di e en om each o he (One-way ANOVA,
LSD es , p< 0.0001). C-: non inocula ion; N10: inocula ion wi h Ensi e sp. N10; CSN: inocula ion wi h
he SynCom o med by Pseudomonas sp. N4, Pseudomonas sp. N8, Ensi e sp. N10, and Ensi e sp. N12.
3. Discussion
In he las decade, ou e o s ha e been ocused on he eco e y o he deg aded
es ua ine soils o sou hwes Spain using au och honous halophy es and hei associa ed
bac e ia [
11
–
15
]. Recen ly, we ha e p oposed he combined use o hizobia and plan
g ow h p omo ing nodule endophy es o inocula e au och honous Medicago spp. plan s in
an a emp o eco e deg aded es ua ine soils by inc easing hei e ili y and educing
me als impac in plan s [
19
]. We selec ed an endophy ic bac e ial SynCom con aining wo
Ensi e sp. and wo Pseudomonas sp. s ains wi h se e al PGP p ope ies ha we e able o
p omo e M. sa i a g ow h and nodula ion on deg aded es ua ine soils. M. sa i a is a c op
o age plan widely used in Spain o cen u ies and, al hough i is no a na i e species, i
is a na u alized one. Ou pu pose in his wo k was o u he elucida e he u ili y o he
designed nodule SynCom o enhance M. sa i a adap a ion and g ow h in es ua ine soils
unde a ple ho a o s esses in a clima e change scena io. Odiel i e es ua ine (SW Spain)
is loca ed in a egion ha expe iences long pe iods o d ough , in which empe a u es can
each 40
◦
C a cen al hou s o he day du ing summe s, unde a hea y sun. The u u e
p ospec i e is e en wo se, wi h longe pe iods o d ough and high empe a u es.
M. sa i a plan s subjec ed o abio ic s essing si ua ions inc eased hei g ow h, nodu-
la ion a e compa ed wi h single inocula ed plan s and imp o ed hei pho osyn he ic
pa ame e s in esponse o inocula ion wi h ou nodule SynCom. These esul s a e ela ed
wi h he p esence in hese endophy es o PGP p ope ies. IAA p oduc ion is in ol ed in
he inc ease o oo s and shoo s leng h [
20
], since his ho mone inc ease cell expansion
and is essen ial in oo de elopmen [
21
]. Bac e ial p ope ies ela ed wi h plan nu ien
acquisi ion also in luenced M. sa i a g ow h. Phospha e solubiliza ion p o ided phospho-
us [
22
,
23
], which helps in he nodula ion p ocess, amino acid and p o eins syn hesis [
24
].
Side opho es p oduced by bac e ia could p o ide essen ial me als such i on and Zn [
25
].
Conce ning ni ogen, ou SynCom enhanced M. sa i a nodula ion hus inc eased ni ogen
con en in plan s. Inc ease in nodula ion a e could be ela ed wi h ACC deaminase ac i i y,
which acili a ed he educ ion o he s ess le els in he plan s by educing e hylene and
Plan s 2023,12, 2083 9 o 16
hen a o ing nodula ion [
26
]. E hylene accumula ion is also esponsible o de olia ion,
cell elonga ion inhibi ion and senescence [
27
]. IAA also in e e es in he hizobia-legume
dialogue in e ac ion, delaying nodule senescence by in e ac ing wi h he bac e oid inside
he nodules [
28
]. Ensi e sp. and Pseudomonas sp. s ains showed ACC deaminase ac i i y
and he abili y o p oduce auxins, solubilize phospha e and o m bio ilms in he p esence
o As, Cd, Cu and Zn. In e es ingly, he p esence o me als inc eased he le els o ACC
deaminase ac i i y in Pseudomonas sp. s ains and induced his ac i i y in Ensi e sp. s ains,
ha lacked i in absence o me als. I is impo an o no e ha , despi e one o he s ess
condi ions, was he high me al con en in soil— he soil om he es ua ine used in his wo k
o assay he o he s esses had mode a e le els o me al con amina ion.
The posi i e e ec o inocula ion wi h he SynCom on M. sa i a g ow h and nodula ion
unde abio ic s ess was also e lec ed in inc eased alues o pho osyn hesis pa ame e s.
