PRO LIGNO - cabi.org fileonline issn 2069-7430 issn-l 1841-4737 pro ligno vol. 8 n°2 2012 pp. 3-18...

ONLINE ISSN 2069-7430ISSN-L 1841-4737

PRO LIGNO Vol. 8 N°2 2012www.proligno.ro pp. 3-18

MONITORIZAREA DIVERSITATIIFUNGICE PENTRU DETERMINAREA

ORIGIN!! GEOGRAFICE A CHERESTELEIDIN SPEC!! EXOTICE

MONITORING FUNGAL DIVERSITY TODETERMINE THE GEOGRAPHICAL

ORIGIN OF EXOTIC TIMBERS

Alba ZAREMSKI*Dr. - UR GFP-Diversite genetique et amelioration des especes forestieres

CIRAD BIOS; TA A-108/C Campus International de BaillarguetAdresa/Address: 34398 Montpellier Cedex 5, France

E-mail: [email protected]

Renaud HENRYResearcher - Universite Europeenne de Bretagne, France

Universite de Brest, EA3882 Laboratoire Universitaire de Biodiversite et Ecologie Microbienne, ESMISAB,Technopole de Brest Iroise

Adresa/Address: 29280, Plouzane, France

Gaetan LE FLOCHResearcher - Universite Europeenne de Bretagne, France

Universite de Brest, EA3882 Laboratoire Universitaire de Biodiversite et Ecologie Microbienne, ESMISAB,Technopole de Brest Iroise

Adresa/Address: 29280, Plouzane, France

Louis GASTONGUAYResearcher - Institut de Recherche d'Hydro-Quebec, Sciences des materiauxAdresa/Address: 1800 Lionel-Boulet, Varennes (Quebec) J3X1S1, Canada

Rezumat:Lemnul este o resursa economics importanta

pentru multe Cari tropicale. Printre beneficiile adusede cele mai multe specii tropicale, se pot mencionadurabilitatea for naturals capacitatea de a rezistain mediul exterior fara a utiliza substance chimice deproteccie. De asemenea multe dintre aceste speciitropicale prezinta proprietaci incontestabile, pe careoamenii le cauta, cum ar fi aspectul esteticproprietaci mecanice bune. Sunt disponibile diferitesisteme de marcare pentru a facilita trasabilitatealemnului dar acestea au unele limite in utilizarea forpe scars larga. Acest studiu are drept scopdezvoltarea unei tehnici viabile pentru a evaluaoriginea geografica a diferitelor specii lemnoase.Lemnul poate fi colonizat de microorganismele dedistrugere fungice cunoscute sub numele endofite.Principalul obiectiv al acestei lucrari este de a utilizaaceste microorganisme ca un marker molecular. S-arealizat o amplificare a regiunii ITS (spacii transcriseintern necodificatoare ce pot fi utilizate indiferencierea speciilor inrudite strans din interiorulunui gen fungic), utilizand tehnica de analizastructurala CE-SSCP pe trei specii tropicale diferite,din 6 Cari: Limba (Terminalia superba), Limbali(Gilbertiodendron dewerei-preussii) Teak (Tectonagrandis). Rezultatele arata o grupare intre diferiteprofile microbiene legate de specia lemnoasadiferenca intre speciile Limba Limbali. Se pune in

* Autor corespondent / Corresponding author3

Abstract:Wood is an important economical resource

for many tropical countries. Among the benefits ofmost of the tropical woody species, we can mentiontheir natural durability and their capacity ofwithstanding outside environment without the use ofchemical preservatives. Also, many of these tropicalspecies show undeniable properties that peoplelooked for such as aesthetic appearance and goodmechanical property. Different marking systems areavailable in order to facilitate the traceability of woodbut they present some limitations in their use at alarger scale. This study aims at developing a reliabletechnique to asses the geographical origin ofdifferent wood species. Wood can be colonized bydestroying fungal microorganisms known asendophyte. The main objective of this work is to usethese microorganisms as a molecular marker. Anamplification of the ITS region has been done andused with CE-SSCP on tree different tropical speciesfrom 6 countries: Limba (Terminalia superba),Limbali (Gilbertiodendron dewerei-preussii) andTeak (Tectona grandis).

