Genetic characterization, evaluation of growth and production of biomass of strains from wild edible mushrooms of Lyophyllum of Central Mexico

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Food

Microbiology

Genetic

characterization,

evaluation

of

growth

and

production

of

biomass

of

strains

from

wild

edible

mushrooms

of

Lyophyllum

of

Central

Mexico

Yolanda

Arana-Gabriel

_,

_Cristina

_{Burrola-Aguilar}

_,

_Roberto

_{Garibay-Orijel}

_,

Noemí

Matías-Ferrer

_,

_Sergio

_Franco-Maass

_,

_Gerardo

_Mata

a_{UniversidadAutónomadelEstadodeMéxico,FacultaddeCiencias,CentrodeInvestigaciónenRecursosBióticos,Toluca,Mexico} b_{UniversidadNacionalAutónomadeMéxico,InstitutodeBiología,CiudaddeMéxico,Mexico}

c_{UniversidadAutónomadelEstadodeMéxico,InstitutodeCienciasAgropecuariasyRurales,Toluca,Mexico} d_{InstitutodeEcología,Veracruz,Mexico}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1September2017 Accepted13December2017 Availableonline10February2018 Associateeditor:GiseleMonteirode Souza

Keywords:

LyophyllumsectionDifformia

Nativegermplasm Culture

Mycelium Pellets

a

b

s

t

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a

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t

Thepresentstudyconductedageneticcharacterizationanddeterminedgrowthrateand biomassproductionin solidand liquidmedia,usingstrainsobtained fromwildedible sporomesof Lyophyllumthatgrow inhighmountains.Vegetative isolationwasusedto obtainatotaloffourstrains,whichweredividedintotwocladeswithinthesection Dif-formia:Lyophyllumsp.andLyophyllumaff.shimeji.Growthrateandbiomassproductionwere influencedbyboththeculturemediaandthestrains.Inapotatodextroseagarmedium, thestrainspresentedahighergrowthrate,whileinamaltextract-peptoneandyeastagar medium,thegrowthratewaslower,butwithahigherbiomassproductionthatwasequal tothatinthemaltextract-peptoneandyeastliquidmedium.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Wildedible mushrooms playan important role in natural ecosystemsandhavegreateconomicpotential.Management oftheir natural populations should therefore be based on sustainabledevelopment,through localknowledgeandthe

∗ _{Correspondingauthor.}

E-mail:[email protected](C.Burrola-Aguilar).

studyofspeciesdomesticationtechniques,thatallows long-term conservationofthe wild germplasm.1–3 _{Technological}

developmentsurroundingstrainsofwildediblemushrooms shouldpreferentiallybefocusedonspeciesthatareadapted tolocalclimaticconditionsandtotheexistingsubstratesof eachregioninordertobesuccessfullycultivated.4

Worldwide,92speciesofwildedibleandmedicinal mush-roomshavebeenreportedthatcanbecultivated.5_Thegenus

Lyophyllumisofgreateconomic,culinaryandmedicinal impor-tance,aswellasbeingusedforauto-consumption,andsome

https://doi.org/10.1016/j.bjm.2017.12.002

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ofitsediblespeciesarecharacterizedbytheirexcellentflavor and texture.6–11 _{These species are phylogenetically}

classi-fiedwithinthe section Difformia, althoughtheir taxonomic positioncanbeconfusingasaresultoftheirmorphological plasticity.12,13_{Ingeneral,theyaresaprobicspecies;however,}

somecanbefacultativeectomycorrhizal.10

CommerciallycultivatedspeciesofLyophyllum includeL. shimeji(Kawam.)Hongo,L.descastes(Fr.)SingerandL. fumo-sum(Pers.)P.D.Orton.5,9,11,14–17_{DespitethefactthatL.shimeji}

formsectomycorrhizae,ithasbeencultivatedsuccessfullyin syntheticmediumthankstothe capacity ofthe mycelium for rapidgrowth in generalist media, such as potato dex-trose agar.13 _{There iscontroversyin thecase ofL.decastes}

and L. fumosum, since both have been reported as sapro-bic or ectomycorrhizal.10,13,14,18,19 _{This could be related to}

the geographic origin ofthe strains and to the habitat in whichtheydevelop.Thisinconsistencycouldalsobea con-sequence ofincorrect speciesidentification.10,12 _{In Mexico,}

bothspecies are considered edible and are ofculturaland dietaryimportance.Theyarecommonlyknownas“clavitos”, “enterrados”or“macoyitas”andarerareanddifficulttofind. TheygrowinforestsofPinus,Pinus-Alnus,Pinus-AbiesandAbies religiosa.6,7,20–23

Commercial cultivationofLyophyllum spp. isa long and complex process.Moststudies are focusedon the acquisi-tionofbiomassinsubmergedcultures,whichisconsidereda viablealternativeforobtaininghighqualitymushrooms,rich inimmunostimulantpolysaccharides.8Thenecessityof gen-eratingknowledgeregardingthegeneticresourcesofnative fungiofcentralMexicoisthereforeproposed.Theobjectives ofthepresentstudyweretocharacterizenativehigh moun-tainstrainsofthegenusLyophyllum,aswellastodetermine theirgrowthrateandbiomassproductioninbothsolidand liq-uidmedia.Itisintendedtousethisinformationtolayabase groundworkthathelptoknow optimumconditionsforthe growthandconservationofgermplasm(strains),forfurther workonfruitingbioassays.