Ne e heless, plan s g owing a high empe a u e showed alues pa icula ly low o pa-
ame e s such as ne pho osyn he ic a e (A
N
), he quan um e iciency o PSII (
ΦPSII
) and
elec on anspo e iciency (ETR), e en unde SynCom inocula ion. The e a e se e al e-
iews epo ing he e ec s o high empe a u e in pho osyn hesis, which is highly sensi i e
o his s ess [
29
]. Among o he s, high empe a u e inhibi s PSII, which e iciency was pa -
ially es o ed by SynCom inocula ion in his wo k (Figu e 2D). Hea y me al and d ough
s esses pa icula ly a ec ed he ne pho osyn he ic a e, while e y low alues o iWUE
we e eco ded in con ol non-inocula ed plan s unde sal s ess. Values o iWUE unde
sal s ess we e comple ely es o ed in plan s inocula ed wi h he SynCom (Figu e 2E). In
his wo k, M. sa i a plan s inocula ed wi h he SynCom inc eased hei chlo ophyl con en
unde he di e en s esses. Conce ning legumes, enhanced amoun s o chlo ophyl we e
also ound in M. sa i a plan s inocula ed wi h wo Va io o ax endophy es unde me al
s ess [
18
], in chickpea plan s inocula ed wi h he endophy e Bacillus sub ilis BERA 71
unde sal s ess [
30
] o in soybean plan s inocula ed wi h he endophy e Bacillus ce eus SA1
unde high empe a u e [
31
]. Alyssum se pylli olium plan s inocula ed wi h Pseudomonas
azo o o mans ASS1, a d ough - esis an endophy e, showed inc eased alues o chlo ophyl
unde d ough s ess [32].
I is well-known ha p oduc ion o an excess o eac i e oxygen species (ROS) by plan
and a dec ease in he an ioxidan de ense mechanisms se e ely damage plan s, al e ing
cell unc ion and educing de elopmen [
33
,
34
]. Plan an ioxidan de ense enzymes, such
as ca alase, guaiacol pe oxidase, asco ba e pe oxidase and supe oxide dismu ase, ac as
p o ec o s agains ROS. In his wo k, ac i i ies o hese enzymes inc eased in oo s and
shoo s o M. sa i a plan s inocula ed wi h he SynCom unde he di e en abio ic s esses.
These esul s sugges ha bac e ial induc ion o plan an ioxidan enzyma ic ac i i ies
is a common and impo an mechanism o plan p o ec ion in abio ic s ess condi ions.
M. sa i a plan s inocula ed wi h izosphe ic bac e ia unde hea y me al s ess [
35
] o
nu ien de iciency [
17
] showed inc eased le els o ca alase, guaiacol pe oxidase, asco ba e
pe oxidase and supe oxide dismu ase. Simila esul s we e also ound in M. sa i a plan s
g owing in me al con amina ed es ua ine soils inocula ed wi h nodule endophy es om
Va io o ax genus [
18
]. Inocula ion o chickpea plan s wi h he endophy ic bac e ium Bacillus
sub ilis BERA 71 unde saline condi ions enhanced supe oxide dismu ase and ca alase
ac i i ies in he plan [
30
]. Soybean plan s inocula ed wi h he endophy e Bacillus ce eus
SA1 showed inc eased le els o asco ba e pe oxidase and supe oxide dismu ase unde
high empe a u e s ess [
31
]. In he same way, he d ough - esis an endophy ic bac e ia
Pseudomonas azo o o mans ASS1 enhanced he ac i i ies o Alyssum se pylli olium supe oxide
dismu ase, pe oxidase and ca alase in inocula ion expe imen s unde d ough s ess [32].
Legumes a e known o be able o accumula e high amoun s o hea y me als in oo s,
wi h low le els o ansloca ion o shoo s, wi hou a ec ing plan g ow h [
36
,
37
]. In his
wo k, M. sa i a plan s inocula ed wi h he SynCom in an es ua ine soil wi h high le els o
me als inc eased he amoun s o As, Cd, Cu and Zn accumula ed in oo s, compa ed wi h
non-inocula ed plan s o single inocula ed wi h hizobia. The concen a ion o me als in
oo s we e simila o hose measu ed in M. sa i a oo s o plan s g owing in an es ua ine
Plan s 2023,12, 2083 16 o 16
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Disclaime /Publishe ’s No e:
The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .