Potential of fungal endophytes isolated from pasture species in Spanish dehesas to produce enzymes under salt conditions

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.