Academic Edi o : Soon-Jae Lee
Recei ed: 25 June 2025
Re ised: 28 Augus 2025
Accep ed: 29 Augus 2025
Published: 8 Sep embe 2025
Ci a ion: Labidi, S.; Se en , A.;
Bouzoumi a, G.; Julien, T.; Cazals, G.;
Ib ahim, M.; Hammami, S.B.M.;
Achi , N. A buscula Myco hizal
Fungi Inocula ion Enhances
Nu i ional Quali y o P ickly Pea
(Opun ia icus-indica) F ui s and
Cladodes. Ag icul u e 2025,15, 1902.
h ps://doi.o g/10.3390/
ag icul u e15171902
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Licensee MDPI, Basel, Swi ze land.
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A icle
A buscula Myco hizal Fungi Inocula ion Enhances
Nu i ional Quali y o P ickly Pea (Opun ia icus-indica)
F ui s and Cladodes
Sonia Labidi
1
, Ad ien Se en
2,3
, Gho an Bouzoumi a
1
, Tina Julien
2,3
, Guillaume Cazals
4
, Manel Ib ahim
1
,
So iène B. M. Hammami 1and Nawel Achi 2,*
1Labo a oi e des Sciences Ho icoles, Ins i u Na ional Ag onomique de Tunisie, LR13AGR01,
Uni e si é de Ca hage, 43 A e Cha les Nicolle, Tunis 1082, Mah ajène, Tunisia
2
UMR Qualisud, Uni Mon pellie , A ignon Uni e si é, CIRAD, Ins i u Ag o, IRD, Uni e si é de La Réunion,
F-34398 Mon pellie , F ance
3CIRAD, UMR Qualisud, F-34398 Mon pellie , F ance
4IBMM, Uni Mon pellie , F-34398 Mon pellie , F ance
*Co espondence: nawel.achi @ins i u -ag o.
Abs ac
The e ec s o a buscula myco hizal ungi (AMF) inocula ion on he chemical composi ion
o he ui s and cladodes o wo Opun ia icus-indica cul i a s, cha ac e ized by hei ed
and yellow ui colo , we e in es iga ed unde ield condi ions. AMF ea men was
ound o signi ican ly in luence he concen a ion o phy onu ien s in he ui s. The
concen a ions o be acyanin and be axan hin inc eased by 1.2- and 1.9- old in ed and
yellow ui s, espec i ely. The polyphenol con en inc eased by 50%, wi h piscidic acid
being he mos abundan polyphenol in he ed ui s. A simila inc ease in asco bic acid
was obse ed in he yellow ui s. Rega ding he cladodes, AMF ea men was ound o
signi ican ly a ec mac onu ien le els, wi h glucose and uc ose con en s being 90% and
34% highe , espec i ely. Addi ionally, cladodes om plan s g own wi h AMF inocula ion
showed a 20% inc ease in asco bic acid and phospho us. These esul s demons a e cul i a -
and pa -o -plan -dependen e ec s o AMF inocula ion and con i m he nu i ional and
sus ainable po en ial o Opun ia icus-indica, pa icula ly when coupled wi h myco hizal
bio e iliza ion p ac ices.
Keywo ds: be axan hin; be acyanin; piscidic acid; ood dye; odde
1. In oduc ion
The Medi e anean basin has been iden i ied as a clima e change “ho spo ” and is
pa icula ly ulne able, acing se e al clima ic cons ain s [
1
]. Among eme ging c ops ha
equi e li le wa e and gene a e a easonable p o i , p ickly pea (Opun ia icus-indica) s ands
ou due o i s many ad an ages [
2
,
3
]. En i onmen ally, i plays a key ole in con olling
soil e osion h ough i s oo sys em, which enhances soil p ope ies [
4
]. In addi ion, i is a
honey plan , and i s ui s a e edible. The global ma ke o p ickly pea ui is expanding,
wi h annual p oduc ion exceeding 400,000 onnes in coun ies such as Mexico and I aly.
This g ow h is d i en by inc easing consume in e es in unc ional and exo ic ui s [
5
,
6
].