Materials

and

methods

Biologicalmaterial

Strains were obtained by vegetative isolation from sporophores of the genus Lyophyllum. Mushrooms were collectedthroughdirectedsampling inforestsofPinusspp. and Abies religiosa within the Nevado de Toluca Flora and FaunaProtection Area(APFFNT,byits Spanishacronym) in StateofMexico,Mexico.Collectionswereconductedwiththe help of key informants, who provided information during the fieldwork about the use of these mushrooms as food. Thespecimenswere herborized,24 _{characterizedwithbasic}

mycologytechniques, identified11,25,26 _{anddeposited inthe}

FungiCollectionoftheNacionalHerbariumofMexicoatthe InstituteofBiology,UNAM(MEXU),withherbariumnumbers 28148,28147 and 28149.Thestrains were deposited inthe Strain Collection of the Institute of Ecology (INECOL) in Xalapa,Veracruz,Mexico,withthekeysIE981,IE982,IE983. Alongwiththestrainsobtained,analysiswasconductedon strainIE975,whichhadalreadybeendepositedinthestrain

collectionafterbeingobtainedinapreviousstudy.27_TheDNA

ofthestrainswasextractedandsequencedandtheirgrowth rate and biomass production in solid media and biomass productioninliquidmediaevaluated.

Geneticcharacterization

AnXNAPkit(Sigma–Aldrich)wasusedtoextracttheDNAfrom the strains. Adissectionneedle was usedtotake 1–2mm2

of aerial mycelium from each strain in the PDA medium. Themyceliumwasincubatedfor10minat65◦Cand10min at 95◦C in 20␮L of extraction solution in a thermocycler (Bio-Rad,modelT100).Followingincubation,20␮Lofdilution solution wasadded and the mixtureincubated atambient temperaturefor30min.28_{Theribosomalinternaltranscribed}

spacer(ITS)regionwasamplifiedwiththeprimersandITS1F and ITS4,29_{followingtheprotocolofIzzoetal.}30 _The

qual-ity ofthePCRproductswasreviewedbyelectrophoresisin 1% agarose gels. Good quality products were selected and cleanedusingamixtureof1␮Lofultrapurewaterand1␮Lof ExoSAP-IT(USB-Affimetrix)foreach3.5␮LofPCRproduct.The sequencingreactionwasconductedwithaBigDye® Termina-torv3.1CycleSequencingKit(AppliedBiosystems),following themanufacturer’sinstructions.TheDNAwassequencedin bothdirections,usingthesameprimersusedinthePCR,in a sequencer ABI 3100(Applied Biosystems) in the Labora-toryofGenomicSequencingofBiodiversityandHealthofthe InstituteofBiology,UNAM.Thesequences wereeditedand assembled inGeneious ProR7 (Biomatters). Thesequences ofeachstrainweredepositedinGenBankwiththeaccession numbersKY195930,KY195931,KY195932andKY195933.

The phylogenetic analyses used the DNA sequences of clade V (section Difformia)for phylogenetic analysis of the familyLyophyllaceae.31_{SequencesofspecimensofLyophyllum}

collectedpreviouslyinStateofMexicowerealsoused(Table1). TheoutgroupwasLyophyllumleucophaeatum(AF357032).The ITSsequenceswerealignedinMuscle32_{andanalyzedusing}

methodsofmaximumlikelihoodandbyBayesianinferencein GeneiousProR7(Bio-matters).Thefinalalignmenthad573bp andanoverallnucleotidepairwiseidentityof94%.Thebest substitutionmodelswerechosenwithMrModelTestversion 2.33 _{Theanalyses ofmaximum likelihood used the PhyML}

algorithms,34 _{searchingforthetreetopologywithBESTand}

GTRasasubstitutionmodel.Bootstrap(MLb),with1000 repli-cations,wasusedtotestthesupportofthebranches.Inthe Bayesiananalysis,thealgorithmsofMrBayeswereapplied,35

usingtheradialoftheGammavariationandtheGTRmodel ofsubstitutionwithfour MonteCarlochainsover1,000,000 generations.Each400generationswassampledwitha tem-perature value of 0.2. In order to test the support of the branches,thebranchlengthvalueswerestoredandposterior probabilities(BPp)calculated.ITSsequenceswereusedinthe Bayesianandthemaximumlikelihoodanalysis.