Results show a grouping among differentmicrobial profiles related to the wood species anddiscernment between Limba and Limbali. We showthat microbial profiles could be used as a marker toensure the proper origin of wood as part of an eco-certification for sustainable management of the


PRO LIGNO Vol. 8 N ° 2 2012www.proligno.ro pp. 3-18

evidenta faptul cä profilele microbiene ar putea fiutilizate ca markeri pentru a garanta originea corectaa lemnului, ca parti ale unei eco-certificari pentru unmanagement sustenabil al padurii. De asemeneaacestea vor ajuta in controlarea traseului produsuluidin lemn de la padure la consumator.

Cuvinte cheie: specii exotice; marker molecular;origine cherestea; amplificare PCR; analiza CE-SSCP; analiza D-HPLC; trasabilitate.

INTRODUCEREDe mai multi ani, mediul industrial a trebuit

sa fie capabil sa ateste originea produselor salepentru a -si satisface clientii. Intr-adevar, pentru unanumit produs, trebuie sa fie posibila urmarireatuturor etapelor din procesul de fabricatie, precum §ioriginea tuturor componentelor sale. Acest sistempoarta numele de trasabilitate. Aceasta serve§te, deexemplu, pentru a gasi furnizorii de materii prime,locurile diferite unde produsul a Post depozitat, modulde manipulare §i echipamentele utilizate infabricarea sa.

Scopul trasabilitatii este, de altfel, stabilireaunei legaturi intre produs, originea sa §i procesul defabricatie. Aceasta implica un sistem care utilizeazacoduri de bare sau alte etichete care °felt' maximumde informatii cu privire la produsul in cauza. Totu§i,aceste metode administrative sunt nepotrivite incazuri in care originea geografica a Post falsificata.Prin urmare, este importanta gasirea unui instrumentanalitic credibil, care sa poata identifica cucertitudine originea geografica a unui produs.

Exportul de cherestea reprezinta una dinprincipalele surse de venit pentru anumite taritropicale. Intr-adevar multe din casele noastre continelemente de lemn din aceste tall, care este utilizatpentru structure, pardoseala sau mobilier, motivulfiind calitatile estetice incontestabile §i rezistentaridicata. Standardul NF EN 350-1 define§te claselede durabilitate naturala a lemnului la atacul deciuperci (cinci clase, de la foarte durabil la nedurabil).Avantajele celor mai multe specii tropicale sunt inmod cert legate de buna lor durata de viata §i

capacitatea de a rezista in exterior fare substantechimice de protectie, care sunt daunatoare pentruom §i mediul inconjurator.

Lemnul din specia Afara (Terminaliasuperba) este folosit in principal pentru placaj,constructii de interior, sau instrumente muzicaleprecum chitara. Lemnul din specia Limbali(Gilbertiodendron dewevrei) este specific structurilortip cadru din lemn datorita rezistentei mecaniceridicate. Se poate de asemenea utiliza pentrurealizarea pardoselilor dace este foarte bine uscat.Lemnul de Teak (Tectona grandis) este consideratca un lemn valoros §i rezistent la putrezire. Estepotrivit de altfel, pentru asamblarea puntilorambarcatiilor sau pentru mobilier de exterior (mesede grading, §ezIonguri, umbrare de grading etc.)

forest. Also, they will help in controlling the itineraryof a wood product from forest to consumer.

Key words: exotic species; molecular marker;timber origin; PCR amplification; CE-SSCP analysis;D-HPLC analyis; traceability.

INTRODUCTIONFor several years, industries have had to be

able to certify the origin of their products in order tosatisfy their customers. Indeed, for a given product, ithas to be possible to track down all the stages of itsmanufacturing process as well as the origin of all itscomponents. This is called traceability. It serves, forexample, to find the suppliers of raw materials, thedifferent places where the product has been stored,and the handling and equipment used in itsmanufacture.

The purpose of traceability is therefore toestablish a link between the product, its origin and itsmanufacturing process. It involves a system usingbar codes or other labels that provide maximuminformation about the product in question. However,these administrative methods seem to be unsuited tocases where geographical origin has apparentlybeen falsified. Therefore it seems important to findan analytical tool that can identify the geographicalorigin of a product with certainty and reliability.