Al hough i is cu en ly only comme cially iable in a ew coun ies, such as Mexico,
I aly, and Sou h A ica, Opun ia icus-indica has he po en ial o gene a e bo h income and
employmen when suppo ed by s uc u ed alue chains and e ec i e ma ke ing. In
Ag icul u e 2025,15, 1902 h ps://doi.o g/10.3390/ag icul u e15171902
Ag icul u e 2025,15, 1902 2 o 15
Mexico, o example, cac us pea cul i a ion p o ides a p ima y o supplemen a y sou ce
o li elihood o a ound 20,000 amilies [
7
]. In Tunisia’s semi-a id zones, ecen s udies ha e
epo ed yields o 10–40 o cladodes and 1–5 o ui pe hec a e. This le el o p oduc ion
co esponds o an es ima ed ne economic e u n o app oxima ely USD 800 pe hec a e o
a me s. Nea ly 50% o he cladodes a e used as li es ock eed, educing eed cos s and
highligh ing he plan ’s ole in imp o ing a m esilience and household income [
3
]. P ickly
pea ui s ha e a swee la o and mild a oma, wi h a soluble solids con en ypically
anging om 12 o 16
◦
B ix, making hem a ac i e o bo h esh consump ion and
indus ial p ocessing. The economic ele ance o bo h he ui and he cladodes con inues
o g ow due o hei nu i ional p ope ies, d ough ole ance, and hei applica ions in
he ood, cosme ic, and pha maceu ical indus ies [
8
–
10
]. The wo mos comme cially
impo an Opun ia icus-indica a ie ies o ui consump ion a e hose wi h o ange skin
and yellow pulp (“Gialla”, meaning “yellow” in I alian) and hose wi h ed peel and pulp
(“Rossa”, meaning “ ed”), widely cul i a ed in Medi e anean coun ies such as I aly [
8
,
11
].
The ai s a ise om he a ious combina ions o be axan hins, which a e yellow-o ange
pigmen s, and be acyanins, which ange om ed o pu ple [
12
]. P ickly pea ui s a e
ecognized o hei in e es ing phy onu ien con en , pa icula ly an ioxidan s such as
la onols (e.g., que ce in de i a i es) and phenolic acids (e.g., piscidic and e ulic acids),
which con ibu e o hei po en ial as unc ional oods [
13
–
16
]. The cladodes (pads) o
Opun ia icus-indica also o e signi ican nu i ional alue o humans and animals. Young
cladodes, known as ‘nopali os’, a e adi ionally ea en as ege ables in La in Ame ica [
17
,
18
].
In li es ock eeding sys ems, pa icula ly in a id and semi-a id egions, ma u e cladodes
a e used as a aluable sou ce o o age, p o iding animals wi h wa e , ca bohyd a es, and
mine als while imp o ing he esilience o ag icul u e in a eas p one o d ough [
19
]. These
mul i ace ed uses highligh he impo ance o Opun ia icus-indica as a sus ainable c op wi h
signi ican ag onomic and nu aceu ical ele ance.
Mo e sus ainable cul i a ion p ac ices, such as myco hizal bio e iliza ion, a e eme g-
ing. This p ac ice consis s o using bio e ilize s o mula ed wi h a buscula myco hizal
ungi (AMF), which a e known o play a key ole in imp o ing wa e egula ion in plan s
h ough se e al mechanisms [
20
]. These ungi s imula e he p oduc ion o phy oho mones
ha enhance s ess ole ance and imp o e plan wa e s a us. Fo example, hey inc ease
he syn hesis o abscisic acid, a key ho mone in wa e s ess managemen ha egula es
s oma al closu e [
21
]. In addi ion, hey s imula e he p oduc ion o osmoly es, egula o s o
osmo ic p essu e, and he egula ion o aquapo ins, which p omo e hyd aulic conduc i -
i y [
22
]. The e ec s o AMF inocula ion on plan g ow h and s ess esponses can a y bo h
in hei na u e and in ensi y, depending on he hos plan . Di e en plan species—and
e en di e en cul i a s wi hin a species—may he e o e exhibi a iable esponses o AMF
coloniza ion [
23
]. Some s udies ha e shown ha his p ac ice can modula e he physiologi-
cal pe o mance o plan s bu also change he chemical composi ion o hei a ious pa s,
especially he ui s [
24
,
25
]. Indeed, i was demons a ed ha myco hizal inocula ion
o oma o plan s subjec ed o wa e s ess mi iga es he nega i e e ec s o d ough . This
esul ed in inc eased suga le els in ui s and s imula ion o ege a i e g ow h, as well
as imp o ed accumula ion o osmoly es and mine al nu ien s, con ibu ing o inc eased
yield [
26
]. As a ha dy c op adi ionally g own wi hou e ilize s, cac us ep esen s a
pa icula ly sui able candida e o bio e iliza ion. Howe e , in es iga ions in o hei in e -
ac ions wi h AMF emain sca ce. P e ious esea ch has p ima ily ocused on physiological
esponse o AMF inocula ion and cladode biomass p oduc ion unde d ough s ess, bu
no s udies ha e in es iga ed he e ec o his ea men on ui chemical composi ion.
Mo eo e , mos s udies ha e concen a ed on nu ien supply and mine al abso p ion,
epo ing educ ions in d ough -induced s ess ma ke s, bu exclusi ely in cladodes [
27
,
28
].
Ag icul u e 2025,15, 1902 3 o 15
The p esen s udy he e o e aims o in es iga e he e ec o AMF inocula ion on bo h
cladode and ui chemical composi ion, p o iding a mo e comp ehensi e unde s anding
o he e ec o AMF inocula ion on he whole plan . Speci ically, his s udy seeks o assess
he impac o AMF inocula ion on key nu ien s and phy onu ien s ha a e ele an o
human and animal nu i ion, as well as hei po en ial applica ions in he ood indus y.