Culturemedia

Vegetativeisolationwasconductedinthepotatodextroseagar (PDA)medium.Forevaluationofthestrains(growthrateand biomassproduction)insolidmedia,maltextract-peptoneand

Table1–StrainsandspecimensofLyophyllumstudied.

ID Strain Species MEXU GenBank Vegetation Site

LyIE975 IE975 Lyophyllumsp. 27406 KY195930 Abiesreligiosa APFFNT

Ly3 IE982 Lyophyllumsp. 28148 KY195932 Pinussp. APFFNT

Ly1 IE981 Lyophyllumsp. 28147 KY195931 Pinussp. APFFNT

AR09641 – Lyophyllumsp. 25876 KT875066 Pinussp. Amanalco

CB08330 – Lyophyllumsp. 25761 KT875064 Abiesreligiosa Amanalco

Ly5.3 IE983 Lyophyllumaff.shimeji 28149 KY195933 Abiesreligiosa APFFNT

GO-2009-278 – Lyophyllumaff.shimeji 26748 KT875068 – Amecameca

HC-PNNT-078 – Lyophyllumaff.shimeji 26594 KT875069 Abiesreligiosa APFFNT

HC-PNNT-246 – Lyophyllumaff.shimeji 26689 KT875070 Abiesreligiosa APFFNT

yeastagar(ME-PYA)andPDAwereused.ThePDAmediumwas preparedwith39%w/vpotatodextroseagarBDBioxonMR_;the

mediumME-PYAwaspreparedwith33.6%w/vmaltextract agarBDDifcoTM_{,2%w/vyeastextractBDBioxon}MR_and1%w/v

gelatinpeptoneBDBioxonR_{.Fortheliquidmedia,maltextract}

withpeptoneandyeast(ME-PY)andpotatodextrose(PD)were used.ThemediumME-PYwaspreparedwith33.6%w/vmalt extractBDBactoTM_{,2%w/vyeastextractBDBioxon}MR_and1%

w/vgelatinpeptoneBDBioxonR_{. ThemediumPDwas}

pre-paredwith 39%w/v potatodextrose brothBDDifcoTM_{. All}

culturemediaweresterilizedinanautoclaveat121◦C,15Ibs psifor15minand0.05gofChloramphenicol(Sigma®)were added.

Evaluationofstrainsinsolidmedia

Straincharacterization

Morphologicalcharacterizationofthestrainswascarriedout accordingtoCruz-Ulloa,36 _{using thecolor key “HTMLcolor}

codes”(html-color-codes.info/).Formicroscopic characteriza-tion,asampleofmyceliumwastakenfromtheperipheryof thestrainandtemporarypreparationsweremadewithCongo reddyeat10%.Inthese,thepresenceorabsenceofclampswas determinedandthediameterofthehyphaemeasuredat100× (20hyphaepertreatment)usingaMotic® opticalmicroscope andthesoftwarefortheMoticDigitalMicroscopeDMB3-223 (MoticChinaGroupCo.,Ltd.,2001–2004).

Growthrateandbiomassproduction

A cork borer was used to take a sample of agar with mycelium5mm indiameter. Five replicates were taken of this agar sampleper culture mediumand strain and each was placed in the center of a Petri dish (9cm in diame-ter). ThePetri dishes were incubated in darkness at 18◦C taken from that reported by a previous study27 _{and every}

thirddaythecolonydiameterwasmeasuredwithavernier (5 metal vernier caliper, Pretul®, China). The growth rate (GR) was obtained from the equation GR=(Fd−Id)/(Ft−It) (where “Fd” is the final diameter of growth, “Id” the ini-tial diameter of growth and “Ft−It”=the days of mycelial growth).37_{Attheendoftheincubationperiodofthestrains,}

theagarwaseliminatedbyimmersioninboilingwaterand themyceliumwasthenrinsedwithhotwater.Oncewashed, it was oven dried at 80◦C for 24h and its dry biomass recorded.27

Evaluationofstrainsinliquidmedium Productionofbiomass

In 250mL Erlenmeyer flasks, 100mLof the ME-PY and PD mediawereplacedwithfivereplicatesperstrainandculture medium.Themediawereinoculatedwithmyceliumofeach strainof0.5cmindiameterandincubatedindarknessfor20 daysat18◦Candat120rpminaPolyScience®agitatingwater bath.Attheendoftheincubationperiod,themyceliumwas filteredandwashedwithdistilledwaterthenovendriedat 80◦Cfor24handthenweighed.ThepHofthemediumwas measuredatthebeginningandendoftheincubationperiod.

