Ci a ion: Ga cía-La o e, C.; Rod igo,
S.; San ama ía, O. Po en ial o Fungal
Endophy es Isola ed om Pas u e
Species in Spanish Dehesas o
P oduce Enzymes unde Sal
Condi ions. Mic oo ganisms 2023,11,
908. h ps://doi.o g/10.3390/
mic oo ganisms11040908
Academic Edi o : Myung-Ji Seo
Recei ed: 20 Janua y 2023
Re ised: 21 Ma ch 2023
Accep ed: 30 Ma ch 2023
Published: 31 Ma ch 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/).
mic oo ganisms
A icle
Po en ial o Fungal Endophy es Isola ed om Pas u e Species
in Spanish Dehesas o P oduce Enzymes unde Sal Condi ions
Ca los Ga cía-La o e 1, Sa a Rod igo 2and Osca San ama ía3,*
1School o Ag icul u al Enginee ing, Uni e si y o Ex emadu a, A da. Adol o Suá ez s/n,
06007 Badajoz, Spain; [email p o ec ed]
2Indehesa Resea ch Ins i u e, Campus de Badajoz, Uni e si y o Ex emadu a, A da. de El as s/n,
06006 Badajoz, Spain; [email p o ec ed]
3
Depa men o Plan P oduc ion and Fo es Resou ces, Uni e si y Ins i u e o Resea ch in Sus ainable Fo es
Managemen (iuFOR), Uni e si y o Valladolid, A da. Mad id 57, 34004 Palencia, Spain
*Co espondence: osca [email p o ec ed]
Abs ac :
Endophy ic ungi ha e been ound o p oduce a wide ange o ex acellula enzymes, which
a e inc easingly in demand o hei indus ial applica ions. Di e en by-p oduc s om he ag i ood
indus y could be used as ungal g ow h subs a es o he massi e p oduc ion o hese enzymes,
speci ically as a way o e alo ize hem. Howe e , such by-p oduc s o en p esen un a o able
condi ions o he mic oo ganism’s g ow h, such as high sal concen a ions. The e o e, he objec i e
o he p esen s udy was o e alua e he po en ial o ele en endophy ic ungi—which we e isola ed
om plan s g owing in a ha sh en i onmen , speci ically, om he Spanish dehesas— o he pu poses
o he
in i o
p oduc ion o six enzymes (i.e., amylase, lipase, p o ease, cellulase, pec inase and
laccase) unde bo h s anda d and sal -amended condi ions. Unde s anda d condi ions, he s udied
endophy es p oduced be ween wo and ou o he six enzymes e alua ed. In mos o he p oduce
ungal species, his enzyma ic ac i i y was ela i ely main ained when NaCl was added o he
medium. Among he isola es e alua ed, Sa ocladium e icola (E025), Ac emonium implica um (E178),
Mic odiplodia hawaiiensis (E198), and an uniden i ied species (E586) we e he mos sui able candida es
o he massi e p oduc ion o enzymes by using g ow h subs a es wi h saline p ope ies (such as
hose ound in he many by-p oduc s om he ag i ood indus y). This s udy should be conside ed
an ini ial app oach by which o u he s udy he iden i ica ion o hese compounds as well as o
de elop he op imiza ion o hei p oduc ion by di ec ly using hose esidues.
Keywo ds:
seconda y me aboli es; by-p oduc s; endophy ic ungi; ag i ood indus y; halo ole ance
1. In oduc ion
The e m endophy e includes mic oo ganisms, mainly ungi and bac e ia, which
colonize he in e nal issues o plan s wi hou causing any isible disease symp oms [
1
].
Al hough many o hese endophy es ha e been desc ibed o ha e a symbio ic ole [
2
],
many o he s a e oppo unis ic species ha a e wai ing o plan senescence in o de o
ake ad an age o he plan issues’ coloniza ion [
3
]. In he case o endophy ic ungi, his
coloniza ion is ca ied ou h ough ex acellula enzymes ha , oge he wi h he p oduc ion
o many o he me aboli es, a e capable o deg ading he cell wall o he plan [
4
] and
coun e ac ing i s chemical de enses [
5
,
6
]. Di e en g oups o hese enzymes, such as
pec inases, xylanases, cellulases, lipases, p o eases, o phenol oxidases ha e been desc ibed
o be in ol ed in his p ocess [
7
]. These compounds ha e been ound o be also in ol ed in
he plan immune sys em by elici ing de ense mechanisms agains pa hogens [
8
], as well as
in he g ow h s a us o he hos , whe eby nu ien up ake h ough he oo s is enhanced [
9
].
Besides hei ecological oles, all hese enzymes a e aluable supplies ha a e used
in nume ous ood, pha maceu ical, o pape indus ies [
10
–
12
]. Fo example, pec inases
play an impo an ole as ining agen s in he juice and wine indus ies by enhancing he
Mic oo ganisms 2023,11, 908. h ps://doi.o g/10.3390/mic oo ganisms11040908 h ps://www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2023,11, 908 2 o 12
cla i ica ion p ocess and by imp o ing he o e all quali y o he inal p oduc [
11
]. Simila ly,
ungal amylases ha e inc easingly been used o s a ch hyd olysis in he p oduc ion o
simple suga s and sy ups, as well as in he op imiza ion o he bee mashing p ocess [
5
,
13
].
Mic obial p o eases and lipases a e also used in he ood indus y, speci ically o he
s abiliza ion o d ied p oduc s in he milk indus y [
11
] and o ex ending he shel li e o
bake y p oduc s [
14
]. These enzymes ha e o he impo an applica ions. Fo example, in
he cosme ic indus y—as in he case o p o eases— hey a e used as a componen in c eams
o he emo al o dead cells [
15
]; lipases, o ob aining a y amides [
16
]; o laccases, which
a e used in he p epa a ion o hai dyes [
17
]. In he case o he pape indus y, ungal
laccases a e used o he polyme iza ion o ibe s [
11
] and cellulases in o de o ha den
he inal p oduc [
18
], which may allow a huge amoun o ene gy o be sa ed du ing he
pape -making p ocess [19].