2. Ma e ial and Me hods
2.1. Chemicals
All p oduc s, including he sol en s (e hanol, me hanol, ace oni ile), acids (sul u ic,
o mic, hyd ochlo ic, ni ic), hyd ogen pe oxide, sodium lau yl sul a e,
α
-amylase, p o ease,
e hylenedini ilo e aace ic acid (EDTA), sodium bo a e, sodium hyd ogen phospha e,
ie hylene glycol, ce yl ime hylammonium b omide, sodium hyd oxide, and s anda ds
(glucose, uc ose, suc ose, piscidic, and asco bic acids), we e bough om Sigma-Ald ich
(Sain -Louis, MA, USA).
2.2. Expe imen al Design
The expe imen al s udy was ca ied ou in wo comme cial p ickly pea o cha ds a
he Go e no a e o Nabeul (no heas e n Tunisia). The expe imen al si es a e si ua ed in
he supe io semi-a id bioclima ic s age acco ding o Embe ge ’s clima e classi ica ion. The
i s o cha d co e s app oxima ely 245.5 m
2
(36.302304
◦
N, 10.3542
◦
E) and is cha ac e ized
by sandy soil. I included 12 p ickly pea plan s, e enly di ided in o six AMF-inocula ed
and six non-inocula ed ones. In his o cha d, he spacing be ween he plan s was 3.5 m.
The second o cha d was la ge , wi h an a ea o 1010.07 m
2
(36.3123
◦
N, 10.3323
◦
E) and
cha ac e ized by sil y clay soil. In his si e, 12 inocula ed and 12 non-inocula ed plan s we e
conside ed. In his o cha d, he spacing be ween he plan s was 5.5 m. Bo h si es ollowed
a comple e block expe imen al design wi h wo ea men s: inocula ed and non-inocula ed
plan s. Inocula ion was pe o med wi h an a isanal myco hizal bio e ilize p epa ed in
he ho icul u al science labo a o y (LSH-INAT, Tunis, Tunisia). I is a g anula o mula ion
(20 p opagules/g) composed o na i e Glomus spp. (G. dese icola and G. cons ic um) mixed
wi h sand and pe li e. In No embe 2022, 250 g o inoculum we e added in a hole a
25 cm
o dep h and 50 cm om he plan s em on bo h sides, so 500 g pe plan we e applied.
Ha es ing was conduc ed one yea la e , in No embe 2023. All plan s we e main ained
unde uni o m cul u al p ac ices wi hin each si e, minimizing o cha d a iabili y.
2.3. Plan Ma e ial
All plan ma e ial was ha es ed om he wo expe imen al ields. Sampling o all he
p ickly pea plan s among he wo ea men s: inocula ed (wi h AMF) and non-inocula ed
(con ols). Fo each plan , i e ui s we e collec ed om all he canopies. The ui s we e all
collec ed om he ex e nal pa o he canopy a a human-leng h le el in o de o gua an ee
he same sun exposu e o all he collec ed ui s. The ui s we e sepa a ed acco ding o
hei colo , and only he la ges ones we e kep (mass
≥
50 g). Since he plan s p oduced
mos ly ed ui s, 18 ed ui s wi h AMF inocula ion we e used o analysis, along wi h
18 ed ui s (Rossa) o he con ol. The yellow ui s being less common, 9 ui s (Gialla)
wi h AMF inocula ion we e used and 9 o he con ol. In addi ion, 12 samples o 100 g
cladodes we e aken om each ea men . Samples we e pu in polye hylene bags and
di ec ly ozen a −20 ◦C.
2.4. Sample P epa a ion
F ui s we e peeled, c ushed, and sie ed h ough a ki chen colande o sepa a e he
juice and he seeds. Fo each sample, he peels, juices, and seeds we e weighed o calcula e
Ag icul u e 2025,15, 1902 4 o 15
he Juice/F ui (J/F) and Seed/F ui (S/F) a ios. The cladodes we e c ushed in o a pu ee
using a The momix (GM6, Vo we k, Wuppe al, Ge many). Juice and pu ee we e sealed in
plas ic bags unde acuum and s o ed in he da k a
−
20
◦
C un il analysis. A po ion o he
juice and c ushed cladodes was eeze-d ied in a eeze-d ye SMH15 (Usi oid, Élancou ,
F ance) o ca y ou some speci ic analyses.