Mycelialcharacterization

Characterizationofthemorphologyofthemyceliumwas con-ductedpriortodryingusingaMotic®stereoscopicmicroscope. Thenumberofpelletsformedwasquantifiedandtheir diame-termeasuredwithavernier.Formicroscopiccharacterization, samplesofthepelletsweretakenattheendoftheperiodof agitationandtemporarypreparationsmadewithCongored dyeat10%forobservationunderaMotic®opticalmicroscope andcorroborationofthepresenceofclamps.

Statisticalanalysis

The statistical analyses were conducted with the program Statgraphics® Centurion XVI (Statpoint Technologies, Inc., 2009).Theeffectoftheinteractionbetweenthestrainsand the culturemediaonthegrowth rateandbiomass produc-tionwasevaluatedusingamultivariateanalysisofvariance (MANOVA).Significantdifferencesamongculturemediawere determinedwithaTukeymultiplerangetest(p≤0.05)and cor-relationanalysiswasperformedbetweenbiomassproduction andgrowthrateinthesolidmedium.Thedatapresenteda normaldistribution.

Results

Biologicalmaterial

Thesporophoresthatwerecollected haddifferent morpho-types.ThesporophoresofstrainIE983(MEXU28149)hadasize ofthepileusof1.9–7cm,convex,smoothsurfaceandincurved margin or plane, dark brown (#2A1B0A) a brown-greyish (#2A1B0A-#1C1C1C).Lamellaeslightlydecurrentorfree,white

(#FFFFFF) to cream (#FBF8EF). Stipite of 2.7–6×0.9–2cm, cylindrical,fibrous-cartilaginousandwhite-cream (#FAFAFA-#F8ECE0).Subglobose spores of 6.2(4.5–7)×3.8 (3.4–4.2)␮m andbasidiaof32(30.4–34.6)×6.5(5.4–7.3)␮m.

Thesporophoresmorphologyofthe strainIE975(MEXU 27406),IE981(MEXU28147)andIE982(MEXU28148)isvaried, thesizeofthepileusrangesfrom2to9cmacross,ofform flat-convex,slightlyumbonate,withsmoothandincurvededge, crema-greyish(#FBF8EF-#D8D8D8), lightbrown(#61380B) to brown(#3B240B).Lamellaefromfreeadheredtoslightly decur-rent,white(#FFFFFF),cream(#FBF8EF)andgrayish(#D8D8D8). Stipecylindricalof3–9cminlength,smoothorslightly stri-ate,cream-grayish(#FBF8EF-#D8D8D8),orbrown(#613F0B).In termsofsizeofthesporesandthebasidia,thespecimensof thestrainsIE981andIE975aredifferenttoIE982.The spec-imensofstrainsIE981andIE975haveglobosetosubglobose sporesof3.5–6.5×3.8–6␮mwithbasidiaof23–36×5–8␮m,the specimenofstrainIE982hassporesof4.9–7×5.3–7␮mand basidiaof36–42×3.4–4.5␮m.

Geneticcharacterization

Thestrainsandsporophoresweredividedintotwolineages ofLyophyllumwithincladeV(sectionDifformia).Thefirstwas formedbythe strain IE983, bysporophores bought inthe marketofAmecameca,StateofMexicoandbysporophores collected in the APFFNT (in the zone from which all the strainswereobtained).Thiscladewasrecognizedbothbythe Bayesianandmaximumlikelihoodanalyseswithahighlevel ofsupport(BPp=0.99,MLb=91.6).Inbothcases, thisisthe brothercladeofL.shimeji.Thesecondcladewasformedby thestrainsIE981,IE982andIE975,togetherwithspecimens collectedinAmanalco,StateofMexico.Thiscladehadgood supportinthe Bayesiananalysis(BPp=0.87)(Fig.1), butno supportintheanalysisofmaximumlikelihood(MLb=31.5). Inbothanalyses,thepositionofthiscladeisuncertainwithin thecladeofL.decastess.l.(L.decastes,L.fumosum,L. congloba-tum).Table1showsthedataofthestrainsgeneratedinthe presentresearch,aswellastheirIDwasusedintheBayesian inferenceanalysis(Fig.1)

Characterizationofthestrains

Four native strains ofLyophyllum were evaluated, ofwhich threewereobtainedinthepresentstudy(IE981,IE982and IE983)andonewaspreviouslydepositedinINECOL(IE975). Thesestrainspresentedmorphologicaldifferenceson cultur-ingindifferentmedia(Fig.2).

Thestrain IE 975 presented prostrate mycelium in ME-PYAandsubmerged-prostratemyceliuminPDA,whileinIE 981,themyceliumwassubmerged inPDAand submerged-prostrateinME-PYA.ThestrainIE982presenteddifferences betweenbothmedia,fromcolortosurfacetype.Thiswasalso trueofIE983,whereonlythecircularformwassimilarinboth media.Exudateswereabsentinallthecases(Table2,Fig.2).