Al hough hese enzymes can be ob ained om many sou ces, hose which a e p o-
duced by ungi and bac e ia ha e been desc ibed o be mo e s able du ing hei p ocess-
ing [
20
]. Howe e , only a ound wen y o hese mic obial enzymes, such as hose ob ained
om di e en Aspe gillus species, a e p oduced on an indus ial scale [
21
]. The e o e, he
use o endophy es, in his case ungal endophy es, migh p o ide a sui able al e na i e
by which o y o expand he ange o o ganisms o massi ely p oduce hese kinds o
enzymes. This massi e p oduc ion would need he u iliza ion o a as amoun o subs a e
o g ow he enzyme-p oducing ungi. In his ega d, he use o by-p oduc s om he
ag i ood indus y may allow one o ob ain he amoun o subs a e equi ed while, a he
same ime, o loading hose ini ially alueless was es [
22
]. Howe e , hese esidues a e
usually no pa icula ly a o able o ungal g ow h, as hey may con ain annins, phenols,
and o he subs ances wi h biocide ac i i y [
23
]. In addi ion, hei chemical p ope ies,
such as salini y, migh no be he mos app op ia e o hei de elopmen [
24
]. Howe e ,
his nega i e conside a ion ega ding he un a o able p ope ies o such subs a es migh
ha e a posi i e coun e pa : he endophy ic ungi ha a e capable o g owing unde hese
di icul condi ions may p oduce a wide a ie y and g ea e numbe o hese enzymes, as
he link be ween enzyme p oduc ion and un a o able g ow h condi ions has been al eady
demons a ed [
25
]. Se e al ag o-was es and by-p oduc s—wi h limi ing cha ac e is ics o
ungal g ow h, such as salini y—ha e al eady been es ed o use as subs a es o ungal
inocula ion, as well as in he p oduc ion o bioac i e compounds, as is he case o oli e-mill
was ewa e s [26]. Howe e , he scien i ic li e a u e in his ma e is s ill qui e limi ed.
Due o he ole o such subs ances in he heal h s a us, pe o mance, and g ow h o
he plan hos , he ungal endophy ic species ha a e isola ed om plan s g owing unde
ha sh condi ions, such as d ough and salini y, may be mo e sui able o p oduce o a g ea e
ex en hese kinds o compounds [
27
]. Spanish dehesas a e g asslands wi h sca e ed ees
and a well-de eloped he baceous unde s o y, which a e used mainly o he ex ensi e
ea ing o li es ock. Fu he mo e, hey a e cha ac e ized by low and i egula ain all
as well as by soil wi h low e ili y and, e en ually, salini y [
28
,
29
]; hese condi ions a e
pa icula ly un a o able o he plan ’s g ow h. Fo his eason, ungal endophy es which
a e isola ed om he pas u es g owing in hese ecosys ems could be especially sui able
candida es in e ms o p oducing such subs ances. In o de o iden i y he endophy ic
s ains wi h he po en ial o p oduce enzymes, especially when g own in saline media, he
use o quali a i e es s could be conside ed one o he bes op ions, as hey migh allow one
o examine a wide ange o isola es o se e al ypes o enzymes [30].
Thus, he aim o his s udy was o e alua e he po en ial o se e al ungal endophy es
ha we e isola ed om he pas u es o dehesas in o de o p oduce enzymes wi h appli-
ca ions in he ields o ag i ood and o he indus ies. A second goal was o e alua e hei
capaci y o colonize and p oduce enzymes in a saline en i onmen , such as hose ha a e
p o ided by many o he indus ial by-p oduc s being conside ed. This would be a i s
s ep o assess i hey could be u he used as solid-s a e e men a ion subs a es ha would
be u ilized o massi ely p oduce hese enzymes.
Mic oo ganisms 2023,11, 908 3 o 12
2. Ma e ials and Me hods
2.1. Fungal Ma e ial
Ele en ungal s ains, p e iously isola ed om di e en heal hy plan s collec ed om
he dehesas o Ex emadu a (in he sou hwes o Spain), we e selec ed o he s udy (Table 1).
The iden i ica ion o hese ele en ungi was i s a emp ed mo phologically by means o
hei ep oduc i e s uc u es, and hen a a molecula le el h ough he compa ison o
hei ITS egion sequence wi h hose included in wo da abases, GenBank (www.NCBI.
nlm.nih.go , accessed on 13 Decembe 2022) and UNITE (h ps://uni e.u .ee, accessed
on 13 Decembe 2022), while using a BLAST sea ch [
31
]. A mo e exhaus i e explana ion
ega ding he iden i ica ion and he species assigna ion p ocess can be consul ed in he
wo ks o Lledóe al. [
32
] and San ama ía e al. [
33
]. The endophy ic s ains we e selec ed
acco ding o hei equency o isola ion om he o iginal plan hos s o h ough he
obse a ion o bioac i i y in p elimina y assays [34,35].
Table 1.
Fungal endophy es used in he expe imen s. Fo e e y s ain, he in e nal code used in he
labo a o y, he plan hos whe e i was isola ed om, he en a i e iden i ica ion, and he Genbank
accession numbe a e shown.
Code Plan Hos Iden i ica ion 1Genbank
Accession #
E025 Bise ula pelecinus Sa ocladium e icola (J.H. Mill., Giddens &
A.A. Fos e ) OK161081
E064 Poa annua D echsle a bisep a a (Sacc. & Roum.) M.J.