2.5. P oximal Composi ion
Mois u e con en , complemen a y o d y ma e , was ob ained by g a ime y, ac-
co ding o he AOAC me hod 925-09. To al soluble solid was de e mined by he AOAC
me hod 932-12 using a digi al e ac ome e (PAL-1, ATAGO, Tokyo, Japan). The pH was
de e mined acco ding o he AOAC me hod 981-12. Finally, he i a able acidi y was
measu ed acco ding o he AOAC me hod 942-15 by po en iome ic i a ion wi h NaOH
(0.1 mol
·
L
−1
) up o pH 8.1 o a 5 mL ( o p ickly pea juice) homogenized sample mixed
wi h 25 mL o deionized wa e . The esul s we e epo ed in g o ci ic acid
·
100 mL
−1
o in
g·100 g−1o he sample [29].
Ash, ni ogen, and ibe s we e de e mined on eeze-d ied samples. The ash con en
was de e mined by igni ing 10 g d ied samples in a mu le u nace (P330, Nabe he m,
Lilien hal, Ge many) a 550 ◦C o 4 h acco ding o he AOAC me hod 942-05.
To al ni ogen con en o all samples was de e mined by he Dumas combus ion
me hod acco ding o he AOAC 990.03 me hod using a ni ogen analyze (T uMac, Leco,
Joseph, MI, USA). A con e sion ac o o 6.25 was used o es ima e he concen a ion
o p o eins.
Fibe con en s we e ob ained by di e ence o he ui s because o he low con en o
ibe expec ed in he ui s, while cladode ibe con en s we e measu ed acco ding o he
me hod o Van Soes . [
30
,
31
]. A p ecise quan i y o pe ec ly homogeneous eeze-d ied
cladode pu ee (be ween 0.7 and 1 g) was weighed in o mac o-po ous bags (F57, Ankom
Technology, Macedon, NY, USA). The ea e , all he me hods we e ca ied ou in a Fibe Sac
Ankom200 equipmen (Fibe Sac Ankom200, Ankom Technology, Macedon, NY, USA). The
samples we e i s subjec ed o consecu i e enzyma ic mace a ions o pu i y he ibe s.
Fi s , he bags we e imme sed in a 1 mL
·
L
−1
FAA he mos able alpha-amylase solu ion o
25 min a 100
◦
C. A e cooling and insing wice wi h ul apu e wa e , he samples we e
imme sed in a p o ease solu ion > 1000 U
·
L
−1
o 15 min a 38
◦
C. The samples we e hen
washed wice wi h ul apu e wa e be o e Neu al De e gen Fibe (NDF) measu emen .
To do so, 60 g o sodium lau yl sul a e was weighed di ec ly in o 800 mL o ul apu e wa e
and mixed gen ly un il comple ely dissol ed. Then, 37 g EDTA, 13.6 g sodium bo a e,
and 9.1 g sodium hyd ogen phospha e we e added and mixed, and he o al olume was
inc eased wi h 800 mL mo e ul apu e wa e . Finally, once he eagen s we e pe ec ly
dissol ed, 20 mL o ie hylene glycol was added, and he solu ion was illed o 2 L wi h
ul apu e wa e and adjus ed o a pH be ween 6.9 and 7.1. The samples we e imme sed
in his solu ion o 1 h 15 min a 100
◦
C, cooled, and insed 5 imes wi h ul apu e wa e .
The humid samples we e d ied a 103
◦
C a nigh and weighed be o e Acid De e gen
Fibe (ADF) analysis. To do so, 40 g o ce yl ime hylammonium b omide was dissol ed
in
1 L
o 0.5 mol
·
L
−1
sul u ic acid. Once dissol ed, he olume was adjus ed o 2 L wi h
0.5 mol
·
L
−1
sul u ic acid. The esidues o NDF we e imme sed in his solu ion o 30 min
a ambien empe a u e o ehyd a e he samples. Then, he empe a u e was inc eased
o 100
◦
C, and he ea men las ed 1 h 15 min. The ea e , he samples we e cooled and
washed
5 imes
wi h ul apu e wa e . The humid samples we e d ied a 103
◦
C a nigh
and weighed be o e Acid De e gen Lignin (ADL) measu emen . In his las s ep, he ADF
esidues we e imme sed in 72% sul u ic acid o 3 h. Samples we e hen insed 5 imes
wi h ul apu e wa e (un il wa e was neu al), d ied a 103 ◦C o e nigh , and weighed.
Ag icul u e 2025,15, 1902 5 o 15
2.6. Suga and Asco bic Acid Analysis
Glucose, uc ose, suc ose, and asco bic acid con en s we e de e mined using an HPLC
sys em (1100 HPLC, Agilen , San a Cla a, CA, USA) equipped wi h RI and UV de ec o s. A
column 300
×
8 mm (SH1011, Shodex, Yokohama, Japan) wi h a mobile phase composed o
H
2
SO
4
(0.1%) in wa e was used a an isoc a ic low o 0.7 mL
·
min
−1
and a 40
◦
C. Injec ion
olume was 10
µ
L, and spec opho ome ic de ec ion was se a 210 and 245 nm. The RI
de ec o was se a 50
◦
C. Opun ia ui juices we e di ec ly injec ed a e il a ion using a
0.45 µm
sy inge il e (Sa o ius, Gö ingen, Ge many). Cladode pu ees we e subjec ed o
ex ac ion wi h e hanol be o e injec ion. To do so, 2 g o pu ee was mixed wi h 10 mL o
80% e hanol
o 20 min a 70
◦
C. A e cen i uga ion a 10,000
×
g o 10 min and a 20
◦
C
(A an i-JE cen i uge, Beckman coul e , B ea, CA, USA), he supe na an was collec ed. The
ex ac ion was pe o med wice, and he supe na an s we e pooled and il e ed h ough
a
0.45 µm
sy inge il e (Sa o ius, Gö ingen, Ge many) be o e injec ion. Suga eco e y
was ≥98%.