Timber exports are one of the main sourcesof income for certain tropical countries. Indeed,many of our homes contain timbers from thosecountries, which are used for structural frameworks,flooring or furniture, as they offer undeniableaesthetic qualities and high resistance. Standard NFEN 350-1 defines classes of natural timber durabilityin relation to wood-decaying fungi (five classesranging from highly durable to not durable). Theadvantages of most tropical timbers are preciselytheir good life expectancy and their ability to resist inan outside environment without chemicalpreservatives that are harmful to human life and theenvironment.

Afara (Terminalia superba) is mostly used forplywood, indoor woodwork or musical instrumentssuch as guitars. Limbali (Gilbertiodendron dewevrei)is often specific to framework timber for its very highmechanical resistance. It can also be used to makeflooring if it is very well dried. And lastly Teak(Tectona grandis) is considered as a valuable androt-proof timber. It is therefore suitable forassembling boat decks or for outside furniture(garden tables, loungers, garden sheds, etc.).

Different systems for physically markingtimber and enabling its traceability are availabletoday, but their uses can be limited. They range frombar codes to radio frequencies, and including paint



Diferite sisteme de marcare fizica acherestelei sunt astazi disponibile, acestea permittrasabilitatea, dar utilizarea lor este limitata. Acesteavariaza de la coduri de bare, la frecvente radio,inclusiv marcaje cu vopsea §i UV. Totu§i acestemetode sunt limitate la anumite parti ale ciclului deviata a lemnului §i prin urmare nu certifica origineageografica in cazul unei fraude. Diferentiereagenetica a fost de asemenea investigate cu ajutorulmarkerilor, poliformism cu lungimi de fragmentamplificat al ADN-ului cloroplastic §i care in generaltine de evaluarile taxonomice traditionale (Cao §.a.2006). Harvard §.a. (2007) au utilizat recent izotopulde strontiu pentru a determina originea geografica alemnului tropical, dar metoda ramane costisitoare §inecesita actualizarea bazei de date.

El Sheikha §.a. (2009) au aratat ca esteposibil sa se determine originea coacazei cupelerina, din patru regiuni diferite ale Egiptului,utilizand ADN-ul 26S de la colonizarea drojdiei defructe ca §i marker microbiologic. Aceea§i abordare afost utilizata pentru trasabilitate in acvacultura.Studiind flora bacteriana de Pangasiushypophthalmus din branhii §i intestine, Le Nguyen(2008) a aratat ca flora este specifics habitatului unuipe§te (regiuni diferite din sudul Vietnamului).

Codul de bare al ADN-ului este un conceptrelativ nou pentru diferentierea speciilor, bazat pesegmentele tinta ale unui genom (nuclear, cloroplast,sau mitocondrial) (Taberlet §.a. 2007, Kress §.a.2006, Hebert §.a. 2003, 2005). Codul de bare ADN,care este in mod curent utilizat de taxonomi§ti pentrucriminalistica, biotehnologie §i de industriaalimentary (Chase §.a. 2005, Janzen §.a. 2004, 2005§i Kress 2004), prezinta un mare potential pentrudiferentierea speciilor in viitor, de§i probleme legatede variatia limitata §i hibridare trebuie sa fie luate inmod serios in considerare.

Regiunea tmH-psbA (Hamilton 1999) permiteamplificarea simple §i robusta a fragmentelor scurte(74500bp). Regiunea este simple la secventiere §i°felt- variatii suficiente pentru diferentieri intre specii.Kress §.a. (2005) au imbogatit fragmentele doarpentru 8 genuri §i 19 specii ale acestei regiunideoarece aceasta arata cea mai mare variatie. In

opinia lor, trnH-psbA locus are cel mai mare potentialpentru distinctia speciilor, fata de toate celelalte zoneale genelor utilizate 'Dana acum pentru diferentiereaspeciilor.

Alte aplicatii sunt prezentate luand exempledin Germania, unde s-au dezvoltat mecanismepentru imbunatatirea controlului comertului cuseminte forestiere §i material saditor §i din comertulcu lemn dintr-o specie importanta din Asia de Sud-Est, familia Dipterocarpaceae (Finkeldey §.a. 2009).