Richa dson & E.M. F ase KP698352
E138 O ni hopus comp essus Embellisia lep inellae E.G. Simmons & C.F. Hill KP698337
E168
T i olium sub e aneum
Fusa ium a enaceum (F .) Sacc. KP698339
E178 Poa annua Ac emonium implica um (J.C. Gilman & E.V.
Abbo ) W. Gams KP899410
E198 O ni hopus comp essus Mic odiplodia hawaiiensis C ous KP899392
E224 Lolium igidum Colle o ichum ce eale Manns KP698357
E528 Lolium igidum Didymella exi ialis (Mo ini) E. Müll. KP899390
E532 Medicago polymo pha Didymella phacae Co baz KP698363
E586 B omus mollis Uniden i ied sp1 KP899447
E635 Medicago polymo pha Penicillium ch ysogenum Thom KP899406
1
Based on mo phological cha ac e is ics and by compa ison wi h ITS sequences in GenBank and UNITE ( e sion
8.3), wi h a simila i y ≥99%.
2.2. E alua ion o Ex acellula Enzyma ic Ac i i y
The six mos used enzymes by indus y (i.e., amylase, cellulase, laccase, lipase, pec i-
nase, and p o ease) we e chosen in o de o quali a i ely e alua e he ex acellula en-
zyma ic ac i i y o he selec ed ungi [
36
]. Fo ha pu pose, a 5 mm diame e plug o
mycelia (ob ained om an ac i ely g owing 7-day-old colony on po a o dex ose aga
medium; PDA) was placed in he cen e o a Pe i dish con aining he speci ic cul u e
medium necessa y o assess he p oduc ion o each enzyme, as indica ed below. Aga plugs
wi hou mycelia we e placed in Pe i dishes wi h he speci ic media o be used as a nega i e
con ol. Once inocula ed, he pla es, p epa ed in iplica e, we e la e incuba ed o 7 days
a 23
◦
C, as his is conside ed he op imal g ow h empe a u e o he selec ed ungi. A e
he incuba ion pe iod, he o ma ion o a hyd olysis halo a ound he colony was conside ed
an indica o o enzyma ic ac i i y (Figu e 1). In posi i e cases, he ex ension o bo h he
colony and he clea a ea a ound i we e measu ed o calcula e he solubiliza ion index (SI)
as SI = (colony diame e + halo zone diame e )/colony diame e [
37
]. The speci ic media
o he iden i ica ion o each enzyme ac i i y is desc ibed as ollows.
Mic oo ganisms 2023,11, 908 4 o 12
Mic oo ganisms 2023, 11, x FOR PEER REVIEW 4 o 12
ungi. A e he incuba ion pe iod, he o ma ion o a hyd olysis halo a ound he colony
was conside ed an indica o o enzyma ic ac i i y (Figu e 1). In posi i e cases, he ex en-
sion o bo h he colony and he clea a ea a ound i we e measu ed o calcula e he solu-
biliza ion index (SI) as SI = (colony diame e + halo zone diame e )/colony diame e [37].
The speci ic media o he iden i ica ion o each enzyme ac i i y is desc ibed as ollows.
Figu e 1. Examples o he hyd olysis halo obse ed in he p esen s udy o posi i e esul s o am-
yloly ic ac i i y (le ) and celluloly ic ac i i y ( igh ).
Amylase ac i i y was assessed by using a yeas mal aga medium (mal ex ac 10.0
g; yeas ex ac 6.0 g; D-glucose 4.0 g; aga 20 g; in 1 L o dis illed wa e ; pH 6.3), which
was amended wi h a 1% soluble s a ch (Pan eac Química SLU, Cas ella del Vallès, Ba -
celona, Spain). A e he incuba ion ime, pla es we e looded wi h a 1% iodine solu ion,
which allowed us o iden i y he clea halo su ounding he colony in he case o posi i e
ac i i y [38].
In he case o cellulase ac i i y, Pe i dishes wi h a yeas mal aga medium we e
supplemen ed wi h 0.5% Na-ca boxy-me hylcellulose (Sigma-Ald ich, San Luis, MO,
USA). A e he g owing pe iod, pla es we e i s looded wi h 0.2% Congo Red (Me ck
KGaA, Da ms ad , Ge many) and hen wi h a 1 M NaCl solu ion, which allowed us o
iden i y posi i e cellulase ac i i y h ough he hyd olysis halo [35,38,39].
Laccase ac i i y was de ec ed by using glucose yeas ex ac pep one aga medium
(glucose 5.0 g; pep one 5 g; yeas ex ac 3.0 g; aga 20.0 g; in 1 L o dis illed wa e ; pH 6.8)
wi h 0.05g 1-nap hol L-1 (pH 6.0) (Sigma-Ald ich, San Luis, MO, USA). As he ungus
p oduced he enzyme, he colo less medium u ned blue due o oxida ion o he subs a e
[20].
Fo lipase ac i i y, he endophy es we e g own in a pep one aga medium (pep one
10.0 g; NaCl 5.0 g; CaCl2·2H2O 0.1 g; aga 16.0 g; in 1 L o dis illed wa e ; pH 6.0) sup-
plemen ed wi h 1% Tween 20 (Me ck KGaA, Da ms ad , Ge many) which was s e ilized
sepa a ely and added be o e pou ing on o he pla es. The hyd olysis halo was di ec ly
isible as he ungi g ew i hey exhibi ed lipase ac i i y [36].
Pec inoly ic ac i i y was de e mined by g owing he ungi in a pec in aga medium
(pec in 5 g; yeas ex ac 1 g; aga 20 g; in 1 L o dis illed wa e ; pH 5.0). A e he incu-
ba ion pe iod, he pla es we e looded wi h a 1% aqueous solu ion o hexadecyl ime-
hylammonium b omide (Pan eac Química SLU, Cas ella del Vallès, Ba celona, Spain) in
o de o de ec he clea zone ha o med a ound he ungal colony in he case o posi i e
ac i i y [20].