Microscopically,thefourstrainsdevelopedbranched, sep-tate,smoothhyphaewithrotundatehyphalterminationand abundantandfrequentpresenceofclamps.Theaverage diam-eterofthehyphaeforthestrainIE975was1.9␮minPDA,and 1.6␮minME-PYA,thestrainIE983presentedanaverage

diam-eterof2.9␮minPDAand3.1␮minME-PYA,thestrainIE981 was2.4␮minPDAand2.2␮minME-PYAandthestrainIE982 was2.3␮minPDAand2.7␮minME-PYA.

Evaluationofthestrainsinsolidmedium

Themyceliumpresentedsignificantdifferencesingrowthrate (F1,39=128.23,p≤0.0001)inthetwoculturemediaused,with

thatofthePDAmediumreachingupto0.4cmdía−1;witha maximumdiameterof9cmreachedinapproximately21days, andthatoftheME-PYAmediumpresentingaminimumrate of0.31cm/day.Differencesingrowthratenotonlydepended on theculture mediumbutalso onthe strain(F3,39=59.69,

p≤0.0001).Table3showsthat,accordingtotheTukeymultiple rangetest,fourgroupswereformed;thehighergrowthinthe mediumPDAwaspresentinthree(IE975,IE982andIE983) ofthefourstrains,followedbyIE982and983inME-PYAwith 0.37and0.34cm/daylowrespectively.ThestrainIE981inboth culturemediaandIE975inME-PYApresented,onaverage, thelowestgrowthrate.Theformationofthegroupsis gener-atedfromtheinteraction(F3,39=22.00,p≤0.0001)betweenthe

factors:culturemediaandstrains,whichaffectsthegrowth rateofthemycelium.Forthisreason,eachstrainrequiresa differentmediumtofavoritsdevelopment.

Intermsofthebiomassproductioninthesolidmedium, significantdifferenceswerealsopresentedamongthestrains (F3,39=54.11, p≤0.0001) and the culturemedia(F1,39=19.73,

p≤0.0001)andtherewasaninteractionbetweenbothfactors (F3,39=7.28,p≤0.05)thatinfluencedbiomassproduction.The

highestbiomassproductionwaspresentedinthestrainIE981 inbothculturemedia(Table3),andinIE982inthemedium ME-PYA,withanaveragemaximumofupto0.383g/Petridish, followedbyIE975andIE983inthemediumME-PYA,IE982 and IE 983 in PDA and a lower production with IE 975 in PDA.Therewasanegativeandstatisticallysignificant correla-tion(p≤0.0001,r=−0.6334)betweengrowthrateandbiomass production. That is,the strains thatdeveloped thehighest biomassweregenerallythosethatpresentedthelowest aver-agegrowthrate.

Evaluationofstrainsinliquidmedium

Forbiomassproductioninliquidmedium,significant differ-enceswere presentedamongstrains(F3,39=9.55,p≤0.0001),

with IE982 the strain that generatedthe highest quantity ofbiomassinbothculturemedia,followedbyIE975,IE982 and IE983 inME-PY and theselatterstrains, withalower production, in PD. These results show that there are sig-nificantdifferencesbetweentheculturemedia(F1,39=15.61,

p≤0.05),withtheME-PYthemostefficientintermsofbiomass production(11.98g/L),whichisuptodoublethatofthePD (5.54g/L) (Table3).Incontrast tothebiomassproductionin thesolidmedium,therewasnorelationshipbetweenthe cul-turemediumandthestrainsintheliquidmedium(F3,39=1.07,

p≥0.05)thatwouldaffectbiomassproduction.

Ingeneral,intermsofbothbiomassproductionandgrowth rate,thestrainsbehaveddifferentlyamongthemselvesand amongculturemedia.InPD,thestrainshadahighergrowth rate, whilein ME-PY the rate wasslow but with agreater biomassproduction,aswasthecaseinME-PY.Intheliquid

AF357032, Lyophyllum leucophaeatum AF357059, Lyophyllum decastes HM572548, Lyophyllum decastes HM572549, Lyophyllum decastes HM572551, Lyophyllum sp. Cultivar Jpn

HM572540, Lyophyllum fumosum KP192574, Lyophyllum sp. PAM-2015a

KP192595, Lyophyllum cf. conglobatum AB08-11-422 KP192601, Lyophyllum decastes

KP192552, Lyophyllum decastes

KP192588, Lyophyllum cf. lanzonii AB05-11-440 KP192599, Lyophyllum cf. conglobatum AB09-09-181 KP192663, Lyophyllum cf. decastes LIP PAM13112212