De-a lungul duratei sale de viata, lemnul estesupus colonizarii cu endofite §i atacului ciupercilor deputrezire. Scopul proiectului a fost prin urmare, de autiliza flora fungica ca un marker microbiologicpentru a identifica originea geografica a cherestelei

and UV markings. However, such methods arelimited to certain parts of the wood's life cycle, andthey do not therefore certify geographical origin in

the event of fraud. Genetic differentiation was alsoinvestigated by using amplified fragment lengthpolymorphism markers of chloroplast DNA andgenerally support the traditional taxonomicassessments (Cao et al. 2006).

Harvard et al. (2007) recently used thestrontium isotope to determine the geographicalorigin of tropical timber, but the method remainsexpensive and requires database updating.

El Sheikha et al. (2009) showed that it ispossible to determine the origin of the capegooseberry from four different regions of Egypt,using 26S DNA of the yeast colonizing the fruit as amicrobiological marker. The same approach hasbeen used for traceability in aquaculture. Whenstudying the bacterial flora of Pangasiushypophthalmus in its gills and gut, Le Nguyen (2008)showed that the flora was specific to fish's habitat(different regions of southern Vietnam).

DNA-barcoding is a relatively new concept forthe differentiation of species based on targetsegments of the genome (nuclear, chloroplast ormitochondria) (Taberlet et al. 2007, Kress et al.2006, Hebert et al. 2003, 2005). DNA-barcoding,which is currently mainly used by taxonomists, forforensics, biotechnology and by the food industry(Chase et al. 2005; Janzen et al. 2004, 2005 andKress 2004) shows great potential for speciesdifferentiation in the future, although issues of limitedvariation and introgression need to be taken intoaccount seriously.

The trnH-psbA region (Hamilton 1999) allowsrobust and simple amplification of short fragments(74500bp). The region is simple to sequence andoffers sufficient variations for a distinction betweenspecies. Kress et al. (2005) enriched fragments foronly 8 genera and 19 species for this region,whereas the region shows the highest variation. Intheir opinion, the trnH-psbA locus has a greaterpotential for species distinction than all the othergene areas used for species differentiation to date.

Other applications are illustrated takingexamples from Germany, where mechanisms havebeen developed to improve the control of the tradewith forest seeds and seedlings, and from the tradewith wood of the important Southeast Asian treefamily Dipterocarpaceae (Finkeldey et al. 2009).

Throughout its life span, wood is subjected tocolonization by endophytes and to attacks by wood-decaying fungi. The aim of the project was thereforeto use fungal flora as a microbiological marker toidentify the geographical origin of timber importedfrom tropical countries. Many molecular tools sharethe common approach of detecting genetic variationswithin microbial communities. The DNA SingleStrand Conformation Polymorphism analysistechnique (SSCP) is considered simple, inexpensive,

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importata din tarile tropicale. Multe instrumentemoleculare impart5§esc abordarea comuna dedetectare a variatiei genetice dintr-o comunitatemicrobiala. Tehnica de analiza SSCP (Single StrandConformation Polymorphism) (utila in detectareapoliformismului determinat de cel putin o nucleotide)este considerate simple, ieftina, rapids §i sensibila lavariatiile de secventa a fragmentelor de ADN(Sunnucks §.a. 2000). Aceasta tehnica poate ficompletata de analiza D-HPLC (cromatografie lichidade inalta performanta de denaturare) §i secventierepentru identificarea speciilor. Regiunea aleasa a fi deinteres taxonomic este regiunea ITS (Spatiitranscrise intern) al ADN-ului ribozomal. Intr-adevaraceasta regiune este suficient de discriminantspentru a identifica ciupercile dup5 specii (White §.a.1990).

In prezenta lucrare se propune identificareaspeciilor de ciuperci existente in cheresteaua din treispecii lemnoase: Afara (Limba), Limbali and Teak.Probele au provenit din §ase tari diferite: RepublicaCentral Africans, Benin, Coasta de Filde§, Congo,Camerun §i Polinezia.