Fo suga s, he LODs (in g/L) we e 0.2 o glucose and 0.3 o bo h uc ose and
suc ose. LOQs we e 0.6 g/L o glucose and 0.9 g/L o uc ose and suc ose. Fo asco bic
acid, he LOD and LOQ (in mg/L) we e 9 and 28, espec i ely.
2.7. Polyphenols and Be alains
Polyphenols, be alains, and pigmen s we e analyzed ollowing he Tamba e al. (2019)
me hod [
32
]. Sepa a ion was ealized using a ch oma og aphy (1260 HPLC, Agilen , USA)
equipped wi h a diode a ay de ec o . A olume o 20
µ
L was injec ed h ough an Up i-
sphe e C18 250 mm
×
4.6 mm
×
5
µ
m column (Up isphe e, In e chim, Mon luçon, F ance).
The pump was con olled as ollows: Phase A, o mic acid 1%; Phase B, ace oni ile. Flow
was se a 0.7 mL
·
min
−1
and empe a u e a 30
◦
C. The g adien was ixed as ollows: a
he ini ial ime, 98% o A and 2% o B; s abilized a 2% B o 10 min; inc eased a 20% o B
om 10 o 30 min; o 40% B om 30 o 50 min; o 60% B om 50 o 70 min; o 80% B om
70 o 80 min
; o 100% B om 80 o 90 min; and e u ned o he ini ial condi ion (2% B) in
5 min
and main ained o 10 min (phase A is always complemen a y o B o each 100%).
The UV- is de ec o was se a 280 nm o polyphenols, 480 nm o be axan hins, and 535 o
be acyanins. Calib a ion cu es o piscidic acid and eucomic acid we e calcula ed be ween
0.1 and 1 g.L
−1
[
32
]. Be acyanin and be axan hin calib a ion was pe o med by ob aining
concen a ions om spec opho ome y (Speco d 600, Jena, Ge many) a 535 and 480 nm,
espec i ely. Be acyanin (MW = 550 g/mol) and Indicaxan hin con en s (MW = 308 g/mol)
we e calcula ed acco ding o he Bee -Lambe law using 60,000 L
·
mol
−1·
cm
−1
as he be a-
cyanin mola ex inc ion coe icien in wa e and
48,000 L·mol−1·cm−1
as he indicaxan hin
one, a e dilu ing un il abso bance was be ween 0.2 and 1. Opun ia icus-indica ui juices
we e di ec ly injec ed a e il a ion using a 0.45
µ
m sy inge il e (Sa o ius, Ge many).
Cladode pu ees we e subjec ed o 2 ex ac ions wi h me hanol be o e injec ion. To do so,
2 g o pu ee was mixed wi h 10 mL o 70% me hanol o 15 min. A e cen i uga ion a
10,000
×
g o 10 min and a 20
◦
C using he A an i-JE cen i uge (c, USA), he supe na an
was collec ed. The ex ac ion was pe o med wice, and he supe na an s we e pooled
and il e ed using a 0.45 µm sy inge il e (Sa o ius, Gö ingen, Ge many) be o e injec ion.
Polyphenol eco e y was ≥96%.
Limi s o de ec ion (LOD) we e as ollows (in mg/L): 6 o bo h piscidic and eucomic
acids, and 1.5 and 4 o be anin and be axan hin, espec i ely. Limi s o quan i ica ion
(LOQ) we e 20 mg/L o piscidic and eucomic acids and 4 and 13 mg/L o be anin and
be axan hin, espec i ely.
Ag icul u e 2025,15, 1902 6 o 15
2.8. Mine al Composi ion
The mine al con en s we e analyzed on eeze-d ied samples by induc i ely coupled
plasma mass spec ome y (The mo Elemen al, X-Se ies, Baden-Baden, Ge many) ollowing
he me hod desc ibed by [
33
]. A ound 50 mg o he eeze-d ied sample was diges ed
wi h HNO
3
:H
2
O
2
(65:35, / ) wi h a MARS Xp ess mic owa e sys em (CEM Co po a ion,
Ma hews, NC, USA). The diges ion condi ions we e as ollows: up o 120
◦
C o 15 min and
hen cons an o 10 min; up o 160
◦
C in 20 min and cons an o 15 min; inally, samples
we e cooled o 22
◦
C o 30 min and dilu ed o 25 mL wi h deionized ul apu e wa e . The
unce ain y o he measu emen is abou 10%. The limi s o de ec ion we e in
µ
g pe g: 22.1
o calcium, 8.6 o po assium, 5.4 o sodium, 9.8 o phospho us, and 0.5 o magnesium.