Finally, p o ease ac i i y was e alua ed by using a casein hyd olysis medium
(skimmed milk powde 28.0 g; pep one 5.0 g; yeas ex ac 2.5 g; glucose 1.0 g; aga 20 g;
Figu e 1.
Examples o he hyd olysis halo obse ed in he p esen s udy o posi i e esul s o
amyloly ic ac i i y (le ) and celluloly ic ac i i y ( igh ).
Amylase ac i i y was assessed by using a yeas mal aga medium (mal ex ac
10.0 g; yeas ex ac 6.0 g; D-glucose 4.0 g; aga 20 g; in 1 L o dis illed wa e ; pH 6.3),
which was amended wi h a 1% soluble s a ch (Pan eac Química SLU, Cas ella del Vallès,
Ba celona, Spain). A e he incuba ion ime, pla es we e looded wi h a 1% iodine solu ion,
which allowed us o iden i y he clea halo su ounding he colony in he case o posi i e
ac i i y [38].
In he case o cellulase ac i i y, Pe i dishes wi h a yeas mal aga medium we e
supplemen ed wi h 0.5% Na-ca boxy-me hylcellulose (Sigma-Ald ich, San Luis, MO, USA).
A e he g owing pe iod, pla es we e i s looded wi h 0.2% Congo Red (Me ck KGaA,
Da ms ad , Ge many) and hen wi h a 1 M NaCl solu ion, which allowed us o iden i y
posi i e cellulase ac i i y h ough he hyd olysis halo [35,38,39].
Laccase ac i i y was de ec ed by using glucose yeas ex ac pep one aga medium
(glucose 5.0 g; pep one 5 g; yeas ex ac 3.0 g; aga 20.0 g; in 1 L o dis illed wa e ;
pH 6.8) wi h 0.05g 1-nap hol L-1 (pH 6.0) (Sigma-Ald ich, San Luis, MO, USA). As he
ungus p oduced he enzyme, he colo less medium u ned blue due o oxida ion o he
subs a e [20].
Fo lipase ac i i y, he endophy es we e g own in a pep one aga medium (pep one
10.0 g; NaCl 5.0 g; CaCl
2·
2H
2
O 0.1 g; aga 16.0 g; in 1 L o dis illed wa e ; pH 6.0) sup-
plemen ed wi h 1% Tween 20 (Me ck KGaA, Da ms ad , Ge many) which was s e ilized
sepa a ely and added be o e pou ing on o he pla es. The hyd olysis halo was di ec ly
isible as he ungi g ew i hey exhibi ed lipase ac i i y [36].
Pec inoly ic ac i i y was de e mined by g owing he ungi in a pec in aga medium
(pec in 5 g; yeas ex ac 1 g; aga 20 g; in 1 L o dis illed wa e ; pH 5.0). A e he
incuba ion pe iod, he pla es we e looded wi h a 1% aqueous solu ion o hexadecyl
ime hylammonium b omide (Pan eac Química SLU, Cas ella del Vallès, Ba celona, Spain)
in o de o de ec he clea zone ha o med a ound he ungal colony in he case o posi i e
ac i i y [20].
Finally, p o ease ac i i y was e alua ed by using a casein hyd olysis medium (skimmed
milk powde 28.0 g; pep one 5.0 g; yeas ex ac 2.5 g; glucose 1.0 g; aga 20 g; in 1 L o
dis illed wa e ; pH 7.0). A clea zone, di ec ly isible a ound he colony, con i med posi i e
ac i i y [39].
Mic oo ganisms 2023,11, 908 5 o 12
2.3. Halo ole ance Tes
To assess he capaci y o he ungi o g ow unde sal s ess condi ions, Pe i dishes
we e p epa ed wi h a PDA medium adjus ed o di e en concen a ions o NaCl (100,
200, and 500 mM) [
40
]. Likewise, pla es con aining he same medium bu wi hou NaCl
(0 mM) we e in oduced as he con ol. A e placing an ac i ely g owing plug (ø = 5 mm)
o mycelia om each o he endophy es on he cen e o he pla e, i s adial g ow h was
measu ed 9 days la e in o de o assess he maximum saline concen a ion ha hey we e
able o ole a e. All samples we e analyzed in iplica e, and he esul s we e exp essed as
he cm o adial g ow h.
2.4. E alua ion o Enzyma ic Ac i i y unde Sal S ess Condi ions
Once bo h he quali a i e enzyma ic ac i i y and he halo ole ance o ungal s ains
we e assessed, ano he es was conduc ed in o de o e alua e hei po en ial o p oduce
he di e en enzymes unde sal s ess. The selec ed endophy es we e placed again in
he basic aga media wi h he speci ic subs a e o he co esponding enzyme, bu in his
case, hey we e adjus ed o a sal concen a ion o 500 mM. A e he incuba ion pe iod, he
ex ension o bo h he colony and he clea a ea a ound i we e measu ed o calcula e he
solubiliza ion index, as indica ed abo e. The es was conduc ed in iplica e and pla es
wi h he speci ic subs a e bu wi hou NaCl we e used as he nega i e con ol.
2.5. S a is ical Analysis
The esul s o all he expe imen s we e analyzed wi h he S a is ix . 8.10 package
(Analy ical So wa e, USA). The e ec o he endophy ic s ain on he enzyma ic p oduc ion
was e alua ed h ough a one-way ANOVA. The e ec o he sal concen a ion on mycelial
g ow h and he e alua ion o he enzyme p oduc ion unde sal condi ions we e analyzed
h ough a mixed-design analysis o a iance (spli plo ANOVA). Fu he mo e, his was
achie ed by conside ing he NaCl con en and he ungal s ain as he main and subplo
ac o s, espec i ely. A Fishe ’s p o ec ed leas signi ican di e ence (LSD) es o mul iple
compa ison was used when signi ican di e ences (p< 0.05) we e ound in he es s.