Prima sectiune a lucrarii prezinta o cercetarea literaturii de specialitate privind utilizarea PCR(reactia in lant a polimerazei) la cherestea, avand invedere ca aceasta matrice particulars poate contineinhibitori de amplificare. Sectiunea a doua descriemetodele utilizate pentru a obtine profilele SSCP,care au fost procesate prin analiza multi-factor.Sectiunea a treia descrie rezultatele obtinute §i

sectiunea finale discuta despre diversitatea fungicaexistents in cherestea in functie de originea lorgeografica.

MATERIALE I METODEAlegerea speciilor pentru acest studiu: Afara (L,Limba), Limbali (Li) §i Teak (T).

Aceste specii provin din §ase tari diferite:Republica Central Africans, Benin, Coasta de Filde§,Congo, Camerun §i Polinezia. Speciile Afara (L) §iLimbali (Li) sunt din padurile africane unde seasigura regenerabilitatea §i sustenabilitatea lor.Lemnul de Teak (T) provine din plantatiile dinPolinezia §i Coasta de Filde§. De obicei speciileutilizate in acest studiu sunt cele mai solicitate pentrumobilier §i constructii. Aceste specii au un bunpotential de a fi utilizate ca lemn pentru constructii(u§or de tratat, durabilitate naturals bunk disponibilerapid).

Speciile analizate in acest studiu au fostaprovizionate din ierbarul CIRAD (Monpellier).Originea §i codul asociat cherestelei sunt prezentatein Tabelul 1.

rapid and sensitive to the sequence variations of aDNA fragment (Sunnucks et al. 2000). Thistechnique can be completed by D-HPLC analysisand sequencing to identify species. The regionchosen to be of taxonomic interest is the ITS region(Internal Transcribed Spacer) of ribosomal DNA.Indeed, this region is sufficiently discriminant toidentify fungi by species (White et al. 1990).

We proposed in this project to identify fungalspecies existing in three timber species: Afara(Limba), Limbali and Teak. The samples came fromsix different countries: Central African Republic,Benin, Ivory Coast, Congo, Cameroon andPolynesia.

The first section presents a literature searchsection on PCR use in timber, as this particularmatrix can contain amplification inhibitors. Thesecond section describes the methods used toobtain SSCP profiles, which were processed bymulti-factor analysis. The third section describes theresults obtained, and the final section discusses thefungal diversity existing in our timbers depending ontheir geographical origin.

MATERIALS AND METHODSChoice of species for this study: Afara (L,Limba), Limbali (Li) and Teak (T).

These species come from six differentcountries: Central African Republic, Benin, IvoryCoast, Congo, Cameroon and Polynesia. Afara (L)and Limbali (Li) are from African forests in whichtheir renewal and sustainability are assured. Finally,Teak (T) comes from plantations in Polynesia andIvory Coast. Usually the species used in this studyare the most requested for furniture andconstruction. These species show good capability foruse in lumber (easily treated, good natural durability,readily available,).

The timbers analysed in this study weresupplied by the CIRAD herbarium (Montpellier). Theorigin of the timbers and their associated codes arelisted in Table 1.

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Tabelul 1 / Table 1Codul ai originea cherestelei analizata in acest studiu /

Code numbers and origin of the timbers analysed in this studReferinta CIRAD /CIRAD reference

Nume comercial /(nume §tiintific)

Trade name(scientific name)

Provenienta / Provenance Cod / Code

18122 Afara(Terminalia superba)

Central African Republic L RCA29151 Benin L Be23730 Ivory Coast L Ci5256 Congo L Co18571 Cameroon L Ca6070 Limbali

Vaa( Gilbertiodendron

dewevrei)

Central African Republic Li RCA30235 Ivory Coast Li Ci18865 Cameroon Li Ca5473 Central African Republic Li RCA

34090 Teak( Tectona grandis)

Polynesia T Pol16476 Ivory Coast T Ci

Extractia ADN din probele de cheresteaProbele de cherestea au fost transformate in

a§chii. Au fost manipulate in cele mai sterile conditiiposibile (arzator cu benzen, alcool etc.) §i cea maimare atentie a fost acordata incalzirii probelor.Reziduurile au fost apoi rafinate prin zdrobire inmojar steril cu un pistil dup5 tratare in azot lichid.