The limi s o quan i ica ion we e in
µ
g pe g: 321.0 o calcium, 39.0 o po assium, 14.0 o
magnesium, 35.0 o sodium, and 97 o phospho us.
2.9. S a is ical Analysis
Due o sample sizes being less han 50, a non-pa ame ic K uskal–Wallis es was used
o compa e g oup means. When signi ican di e ences we e de ec ed, pos hoc pai wise
compa isons we e pe o med using he Bon e oni co ec ion. To compa e esul s be ween
ea ed and un ea ed cladodes, a Mann–Whi ney U es was used. All analyses we e
pe o med wi h S a is ica so wa e e sion 14.1.0. (S a so , Tulsa, OK, USA).
3. Resul s and Discussion
3.1. F ui s
3.1.1. Mac ocons i uen s o F ui s om he Two P ickly Pea Pheno ypes and E ec o
Myco hizal Bio e iliza ion
The d y ma e (DM) con en o p ickly pea ui s anged om 8.23 o 9.59% o he
Gialla a ie y and om 9.02 o 10.69% o he Rossa a ie y (Table 1). These alues closely
aligned wi h he o al soluble solids (TSS) con en , which anged om 8.64 o 9.80% o
he yellow a ie y and om 9.08 o 10.27% o he ed a ie y. This co ela ion is u he
suppo ed by indings in p e ious s udies [
34
]. This simila i y indica es ha he majo i y
o he d y ma e consis s o soluble molecules. This composi ion was aligned wi h he
obse a ion o he low iscosi y o he juice ex ac ed om he ui s. The alues ob ained
in his s udy all wi hin he lowe ange o DM and TSS epo ed in p e ious s udies. Fo
example, TSS alues anging om 11.8 o 15.6% we e obse ed in 25 ui accessions om
Texas and A gen ina [
35
]. S in zing e al. (2003) also epo ed TSS alues o 12.3% and
13.6% o he Gialla and Rossa a ie ies, espec i ely, indica ing highe TSS and DM le els
in he Rossa a ie y, which is consis en wi h ou obse a ions [
9
]. Ou lowe DM alues
could be a ibu ed o a iabili y in e oi o accessions o ag icul u al p ac ices, as no ed
in p e ious s udies [
8
,
35
]. Howe e , he pulp con en ound by Felke e al. (2005) was
app oxima ely 45%, which aligns wi h ou indings [35].
As expec ed, he main cons i uen s o he d y ma e o ui s we e suga s and, mo e
pa icula ly, glucose and uc ose in simila amoun s. S in zing e al. (2003) and Melga
e al. (2017) also ound ha he main suga s in cac us pea s we e glucose and uc ose, wi h
glucose being sligh ly mo e p edominan [
9
]. They also ound a glucose/ uc ose a io o 1.3,
iden ical o bo h Gialla and Rossa a ie ies. Fo he wo cul i a s, ash, p o eins, and ibe s
each accoun ed o app oxima ely 6% o he d y ma e . The main signi ican di e ences
be ween he cul i a s, as p esen ed in Table 1, we e hei acid p o iles. The Rossa a ie y
exhibi ed a lowe pH (5.63–5.74) compa ed o Gialla (6.01–6.37) and a signi ican ly highe
i a able acidi y, wi h alues o 0.11–0.14%, e sus 0.01–0.02% o Gialla. Addi ionally,
suc ose le els we e highe in he Rossa a ie y. The mean suga - o-acid a io, as shown in
Table 1, was close o 80 o Rossa and exceeded 400 o Gialla. This esul was simila o
Ag icul u e 2025,15, 1902 7 o 15
o he s udies ha ha e demons a ed highe acidi y in Rossa cul i a s. This highe suga -
o-acid a io con ibu es o explaining he swee as e o p ickly pea ui and highligh s he
need o blend hem wi h mo e acidic ui s be o e consump ion o achie e a suga - o-acid
a io o a ound 10, which is o en necessa y o consume p e e ences o achie e a balanced
swee -sou la o [
9
]. Acco ding o li e a u e, he main acids a e ci ic acids, ep esen ing
90% o he o ganic acids, ollowed by malic and lac ic acids [
15
,
36
,
37
]. Succinic acid was
also ound as a dominan acid along wi h ci ic acid in o he s udies, showing ha he acid
p o ile may be egion-dependen [38].