KP192600, Lyophyllum cf. decastes AB09-09-204

KP192602, Lyophyllum conglobatum var. albidopallidum KP192546, Lyophyllum cf. lanzonii LIP PAM060928030 AF357060, Lyophyllum decastes

HM572537, Lyophyllum fumosum JX280411, Lyophyllum decastes AR09641 CB08330 Ly3 Ly1 LyIE975 GO-2009-278 Ly5.3 HC-PNNT-078 HC-PNNT-246

AB301604, Lyophyllum shimeji HM572529, Lyophyllum shimeji AF357057, Lyophyllum ambustum

KP192638, Tephrocybe cf. carbonaria GC98041104 KP192641, Tephrocybe cf. carbonaria GC08103106 KP192645, Tephrocybe anthracophila KP192636, Lyophyllum ambustum KP192643, Tephrocybe cf. carbonaria GC98102502 KP192648, Lyophyllum ambustum 0.05 28.3 97.3 27.8 93.2 79.1 96.2 6.5 13.4 36.3 19.1 38.5 87.1 13.2 12.2 4.4 12.7 62 12.1 62 6.2 22.255.2 20.4 41.8 99.5 13.5 6.3 7.3 98.5 90.9 96.2 83.5 KP192615, Lyophyllum tenebrosum

JX280407, Lyophyllum loricatum f. subxanthum

Fig.1–AnalysisofBayesianinferencebasedonITSsequenceofLyophyllumsectionDifformiathatincludesthestrainsand specimensofLyophyllumcollectedintheStateofMexico.LyIE975(IE975),Ly3(IE982),Ly1(IE981),Ly5.3(IE983).

media, the four strainsformed pellets; however, inME-PY, wheretherewas greaterbiomassproduction(Table3)after 20daysinagitation,thepelletsweresaturatedandformeda conglomerateofgelatinousconsistency.

InPD,thepelletswerespongywithshortfilamentsonthe periphery,acharacteristicknownasthe“hairylength”.38_The

numberanddiameterofpelletsdifferedineachstrain;inIE 975,aminimumof2andamaximumof9wereformed,with diametersfrom1to3.2cm;inIE982,therewerefrom6to24,

withdiametersof0.6cmto4.2cm;inIE983,from8to12with diametersof1.6to4cm,andinIE981,fromfiveto11,with diametersof0.9to4.5cm.

TheinitialpHoftheculturemediawas5forME-PYand5.7 forPD.Attheendofthe20daysofincubationandagitation, thiswasdifferentinthefourstrainsandinthetwoculture media.ThestrainIE975presentedafinalpHof4.3inME-PY and4.8inPD,thestrainIE983was4.8inbothmedia,IE981 was4.2inME-PYand5.5inPDandthestrainIE982was5.9in

Fig.2–StrainsofLyophyllumsecc.Difformiaat18◦C.(a)–(d)StrainsonPDA,(e)–(h)strainsonME-PYA;(a),(e)strainsIE975; (b),(f)strainsIE981;(c),(g)strainsIE982;(d),(h)strainsIE983.

Table2–MacromorphologicalcharacterizationoffourLyophyllumstrains.

Strain Culturemedium Color Form Edge Mycelialgrowth Texture Surface

IE 975

PDA Lightyellow(#FBFBEF) Circular Fimbriate Submerged–prostrate Velvet Concave

ME-PYA Lightyellow(#BFBBEF) Circular Fimbriate Prostrate Velvet Concave

IE 981

PDA Lightyellow(#BFBBEF) Circular Fimbriate Submerged Smooth Flat

ME-PYA Lightyellow(#BFBBEF) Circular Fimbriate Submerged–prostrate Smooth Flat

IE 982

PDA Lightyellow(#BFBBEF) Circular Smooth Submerged–prostrate Smooth Flat

ME-PYA White(#FFFFFF) Circular Fimbriate Prostrate–aerial Cotton Concave

IE 983

PDA White(#FFFFFF) Circular Smooth Submerged–prostrate Smooth Flat

ME-PYA White(#FFFFFF) Circular Fimbriate Prostrate–aerial Cotton Concave

PDA,potatodextroseagar;ME-PYA,maltextract-peptoneandyeastagar.

Table3–Growthrateandbiomassproductioninsolidandliquidmedia.

Strains Solidmedium Liquidmedium

Culturemedium Growthrate(cm/day) Biomass(g/Petridish) Culturemedium Biomass(g/l)

IE 975

PDA 0.40aa _{0.09c PD 5.79cd}

ME-PYA 0.32cd 0.22b ME-PY 8.52abcd

IE 981 PDA 0.32cd 0.38a PD 11.12ab ME-PYA 0.31d 0.36a ME-PY 11.98a IE 982 PDA 0.38ab 0.20b PD 5.54d

ME-PYA 0.37b 0.30a ME-PY 9.62abcd

IE 983

PDA 0.40a 0.16bc PD 7.48bcd

ME-PYA 0.34c 0.18b ME-PY 9.92abc

PDA,potatodextroseagar;ME-PYA,maltextract-peptoneandyeastagar;PD,potatodextrose;ME-PY,maltextractwithpeptoneandyeast.