DNA-ul a fost extras din aprox. 200mg a§chiiutilizand kitul Fast DNA SPIN Kit pentru sol (QiagenMP Biomedicals). Inainte de acesta, probele au fostsupuse unei faze de liza (actiune combinata §i

agitare puternica in prezenta unei solutii tampon).Proteinele au fost apoi precipitate prin PPS (Solutiede Precipitare a Proteinei) §i eliminate princentrifugare. ADN-ul a fost fixat intr-o matricesolubila (Binding matrix) retinuta de un filtru dup5trecerea solutiei continand ADN-ul prin centrifugare.In cele din urm5 ADN-ul a fost eluat de 60pL DESAstfel fractia recuperate ar putea fi utilizata apoipentru diferite operatii cum ar fi PCR (reactie in lanta polimerazei)sau cuantificare prin spectrofotometrie.Poate fi depozitata de asemenea la -20°C.

Amplificarea PCR §i analiza CE-SSCPRegiunea ITS al ADN-ului ribozomal a fost

amplificata cu primerii ITS 1 §i ITS 2. Primerul ITS 2a fost etichetat la 5' cu 6-carboxyfluoresceine (FAM)pentru a analiza produsele PCR prin SSCP. S-aurealizat dou5 tipuri de amplificare:- fie intr-un volum total de de 15 pL, cuprinzand 3pLde 5X solutie tampon, 1.17pL de MgCl2 la 25mM,1.2pL de dNTP at 2.5mM, 14 din fiecare primer la10pM, 0.75 unitati de GoTaq Polymerase, 6.48pL4)5 distilata §i 14 de AND;- fie intr-un volum de 50pL, cuprinzand 10pL de 5Xsolutia tampon, 4pL de MgCl2 la 25mM, 4pL dedNTPs la 2.5mM, 11_1L din fiecare primer la 10pM,2pL de DMSO, 25.8pL de ape distilata, 1 unitate deGoTaq Polymerase §i 2pL de ADN (20ng.pL-1).

DNA extraction from timber samplesThe timber samples were reduced to chips.

They were handled under the most sterile conditionspossible (benzene burner, alcohol etc.) and thegreatest attention was paid to sample heating. Theresidues were then refined by crushing in liquidnitrogen with a pestle and sterile mortar.

DNA was extracted from around 200mg ofchips using a Fast DNA SPIN Kit for Soil (QiagenMP Biomedicals). Prior to that, the samplesunderwent lysis (combined action of the beads in thekit and vigorous stirring of the FastPrep in thepresence of buffer). Proteins were then precipitatedby PPS (Protein Precipitation Solution) andeliminated by centrifugation. DNA was fixed on asoluble matrix (Binding Matrix) retained by a filterafter passage of the solution containing DNA bycentrifugation. Lastly, DNA was eluted by 60pL ofDES. Thus, the fraction recovered could then beused for different operations such as PCR orquantification by spectrophotometry. It could also bestored at -20°C.

PCR amplification and CE-SSCP analysisThe ITS region of ribosomal DNA was

amplified with ITS 1 and ITS 2 primers. The ITS 2primer was labelled at 5' with 6-carboxyfluoresceine(FAM) to analyse the PCR products by SSCP. Twotypes of amplification were carried out:- either in a total volume of 15 pL, comprising 3pL of5X buffer, 1.17pL of MgCl2 at 25mM, 1.2pL of dNTPat 2.5mM, 14 of each of the primers at 10pM, 0.75Units of GoTaq Polymerase, 6.48pL of sterile waterand 14 of DNA;- in a volume of 50pL, comprising 10pL of 5X buffer,4pL of MgCl2 at 25mM, 4pL of dNTPs at 2.5mM, 14of each of the primers at 10pM, 2pL of DMSO,25.8pL of sterile water, 1 Unit of GoTaq Polymeraseand 2pL of DNA (20ng.pL-1).

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Apoi a urmat un program de amplificareincepand cu trei minute de denaturare initiala la94°C, apoi 35 cicluri de denaturare la 95°C pentru unminut, hibridizare la 60°C un minut §i alungire la72°C un minut. Amplificarea s-a incheiat cu 10minute de alungire la 72°C. Calitatea amplificarii afost apoi controlata prin transfer electroforetic cu 8%gel de agar la 100 V o ora §i imersie in BET, 20minute pentru a releva ADN-ul.