3. Resul s
3.1. E alua ion o Ex acellula Enzyma ic Ac i i y
The selec ion o endophy es included s ains o some o he mos ep esen a i e gene a
o ungi, such as Ac emonium, Didymella, D echsle a, Fusa ium, Penicillium o Podospo a,
among o he s. The sc eening o hei ex acellula enzyma ic ac i i y showed ha all he
isola es p oduced a leas wo o he enzymes (Table 2). The endophy es ha p oduced a
wide ange o enzymes we e E064 (D echsle a bisep a a), E198 (Mic odiplodia hawaiiensis),
and E635 (Penicillium ch ysogenum), which p oduced ou ypes o enzymes, h ee o hem
being in common: amylases, cellulases, and lipases.
Rega ding he obse ed equency o each enzyme, lipase ac i i y was he mos e-
quen , being ound in nine o he ele en s ains (81.82%); only in E168 (Fusa ium a enaceum)
and E224 (Colle o ichum ce eale) we e hese no de ec ed. The endophy e E586 showed he
highes solubiliza ion index (SI = 2.07), ollowed by E178 (Ac emonium implica um) wi h
SI = 1.90. As he second mos common enzyma ic ac i i y, cellulase ac i i y was de ec ed
in 8 ou o he 11 isola es. In his case, he endophy es E586 (Uniden i ied sp1) and E198
(Mic odiplodia hawaiiensis) p esen ed he highes alues (Table 2). P o ease and amylase
ac i i y was ound in 4 ou o he 11 isola es in bo h cases. The endophy es E178 (Ac emo-
nium implica um) and E635 (Penicillium ch ysogenum) p oduced he highes p o ease and
amylase ac i i y, espec i ely. Finally, only h ee endophy es o he selec ed ungi p oduced
ei he laccase (E138, Embellisia lep inellae; E528, Didymella exi ialis; and E635, Penicillium
ch ysogenum) o pec inase (E138, Embellisia lep inellae; E198, Mic odiplodia hawaiiensis; and
E532, Didymella phacae) ac i i y.

Mic oo ganisms 2023,11, 908 6 o 12
Table 2.
Solubiliza ion index (mean
±
s anda d e o ) p oduced by each endophy e o each enzyme.
A summa y o he ANOVA, showing he e ec o he endophy e, wi h he DF (deg ee o eedom)
and F alues, including he le el o signi icance (*** p
≤
0.001), indica ed in he second ow o each
enzyme ac i i y.
Endophy e DF Amylase Cellulase Laccase Lipase Pec inase P o ease
10 51.64 *** 52.84 *** 781.87 *** 37.69 *** 7362.28 *** 541.19 ***
E025 WA WA WA 1.55 ±0.13 c WA 1.12 ±0.02
E064 1.27 ±0.04 b 1.38 ±0.14 bc WA 1.56 ±0.25 c WA 1.28 ±0.08
E138 WA WA 1.16 ±0.02 b 1.10 ±0.01 d 1.17 ±0.00 a WA
E168 WA 1.39 ±0.05 bc WA WA WA 1.05 ±0.00 c
E178 WA WA WA 1.90 ±0.09 ab WA 1.49 ±0.03 a
E198 1.34 ±0.06 b 2.27 ±0.20 a WA 1.23 ±0.03 d 1.17 ±0.02 a WA
E224 1.30 ±0.05 b 2.05 ±0.09 a WA WA WA WA
E528 WA 1.63 ±0.09 b 1.36 ±0.06 a 1.67 ±0.19 bc WA WA
E532 WA 1.22 ±0.06 c WA 1.19 ±0.05 d 1.10 ±0.01 b WA
E586 WA 2.31 ±0.28 a WA 2.07 ±0.07 a WA WA
E635 1.78 ±0.33 a 1.33 ±0.01 bc 1.09 ±0.02 c 1.19 ±0.02 d WA WA
WA: wi hou ac i i y. In he cases wi h posi i e ac i i y, means in he same column wi h di e en le e s a e
signi ican ly di e en acco ding o he LSD es . Th ee epe i ions we e pe o med o each ea men (n = 3).
3.2. Halo ole ance Tes
The g ow h esponse o saline ea men s a ied widely depending on he ungal
species, as can be obse ed in Figu e 2. Thus, a e nine days, wo o he ele en s ains,
E178 (Ac emonium implica um) and E198 (Mic odiplodia hawaiiensis), showed a signi ican ly
highe adial g ow h in 500 mM o NaCl han hose which we e ound when g own
wi hou sal s ess. A he same ime, wo o he ungi, E168 (Fusa ium a enaceum) and
E224 (Colle o ichum ce eale), showed highe g ow h unde saline condi ions, bu only a
he lowe alues o NaCl concen a ions. On he o he hand, six o he isola es (E064, E138,
E528, E532, E586, and E635) showed educed g ow h when hey we e incuba ed in he
medium wi h he highes concen a ions o NaCl. In any case, he ele en s ains we e able
o g ow, e en unde ha sh saline condi ions.
Mic oo ganisms 2023, 11, x FOR PEER REVIEW 7 o 12
Figu e 2. In luence o he NaCl concen a ion on he adial g ow h o he s udied endophy es a e
9 days o incuba ion. The esul s a e gi en as mean ± s anda d e o (n = 3). The leas signi ican
di e ence (LSD) o he sal concen a ion*isola e in e ac ion is shown in he uppe le co ne (c i -
ical alue o compa ison o he same and di e en le els o sal a e shown as le and igh ba s,
espec i ely).