Table 1. P oximal composi ion (we basis) o ed and yellow ui s om p ickly pea plan s inocula ed
wi h AMF o no (con ol). Da a be ween b acke s ep esen he s anda d de ia ion ( o Rossa,
n = 18
, and o Gialla, n = 9). Values in bold and bea ing di e en le e s o he same pa ame e a e
signi ican ly di e en (p< 0.05).
T ea men Wi h AMF Con ol
Pheno ype Gialla Rossa Gialla Rossa
Juice/F ui a io 0.44 (0.03) a0.53 (0.08)a0.37 (0.03) b0.43 (0.11) a
Seeds/F ui a io 0.08 (0.01) 0.08 (0.02) 0.08 (0.01) 0.10 (0.02)
pH 6.01 (0.17) b5.63 (0.16) a6.37 (0.09) b5.74 (0.16) a
To al soluble solids (%) 9.80 (0.75) 10.27 (2.71) 8.64 (0.25) 9.08 (0.91)
D y ma e (%) 9.59 (0.95) 10.69 (1.64) 8.23 (0.09) 9.02 (0.98)
Glucose (%) 4.34 (0.52) 3.29 (1.06) 3.70 (0.08) 3.77 (0.89)
Suc ose (%) 0.02 (0.01) b0.10 (0.02) a0.01 (0.01) b0.08 (0.01) a
F uc ose (%) 4.19 (0.61) 4.76 (1.49) 3.38 (0.10) 3.71 (0.79)
Ti a able Acidi y (%) 0.01 (0.01) b0.14 (0.02) a0.02 (0.01) b0.11 (0.02) a
Ash% 0.81 (0.38) a0.80 (0.08) a0.44 (0.06) b0.64 (0.06) a
P o ein (%) 0.70 (0.07) 0.80 (0.08) 0.61 (0.06) 0.57 (0.06)
Es ima ed Fibe s (%) 0.45 (0.29) 1.22 (0.55) 0.33 (0.12) 0.86 (0.50)
The e ec o myco hiza ion on he mac ocons i uen s o he ui s was mino and was
only obse ed in e ms o ash con en and he juice- o- ui a io o he Gialla pheno ype.
The ash con en o myco hized ui s was highe , a 0.8%, compa ed o 0.44% o Gialla
ui s ha we e no ea ed. This sugges s ha myco hiza ion enhances he mine al con en
o he yellow pheno ype wi hou causing a concen a ion e ec , as he ui ’s juiciness
inc eases. This e ec is likely due o imp o ed oo sys em de elopmen acili a ed by
ilamen ous ungi [39].
3.1.2. E ec o Myco hizal Bio e iliza ion on he Mic onu ien Con en s
Figu e 1and Table 2illus a e he e ec o a ie y and myco hizal bio e iliza ion
on he main phy o-mic onu ien s. These phy onu ien s include pigmen s (be axan hins
o he Gialla a ie y and be acyanins and be axan hins o he Rossa a ie y), as well as
asco bic acid and polyphenols.
The mean o he o al pigmen con en was 45 mg
·
L
−1
o Rossa and 75 mg
·
L
−1
o
Gialla. The yellow pheno ype exhibi ed a highe amoun o be alains han he ed ones.
These alues a e close o ha ound p e iously o a o al o 75 mg
·
kg
−1
and 95 mg
·
kg
−1
o edible pulp o he ed and yellow a ie ies, espec i ely [
36
]. The main be axan hin
o p ickly pea s is indicaxan in. Acco ding o Bu e a (2002) [
36
], he a io o be anin o
indicaxan hin is a ound 1:8 (w:w) in he yellow ui o 2:1 (w:w) in he ed one. In ou case,
Ag icul u e 2025,15, 1902 8 o 15
he yellow a ie y did no con ain any be acyanin, while he ed cul i a con ained a ound
15 mg
·
L
−1
be axan hin, which makes a be anin- o-indicaxan hin a io o 2:1, as obse ed
by Bu e a e al. (2002) [36].
Figu e 1. Phy o-mic onu ien con en s (mg
·
L
−1
, we basis) in ed and yellow ui s om p ickly
pea plan s inocula ed wi h AMF o le un ea ed (Con ol).
Table 2. Phy o-mic onu ien con en (we basis) o ed and yellow ui s om p ickly pea plan s,
inocula ed wi h AMF o le un ea ed (Con ol). Da a be ween b acke s ep esen he s anda d
de ia ion ( o Rossa, n = 18, and o Gialla, n = 9). Values in bold and bea ing di e en le e s o he
same pa ame e a e signi ican ly di e en (p< 0.05).