ME-PYand6.1inPD.Aswasthecaseinthesolidmedium,the fourstrainsintheliquidmediumpresentedabundantclamps.

Discussion

Thetwo cladesinto whichthe strains and sporophores of

Lyophyllum collectedinthe StateofMexicowere differenti-ated,arefoundinthesectionDifformia,whichislocatedinthe cladeVbthatresolves5phylospecies,9morphospeciesand5 morphogeneticspecies.4_{Clade1,formedbythestrainIE983}

(Ly5.3)andspecimensobtainedinmarketsandbycollection in the APFFNT(GO-2009-278, HC-PNNT-078, HC-PNNT-246), isbrother tothe clade/Vb-6 shimeji, which comprises one strain31,39 _{and awild specimen associatedwith coniferous}

forest,12,31 _{withahighlevelofsupportbothintheBayesian}

analysisandinthemaximumlikelihood.Themorphological characteristics ofthe specimenfrom which the strainwas obtained,aswellasitssporesize,matchthecharacteristics reportedforL.shimeji.11_{Thepositionasabrothercladetothat}

ofL.shimeji(Fig.1)couldbeattributedtodifferentconditions suchasprobablerecentevolutionor tothegeographic ori-ginorhabitatinwhichtheydevelop,sinceL.shimejihasbeen reportedinassociationwithPinusinAsia,10,12_whilethe

spec-imensand strainsinthe presentstudy areassociatedwith

Abiesreligiosa. Thisagreeswiththat reported fortheclade ofL.fumosumandL.decastes,who separatedtheseinto two subcladesbecausetheydevelopindifferentvegetationtypes (deciduousandconiferousforests,respectively).12

Thepositionofclade2formedbythethreeotherstrains:IE 975,IE982andIE981andtwospecimenscollected(AR09641 andCB08330)isuncertain(MLb=31.5)withincladeVb,and themorphologicalcharacterizationofthesporophoresdoes notprovidethedatathatwouldallowamoreaccurate clas-sification. The specimens of those that were obtained are geneticallysimilar amongthemselves; however, their mor-phologyandmicroscopyisvaried,butthesizeofthespores is similar to that ofL. decastes.26 However, the basidia do notmatchthoseofanyoftheotherspecieswithinthe sec-tion Difformia. Likewise, there are differences in terms of habitat(Table1),sincetheyarefoundbothintheforestof

AbiesreligiosaandPinussp.Confusionintheidentificationof thespeciesisgeneratedbytheintraspecificplasticityofthe speciesclassifiedinthecomplexL.decasteswithinthesection

Difformia.11,12,26

Thespecificcladeswithintheclade/Vbarenotclearly dif-ferentiated,duetotheparaphyleticcharacteristicoftheclade Vb-7 supported by a cryptic species (AF357060), while the cladeVb-8 ismonophyletic andphylogeneticallymore dis-tantfromVb-7.31_{Otherstudieshavefoundthesameproblem}

indelimitation ofspecies withinthis clade.12 _The

identifi-cationmustbeaccompaniedbyaphylogeneticanalysisand examinationofecologicalaspects,aswellasa characteriza-tionatstrainlevel.10,40,41_{Forthisreason,delimitationofthese}

speciesrequiresacomprehensivetaxonomicstudy,inwhich thepossibilityofdescribingthemasnewspeciesisgiven con-sideration.

Regarding the characteristics of the strains of the two species,theseare differentiated mainlybythe colorofthe mycelium(Table2)whilesharingsomeothercharacteristics,

suchasthe absenceofanamorphicstructures,the formof myceliumgrowthandthepresenceofabundantclampsand afimbriateedge.Thisagreeswiththatreportedforstrains of different Lyophyllum specieswithin the sections Lyophyl-lum, Difformia and Tephrophana.41 _{A detailed study of the}

strains couldgeneratemoreaccurate informationfor iden-tification of the species. However, the description of the strainsinthisstudydoesnotprovidedataregarding distinc-tivecharacteristicsthatwouldpermitdifferentiationamong the species.40,41 _{The characteristics ofthe strains}

(physiol-ogy,diameter,growthrate)alsoprovideinformationaboutthe influenceoftemperature orpHor eventhechemical com-position of the culture mediaon the mycelialgrowth and production; theseare parametersused inbioassaysand in physiologicalstudiesthatcouldprovidedataregarding suit-ableconditionsforcultivation.42,43_{Inthiscase,theincubation}

temperatureofthestrainswas18◦C,takenfromthatreported byapreviousstudy.27_{Inthisrespect,thetemperatureusedis}

lowerthanthatreportedforL.decasteswhichgrowsat20◦C,44

L.fumosumat25◦C17_{andLyophyllumspp.from20to25}◦_C.41

Optimum incubation temperaturedepends on the climatic conditionsoftheregionsoforiginofthestrains4_;forthis

rea-son, itislogical thatourstrainswould haveloweroptimal temperaturessincetheydevelopinhighmountain environ-mentsandareadaptedtolowertemperatures.