3.3. E alua ion o Enzyma ic Ac i i y unde Sal S ess Condi ions
This expe imen showed ha he solubiliza ion index signi ican ly changed wi h he
salini y o he cul u e medium o all he pa ame e s, excep wi h espec o he amyloly-
ic ac i i y (Table 3). The p oduc ion o amylase was no signi ican ly a ec ed by he sal
concen a ion in any o he isola es (as he sal concen a ion*endophy e in e ac ion did
no signi ican ly a ec he p oduc ion ei he ), wi h alues ha anged be ween 1.65, o
he s ain E635 (Penicillium ch ysogenum) g owing in he non-saline medium, and 2.72, o
he s ain E064 (D echsle a bisep a a).
Rega ding he o he enzymes, he e ec o sal concen a ion on he solubiliza ion
index changed signi ican ly depending on he ungal s ain. In he case o cellulase, ou
o he eigh bioac i e s ains ha p oduced i (E198, E224, E586 and E635) did no educe
such ac i i y when g owing in a saline medium. A be e end was ound o he lipo-
ly ic ac i i y, whe e 89% o he s ains, i.e., all o hem o he han Didymella phacae (E532),
main ained he p oduc ion o his enzyme unde sal condi ions. In he case o pec inase
p oduc ion, he esul o he ungus E198 (Mic odiplodia hawaiiensis) s ood ou , since i
was able o signi ican ly inc ease i s ac i i y unde saline g ow h condi ions (wi h solu-
biliza ion indexes o 1.33 and 1.83 o he con ol and sal -amended media, espec i ely).
The same esul was ound in he case o p o eoly ic ac i i y o he endophy es E025
(Sa ocladium e icola) and E064 (D echsle a bisep a a), wi h signi ican inc eases o 23% and
15%, espec i ely, when compa ed wi h he co esponding con ols wi hou sal added.
Only in ega d o laccases did he sal condi ions signi ican ly educe he enzyma ic ac-
i i y o he h ee s ains. Howe e , e en in his case, he h ee endophy es main ained
hei capaci y o p oduce such compounds.
E025
E064
E138
E168
E178
E198
E224
E528
E532
E586
E635
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 mM 100 mM 200 mM 500 mM
Radial g ow h (cm)
NaCl concen a ion
Figu e 2.
In luence o he NaCl concen a ion on he adial g ow h o he s udied endophy es
a e 9 days o incuba ion. The esul s a e gi en as mean
±
s anda d e o (n = 3). The leas
signi ican di e ence (LSD) o he sal concen a ion*isola e in e ac ion is shown in he uppe le
co ne (c i ical alue o compa ison o he same and di e en le els o sal a e shown as le and
igh ba s, espec i ely).
Mic oo ganisms 2023,11, 908 7 o 12
3.3. E alua ion o Enzyma ic Ac i i y unde Sal S ess Condi ions
This expe imen showed ha he solubiliza ion index signi ican ly changed wi h he
salini y o he cul u e medium o all he pa ame e s, excep wi h espec o he amyloly ic
ac i i y (Table 3). The p oduc ion o amylase was no signi ican ly a ec ed by he sal
concen a ion in any o he isola es (as he sal concen a ion*endophy e in e ac ion did no
signi ican ly a ec he p oduc ion ei he ), wi h alues ha anged be ween 1.65, o he
s ain E635 (Penicillium ch ysogenum) g owing in he non-saline medium, and 2.72, o he
s ain E064 (D echsle a bisep a a).
Table 3.
Enzyma ic p oduc ion unde di e en sal condi ions by he selec ed s ains. The posi i e
esul s (mean
±
se) a e shown as he solubiliza ion index ((colony + halo zone diame e s)/colony
diame e ). A summa y o he ANOVA (DF: deg ee o eedom; F alues; and le el o signi icance
(** p
≤
0.01, *** p
≤
0.001)), showing he e ec o he endophy e, he sal concen a ion, and hei
in e ac ion, is shown o each pa ame e .
Sou ce Amylase Cellulase Laccase Lipase Pec inase P o ease
Sal (S) DF 1 1 1 1 1 1
F 0.02 231.60 *** 296.22 *** 13.90 ** 24.75 *** 62.71 ***
Endophy e (E) DF 3 7 2 8 2 3
F 33.52 *** 435.09 *** 108.34 *** 195.6 *** 56.85 *** 151.67 ***
S*E DF 3 7 2 8 2 3
F 2.23 43.57 *** 27.90 *** 7.37 *** 111.59 *** 198.63 ***
E S Amylase Cellulase Laccase Lipase Pec inase P o ease
E025 0 mM WA WA WA 2.58 ±0.14 b WA 1.78 ±0.03 d
500 mM
WA WA WA 2.53 ±0.15 b WA
2.19
±
0.06 bc
E064 0 mM 2.72 ±0.20 1.33 ±0.04 e WA 3.29 ±0.18 a WA 2.22 ±0.05 b
500 mM
2.46 ±0.03 1.09 ±0.02 h WA 3.08 ±0.25 a WA 2.56 ±0.10 a
E138 0 mM WA WA 2.09 ±0.01 a 1.12 ±0.02 g 2.08 ±0.02 b WA
500 mM
WA WA 1.52 ±0.03 b 1.07 ±0.01 g 2.04 ±0.02 b WA
E168 0 mM WA 1.41 ±0.03 e WA WA WA 2.22 ±0.09 b
500 mM
WA 0.00 ±0.00 j WA WA WA 0.00 ±0.00 e
E178 0 mM WA WA WA 2.25 ±0.05 c WA 2.01 ±0.09 c
500 mM
WA WA WA 2.03 ±0.03 c WA 1.62 ±0.02 d
E198 0 mM 2.71 ±0.10 2.61 ±0.03 b WA 3.27 ±0.10 a 1.33 ±0.05 e WA
500 mM
2.65 ±0.10 2.58 ±0.08 b WA 3.23 ±0.08 a 1.83 ±0.06 c WA
E224 0 mM 2.44 ±0.06 2.59 ±0.07 b WA WA WA WA
500 mM
2.48 ±0.09 2.52 ±0.06 b WA WA WA WA
E528 0 mM WA 2.86 ±0.12 a 1.48 ±0.03 b 1.48 ±0.03 de WA WA
500 mM
WA 2.28 ±0.09 c 1.30 ±0.02 c 1.30 ±0.02 e g WA WA
E532 0 mM WA 1.25 ±0.03 g WA 1.23 ±0.03 g 2.35 ±0.07 a WA
500 mM
WA 0.46 ±0.04 i WA 0.17 ±0.01 h 1.48 ±0.04 d WA
E586 0 mM WA 1.88 ±0.06 d WA 1.72 ±0.03 d WA WA
500 mM
WA 2.00 ±0.02 d WA 1.48 ±0.03 de WA WA
E635 0 mM 1.65 ±0.04 1.2 ±0.03 gh 2.13 ±0.06 a 1.33 ±0.03 e g WA WA
500 mM
1.90 ±0.02 1.12 ±0.03 gh 1.50 ±0.02 b 1.55 ±0.02 de WA WA
WA: wi hou ac i i y. In he cases wi h posi i e ac i i y, means in he same column wi h di e en le e s a e
signi ican ly di e en acco ding o he LSD es . Th ee eplica es we e pe o med o each ea men (n = 3).