T ea men Wi h AMF Con ol
Pheno ype Gialla Rossa Gialla Rossa
Asco bic Acid mg·L−1206 (30) a26 (6) b136 (15) b64 (30) b
Piscidic Acid mg·L−133 (4) b99 (13) a35 (4) b55 (15) b
Eucomic Acid mg·L−110 (4) 21 (10) 7 (1) 10 (5)
Polyphenols mg·L−1130 (12) b232 (35) a121 (14) b159 (35) b
Be acyanin mg·L−1ND 46 (19) aND 21 (10) b
Be axan hin mg·L−1111 (20) a18 (7) b38 (8) b13 (3) b
P (mg·100 mL−1)23 (1) 22 (7) 22 (1) 21 (7)
K (mg·100 mL−1)237 (9) b345 (49) a250 (16) a,b 326 (74) a
Ca (mg·100 mL−1)36 (2) b84 (21) a51 (3) a,b 80 (32) a
Mg (mg·100 mL−1)24 (4) 28 (8) 37 (2) 27 (7)
Na (mg·100 mL−1)1.00 (0.01) 0.90 (0.01) 1.10 (0.01) 0.80 (0.01)
The e ec o myco hiza ion on pigmen con en s was signi ican . Indeed, o be a-
cyanin in Rossa, he con en was highe (46 mg
·
L
−1
) in ui s o myco hized plan s com-
pa ed o non-myco hized ones (21 mg
·
L
−1
). Fo indicaxan hin in Gialla, i was also highe
in ui s om myco hized plan s (111 mg
·
L
−1
) compa ed o hose om non-myco hized
ones (38 mg
·
L
−1
). The e ec o inc eased pigmen con en due o myco hiza ion has ne e
been s udied o p ickly pea ui s. This inc ease in pigmen s due o myco hiza ion has
been p o en on o he ypes o pigmen s, i.e., an hocyanin in s awbe ies [
25
]. The e o e,
myco hiza ion could inc ease pigmen con en , whe he di ec ly o indi ec ly, by s imula -
Ag icul u e 2025,15, 1902 9 o 15
ing he me abolic pa hways o pigmen syn hesis in he plan [
25
]. This e ec o enhancing
pigmen accumula ion is in e es ing. Indeed, p ickly pea s may be o in e es o na u al
ood dye applica ions, as hey lack he ea hy as e associa ed wi h bee oo , he main
sou ce o be acyanin used in he ood indus y o colo low-acid oods [9,40].
A highe asco bic acid con en was obse ed in Gialla ui s. While S inzing e al.
(2003) epo ed simila le els in ed and yellow a ie ies, Reis e al. (2017) ound highe
le els o asco bic acid in whi e, yellow, and o ange O. icus-indica pheno ypes [
9
,
41
]. AMF
ea men inc eased he asco bic acid con en o he Gialla a ie y by 50%.
Rega ding polyphenols, he ed a ie y exhibi ed a highe phenolic con en , a e aging
200 mg
·
L
−1
compa ed o 120 mg
·
L
−1
in he Gialla a ie y. These alues a e lowe han
ha ound in he li e a u e (350 o 650 mg
·
L
−1
) [
42
]. Howe e , hese las alues we e
ob ained by he Folin–Coical eu me hod, which usually gi es o e es ima ed polyphenol
con en s in compa ison o ha ob ained by HPLC [
43
]. Indeed, he spec opho ome ic
Folin–Ciocal eu me hod is no en i ely speci ic o polyphenols; i also eac s wi h o he e-
ducing compounds such as asco bic acid, suga s, and amino acids. Myco hiza ion also had
a signi ican impac on polyphenols, wi h a posi i e e ec on he Rossa a ie y (
p< 0.05
).
The inc ease in phenolic compounds due o bio e iliza ion was al eady epo ed on oma-
oes [
44
]. No ably, asco bic acid is he p ima y an ioxidan in yellow a ie ies, whe eas
polyphenols p edomina e in ed a ie ies. Fu he mo e, i is wo h no ing ha ou AMF in-
ocula ion enhanced he concen a ions o bo h. This end may be due o hese compounds
ac ing as de ense molecules ollowing he es ablishmen o myco hizal coloniza ion in
he plan . Rega ding he polyphenol p o ile, he main compounds we e phenolic acids,
pa icula ly piscidic acid, accoun ing o 50% o he o al polyphenols, as can be seen in
Figu e 2.
Mena e al. (2018) [15]
conduc ed an exhaus i e analysis o
six Spanish cul i a s
,
iden i ying 16 la onols and 12 phenolic acids.
Ga cía-Cayuela e al. (2019)
iden i ied
17 phenolic
compounds, p ima ily la onoid glycosides, wi h piscidic acid being he mos
abundan [
45
]. Simila ly, Ramí ez-Pé ez e al. (2024) epo ed he p esence o eucomic
acid [
37
]. Logically, piscidic and eucomic acids we e signi ican ly highe o he myco -
hized ed cul i a , wi h alues o 99 and 21 mg
·
L
−1
, espec i ely. This esul is in e es ing,
knowing ha piscidic acid is an an ioxidan and has shown a p o ec i e e ec agains
UVA-induced oxida i e s ess [46].
Figu e 2. (A) Example o ch oma og am o polyphenols in Rossa a ie y. Mass spec um and s uc u e
o (B) piscidic and (C) eucomic acids.