Fiecare produs amplificat (1pL) a fostamestecat cu 18.5pL de formamida §i 0.5pL markerGene scan -400 ROX (Applied Biosystems) inaintede tratamentul de incalzire la 95°C cinci minute, apoiracit in gheata, 10 minute. Moleculele ADNdenaturate in forme de fa§ii singulare au fostseparate prin electroforeza capilara utilizand unsecventier automatic ABI PRISM 310 (AppliedBiosystems). Conditiile de migrare au fost 15kV la32°C. Fa§iile ADN la ie§irea din capilare au fostdetectate prin etichetarea ITS 2 cu FAM fluorofora.Diferitele profile obtinute au fost calibrate cusoftware-ul GeneMapper 4.0 prin marimeamarkerului continut in fiecare proba. Profilele SSCPau fost convertite in valori numerice de software-ulSAFUM 2.0.

Analiza D-HPLCToate probele ADN (lemn, tulpina pura §i sol)

au fost analizate prin D-HPLC (Denaturing-HighPerformance Liquid Chromatography - cromatografielichida de inalta performanta cu denaturare).Conditiile utilizate pentru analiza D-HPLC, pentruacest studiu, pe diferite comunitati fungice, suntprezenate in Tabelul 2.

D-HPLC este o varianta a tehnicii standardHPLC utilizata pentru separarea ADN-ului. ADN-uleste introdus intr-o coloana termoreglata. Variatiatemperaturii aplicate in coloana a permis sa selucreze in conditii de denaturare §i ne-denaturare.Nulul §i faza stationara hidrofobica sunt compuse dinparticule alchil neporoase (stiren/vinil benzen). TEAAeste amfifilica (lipofila §i hidrofila) §i adsorbita u§or lasuprafata fazei stationare cu sarcina pozitiva pesuprafata ei. Aceste particule vor atrage §i fixanegativ sarcina AND-ului. Elutia ei este realizata prinutilizarea acetonitrilelor. Un detector UV este utilizatpentru masurarea semnalului.

Then it was followed by an amplificationprogramme starting with three minutes of initialdenaturation at 94°C, then 35 denaturation cycles at95°C for one minute, hybridization at 60°C for oneminute and elongation at 72°C for one minute.Amplification ended with ten minutes of finalelongation at 72°C. Amplification quality was thenchecked by electrophoretic transfer to 8% agarosegel at 100 V for one hour and immersion in BET fortwenty minutes to reveal the DNA.

Each amplification product (1pL) was mixedwith 18.5pL of formamide and 0.5pL of Gene*scan-400 ROX size marker (Applied Biosystems) beforeundergoing heat treatment at 95°C for five minutesthen cooling in ice for ten minutes. The denaturedDNA molecules in single strand forms wereseparated by capillary electrophoresis using an ABIPRISM 310 Applied automatic sequencer (AppliedBiosystems). The migration conditions were 15kV at32°C. DNA strands exiting the capillary weredetected by labelling the ITS 2 primer with FAMfluorophore. The different profiles obtained werecalibrated by GeneMapper 4.0 software by way ofthe size marker contained in each sample. TheSSCP profiles were converted into numerical valuesby SAFUM 2.0 software.

D-HPLC analysisAll the DNA samples (wood, pure strain and

soil) were analysed by D-HPLC (Denaturing-HighPerformance Liquid Chromatography). Conditionsused for D-HPLC analysis, for this study on differentfungal communities, are presented in Table 2.

D-HPLC is a variation of the standard HPLCtechnique used for the separation of the DNA. DNAis introduced in a thermoregulated column. Thevariation in the applied temperature of the columnallows us to work in denaturing as well as non-denaturing conditions. The electrically neutral andhydrophobic stationary phase is composed of nonporous alkyl particles (styrene/di vinyl benzene).TEAA is amphiphilic, and adsorb easily on thesurface of the stationary phase with a positivecharge on its surface. These particles will thenattract and fix negatively charge DNA. Its elution isdone by using acetonitrile. A UV-detector is used tomeasure the signal.