Rega ding he o he enzymes, he e ec o sal concen a ion on he solubiliza ion
index changed signi ican ly depending on he ungal s ain. In he case o cellulase, ou o
he eigh bioac i e s ains ha p oduced i (E198, E224, E586 and E635) did no educe such
ac i i y when g owing in a saline medium. A be e end was ound o he lipoly ic ac i i y,
whe e 89% o he s ains, i.e., all o hem o he han Didymella phacae (E532), main ained
he p oduc ion o his enzyme unde sal condi ions. In he case o pec inase p oduc ion,
he esul o he ungus E198 (Mic odiplodia hawaiiensis) s ood ou , since i was able o
signi ican ly inc ease i s ac i i y unde saline g ow h condi ions (wi h solubiliza ion indexes
o 1.33 and 1.83 o he con ol and sal -amended media, espec i ely). The same esul was
Mic oo ganisms 2023,11, 908 8 o 12
ound in he case o p o eoly ic ac i i y o he endophy es E025 (Sa ocladium e icola) and
E064 (D echsle a bisep a a), wi h signi ican inc eases o 23% and 15%, espec i ely, when
compa ed wi h he co esponding con ols wi hou sal added. Only in ega d o laccases
did he sal condi ions signi ican ly educe he enzyma ic ac i i y o he h ee s ains.
Howe e , e en in his case, he h ee endophy es main ained hei capaci y o p oduce
such compounds.
4. Discussion
All he s udied ungi p oduced a leas wo o he six di e en enzymes ha we e
analyzed. F om hem,
≈
27% o he s ains (3 ou o 11) p oduced h ee o hem, and he
same pe cen age p oduced ou o hese compounds. The ac ha none o he isola es
showed he po en ial o p oduce all he es ed enzymes is suppo ed by he li e a u e, whe e
i is epo ed ha his ou come is e y a e [
7
,
26
]. Rega ding he equency o enzyme
occu ence, lipases and cellulases we e he mos equen enzymes since hey we e ound
in ≈82% and ≈72%, espec i ely, o he selec ed s ains.
The high equency o lipoly ic ac i i y may be conside ed an expec ed ou come, as
endophy ic ungi usually p oduce hese compounds in o de o o e come he de ense
mechanisms o hei hos s [
41
]. This g oup o enzymes, oge he wi h p o eases, acili a es
he hyphal pene a ion o endophy es h ough he plan cell wall [
42
]. In his sense, h ee
s ains could p oduce bo h lipases and p o eases, i.e., Sa ocladium e icola (E025), D echsle a
bisep a a (E064), and Ac emonium implica um (E178). In addi ion, en ou o ele en could
p oduce a leas one o bo h g oups o compounds. The solubiliza ion index o lipases
anged om 1.10 o E138 (Embellisia lep inellae) o 2.07 o he uniden i ied s ain E586,
which a e alues ha a e qui e simila o hose ound by o he au ho s who wo ked wi h
o he ungal species [
39
] and who p oduced hese kinds o enzymes. The e o e, he esul s
ob ained in he p esen s udy a e p omising enough o jus i y being u he es ed in o he
di e en condi ions in o de o maximize such a ype o enzyme p oduc ion.
Cellulases we e p oduced by 80% o he isola es s udied. I is known ha celluloly ic
ac i i y is widesp ead among pa hogenic and sap ophy ic mic oo ganisms [
43
]. Endo-
phy es can beha e as bo h pa hogens o as sap ophy es a imes du ing hei biological
cycle. This ac could explain he high p opo ion o endophy es which p oduced cellulases
in he p esen s udy, al hough he ype o ela ionship es ablished be ween ou ungi and
plan hos should be u he in es iga ed in o de o con i m his. This is suppo ed by
he esul s o o he s udies, whe e a simila p opo ion o ungal endophy es p oducing
cellulases was ound [
27
]. Ne e heless, u he s udies should be pe o med o con i m
his ac as o he qui e di e en esul s ha e also been ound when analyzing o he hos s,
such as hose eco ded by Uzma e al. [
44
], whe e only
≈
28% o he endophy es ha we e
s udied we e capable o p oducing cellulase.
