Nodule Synthetic Bacterial Community as Legume Biofertilizer under Abiotic Stress in Estuarine Soils.
Abstract
Estuaries are ecologically important ecosystems particularly affected by climate change and human activities. Our interest is focused on the use of legumes to fight against the degradation of estuarine soils and loss of fertility under adverse conditions. This work was aimed to determine the potential of a nodule synthetic bacterial community (SynCom), including two Ensifer sp. and two Pseudomonas sp. strains isolated from Medicago spp. nodules, to promote M. sativa growth and nodulation in degraded estuarine soils under several abiotic stresses, including high metal contamination, salinity, drought and high temperature. These plant growth promoting (PGP) endophytes were able to maintain and even increase their PGP properties in the presence of metals. Inoculation with the SynCom in pots containing soil enhanced plant growth parameters (from 3- to 12-fold increase in dry weight), nodulation (from 1.5- to 3-fold increase in nodules number), photosynthesis and nitrogen content (up to 4-fold under metal stress) under all the controlled conditions tested. The increase in plant antioxidant enzymatic activities seems to be a common and important mechanism of plant protection induced by the SynCom under abiotic stress conditions. The SynCom increased M. sativa metals accumulation in roots, with low levels of metals translocation to shoots. Results indicated that the SynCom used in this work is an appropriate ecological and safe tool to improve Medicago growth and adaptation to degraded estuarine soils under climate change conditions.
Full text
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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