Regardingthegrowthrate,thestrainIE983,which corre-spondstoL.aff.shimejiandIE975ofLyophyllumsp.presented ahighergrowthratethanthatreportedforL.shimejiandL. decastes, withgrowths of47–60mm and 22–85mm, respec-tively, after four weeks of incubation.41 _{Growth rate and}

biomassproductionarerelatedtonutrientsourcesanda suit-ableC:Nratio.45

Intheculturemediawherethemyceliuminvadedmore rapidly,biomassproductionwasmuchlowerandthestrains thatdevelopedmorebiomasswerethosethathad,on aver-age,alowergrowthrate.Thissuggeststhatthebestnutritional conditionsareprovidedbythemediumbasedonmaltextract, allowingincreasedhyphalbranchingandthusahigher quan-tityofbiomass.Incontrast,inthepotatodextrosemedium, therapidgrowthrateshowsthat,undernutrientlimiting con-ditions,thehyphae tendtobelessbranchedandgrowata higherrateinordertomaximizeexplorationofthemedium insearchofnutrients.45–47

In liquid medium, biomass production, production of polysaccharides and the formofgrowth (pellets) are influ-encedbythestrainandtheculturemediumaswellasbythe methodofcultureinitiation,thesizeofspawnofthestrainand theintensityofagitation.48–51_{Theculturemedium(sourceof}

carbonandnitrogen)alsointervenesinthedensityand diam-eterofthepellets.49,52_{Thestrainthathadthegreatestbiomass}

developedahighernumberofpellets,aresultthatwasvery similartothatofSparassislatifoliaY.C.Dai&ZhengWang.53

Themediumbasedonmaltextractsupplementedbypeptone andyeastwasthemostsuitableforallfourstrainsof Lyophyl-lum. Thismediumisconsidered oneofthebest sourcesof nitrogenforhighmycelialgrowth.Thepeptoneand/oryeast extractare alsorecognizedefficientsourcesofnitrogenfor the production ofbiomass inGanoderma lucidum (Curtis)P. Karst.,Lentinulaedodes(Berk.)Pegler,Pleurotusostreatus(Jacq.) P.Kumm.andL.decasteswithmaltoseandglucoseservingas

sourcesofcarbon.8,54 _{InthecaseofL.decastes,therewasa}

greateryieldandproductionofpolysaccharidesinglucose,as asourceofcarbon,andyeast,8_{incontrasttothefourstrains}

ofLyophyllum,thatpresent greaterbiomassproductionina mediumthatisrichinnitrogen.

Thebiomassgeneratedinaliquidmediumcanbeused asaliquidspawn,asafoodanddieteticsupplement,andfor pharmaceuticalapplicationsandenzymeproduction54,55_due

totheproductionofpolysaccharides.8_{ThepHofthemedia}

wasnotadjusted,butattheendoftheperiodofagitationit hadchangedineachofthestrainsinbothculturemedia.This couldbeduetothehighbiomassproductionand,whilethis wasnotmeasured,theproductionofmetabolites;factorsthat modifythepHattheendoftheincubationperiod.52,53

Mushroomscultivation usingnative strains, particularly ofLyophyllum,requiresstudiesthatcomprisethe characteri-zationandidentification(morphologicalandgenetic)ofthe sporophores and strains utilized; knowledge of ecological aspects,lifecycles,productionandcompositionof metabo-litesandidentificationofsubstratetypesandenvironmental conditionssuitableforfructification.1,3,4

Lyophyllumisagenuswithpotentialforcommercialculture andhasfunctionalandmedicinalproperties.9_ThestrainsIE

982ofLyophyllumsp.andIE983ofL.aff.shimeji,thatpresented thebestconditionsintheinvitroculture,representan excel-lentoptionforstudiesfocusedontheacquisitionofsecondary metabolitesandtheirexperimentalcultureatsubstratelevel. Workingwithnativestrainsconstitutesagoodalternativefor theregionalmanagementofgermplasmadaptedtothe cli-maticandsubstrateconditionswheretheydevelopnaturally, eliminatingdependenceonstrainsfromothercountries.

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