Amyloly ic ac i i y was ound in
≈
36% o he isola es, which is a lowe equency
wi h espec o o he a icles in which he pe cen age o occu ence o his enzyme anged
om 78% o 100% [
8
,
37
,
38
,
45
]. This ou come may be ela ed o he ac ha ungal amylases
occu mo e commonly in sap ophy ic gene a, such as hose ound in Aspe gillus and
Rhizopus [
46
], which we e no p esen in ou s udy. Among ou selec ion, Penicillium
ch ysogenum (E635), belonging o a genus o known sap o ophs, was he endophy e
which p esen ed a signi ican ly highe amylase concen a ion, as was also obse ed by
Fouda e al. [
38
]. This isola e (E635) was one o he mos p oli ic enzyme p oduce s in he
p esen pape , p oducing ou di e en enzymes. Howe e , no pec inoly ic abili y was
obse ed, which is in con as o ha which was obse ed in he a o emen ioned a icle.
The p opo ion o isola es wi h pec inoly ic ac i i y in ou s udy (
≈
27%) was in ag eemen
wi h he esul s ob ained by Shubba and S ini as [
27
], albei a li le bi highe han he 19%
ha was obse ed in he endophy es isola ed om di e en medicinal plan s o India [
44
];
mo eo e , i was also lowe han he 49% de ec ed in he ungi ha we e isola ed om Thai
o chids [7].
Mic oo ganisms 2023,11, 908 9 o 12
Rega ding laccases, only h ee o ou isola es (
≈
27% o he o al) p oduced hem. This
is in ag eemen wi h he common end ound in o he simila s udies, whe e he equency
o ungal endophy es p oducing his enzyme was also e y low [
27
]. As poin ed ou by
Uzma e al. [
44
], laccases a e able o deg ade lignin, which migh ha e a de imen al e ec
on he plan hos in e ms o limi ing hei in e - ela ionships. Such a ea u e migh be mo e
common in sap ophy ic species. The e o e, he occu ence equency o he p oduc ion o
laccases in endophy ic species may be e y low, appea ing only in species ha can also ac
as sap ophy es a a speci ic momen o hei li e cycle. The e o e, he iden i ica ion o h ee
di e en isola es, E138 (Embellisia lep inellae), E528 (Dydimella exi ialis), and E635 (Penicillium
ch ysogenum) wi h he po en ial o p oduce his sca ce enzyme may also allow u he
de elopmen o i s p oduc ion o indus ial applica ion. In he same way, he esul s
ob ained by ungi E064 (D echsle a bisep a a) and E198 (Mic odiplodia hawaiiensis) should be
highligh ed, along wi h E635, due o hei g ea e sa ili y, as hese h ee endophy es we e
able o p oduce ou o he enzyma ic g oups ha ha e been men ioned.
Rega ding he halo ole ance es s, he p elimina y hypo hesis ha plan s om he
dehesa would be sui able o he iden i ica ion o endophy es ha a e capable o g owing
in ela i ely ha sh en i onmen s has been suppo ed by he e idence. In his ega d,
≈
45%
( i e ou o ele en) o he isola es showed g ea e mycelial g ow h in pla es ha we e
supplemen ed wi h 500 mM NaCl han in hose which we e in a non-saline PDA medium
a e nine days. In addi ion, and mo e impo an ly, none o he isola es ceased hei g ow h
unde saline condi ions. These da a may also explain he highe enzyma ic ac i i y o
se e al endophy es unde high salini y condi ions. This was he case o E198 (Mic odiplodia
hawaiiensis) wi h espec o pec inoly ic ac i i y, as well as in E025 (Sa ocladium e icola)
and E064 (D echsle a bisep a a) o p o ease p oduc ion. These endophy es may be he mos
sui able candida es in e ms o p oducing he in ol ed enzymes ia by-p oduc s gene a ed
by o he indus ies wi h saline p ope ies, such as g ape by-p oduc s, which a e well-known
o hei salini y and sodici y p oblems [
47
], o oli e oil mill was e, which may con ain up
o 2% o sal [
48
]. This enzyma ic ac i i y unde saline condi ions has also been obse ed
in o he endophy es, such as Mic osphae opsis a undinis ha a e isola ed om mang o e
ees and which showed a highe ligninoly ic ac i i y unde saline condi ions [
49
]. Fo he
massi e p oduc ion o such enzymes wi h hese endophy es, he ollowing s ep may be
he e alua ion o hei p oduc ion by di ec ly using di e en by-p oduc s as he g owing
subs a e medium in o de o con i m hei sui abili y and hei op imiza ion o he g ow h
condi ions. The speci ic hyd oly ic compounds p oduced and hei quan i ica ion may also
equi e u he in es iga ion. O he enzyma ic ac i i ies, such as he p oduc ion o amylases,
al hough no inc eased, we e no a ec ed by he salini y o he media. Addi ionally, e en
in he cases whe e he p oduc ion o enzymes was signi ican ly educed, such as he
p oduc ion o laccases in he h ee es ed isola es, only one endophy e, Fusa ium a enaceum
(E168), los i s enzyma ic ac i i y unde saline condi ions. The e o e, hese endophy es,
al hough no so p omising, could also be conside ed in u he s eps.
5. Conclusions
The expe imen al esul s e idenced ha he ungal endophy es ha we e isola ed om
plan s na u ally p esen in Spanish dehesas can p oduce a wide ange o enzymes ha a e
g ea ly alued o nume ous indus ies. Among hem, some o he s ains—especially Sa o-
cladium e icola (E025), Ac emonium implica um (E178), Mic odiplodia hawaiiensis (E198), and
an uniden i ied species (E586)—could be sui able o he p oduc ion o such enzymes unde
saline condi ions, which may allow he u iliza ion o by-p oduc s as g ow h subs a es
o la ge-scale p oduc ion. Howe e , since he enzyma ic ac i i y is somehow modula ed
by he subs a e, his s udy should only be conside ed an ini ial app oach, which was
conduc ed in o de o con inue wo king on he iden i ica ion o he compounds and o
de elop he op imiza ion o hei p oduc ion by di ec ly using hose esidues.