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Tabelul 2 / Table 2Condiriile D-HPLC utilizate pentru acest studiu pe comunitati fungice /

D-HPLC conditions used for this study on fungal communitiesTimp / Time Solutie Solutie

(min) tampon A/ tampon B /Buffer A Buffer B

Incarcare / Loading 0 55 45

Start gradient 0,1 48 52

Stop gradient 18,1 61

Start curatire/ Startclean

18,2 55 45

Stop curatire/ Stopclean

18,7 Will. 45

Start echilibrare/ Startequilibrate 18,8 55 45

Stop echilibrare/ Stopequilibrate 20,3 55 45

Pentru analiza PCR a produsului, 5pL suntinjectate in coloana la temperatura de 50°C la un fluxal solventului de 0,5mL/min. Solutia tampon Acontine 0.1M TEAA iar solutia tampon B contine0.1 M TEAA §i 25% acetonitrile (faza mobila).

Analiza statisticaDatele colectate in acest mod au fost

standardizate, adica toate valorile unei probe au fostimpartite la media acelei probe. S-au realizat apoiprofilurile §i s-a determinat OTUs. Treizeci §i unuOTUs au fost caracterizate, luand in consideraretoate varfurile cu o intensitate fluorescents de peste2 (Fig. 6). S-a obtinut un tabel reprezentand diferiteprobe §i OTUs. Tabelul a fost transpus §i s-auinterpretat valorile utilizand software-ul StatBox 6.6pentru a efectua analiza principalelor componente.

S-au obtinut astfel toate datele necesarepentru utilizarea rezultatelor (cercul corelatiei,distribuirea indivizilor etc).

REZULTATECUANTIFICAREA AND-ULUI TOTAL

Au fost analizate unsprezece probe decherestea (Tabelul 3). Doua operatiuni de extractieindependente au fost efectuate §i unele probe au fostextrase, in doua exemplare (un A §i un B au fostadaugate la codul lor).

For the PCR products analysis, 5pL areinjected in the column at a temperature of 50°C at asolvent flow rate of 0,5mUmin Buffer A contains0.1M TEAA and buffer B de 0.1M TEAA and 25% ofacetonitrile (mobile phase).

Statistical analysisThe data collected in this way were

standardized, i.e. all the values for a sample weredivided by the mean of that sample. We thenproduced the profiles and determined the OTUs.Thirty-one OTUs were characterized, considering allthe peaks with fluorescence intensity over 2 (Fig. 6).A table representing the different samples and OTUswas obtained. The table was transposed and weexploited the values using StatBox 6.6 software tocarry out the principal components analysis.

We thus obtained all the data needed tomake use of the results (correlation circle,distribution of individuals etc.).

RESULTSTOTAL DNA QUANTIFICATION

Eleven timber samples were analysed (Table3). Two independent extraction operations werecarried out and some samples were extracted in

duplicate (an A and a B were added to their code).

9


PRO LIGNO Vol.8 N°2 2012www.proligno.ro pp. 3-18

Tabelul 3 / Table 3Rezultatele extractie ADN-ului si cuantificarea cu spectrofotometru /

Results of DNA extraction and quantification on the spectrophotometerExtractia 1 / Extraction 1 Extractia 2 / Extraction 2

Referinte /References

Coduri /Codes

ADN /[DNA] inng.uL -1

260/280(nm)

ADN /[DNA] inng.uL 1

260/280

34090 A T Pol A 11.3 1.62 77.09 1.455473 Li RCA 2 12.34 2.56 12.45 2.1818571 L Ca 147.52 0.93 32.95 1.1723730 L Ci 324.8 0.96 31.43 1.59

34090 B T Pol B 9.16 3.5 nd nd30235 Li Ci A 11.56 1.85 11.24 5.718865 Li Ca 13.56 1.94 10.27 2.8516476 T Ci 14.25 2.13 12.94 2.266070 Li RCA 1 10.87 1.96 111.30 1.5118122 L RCA 184.09 0.81 23.48 1.335256 L Co 33.1 1.24 27.11 1.39

29151 L Be 150.63 0.82 8.77 0.9823730B L Ci B nd nd 26.93 1.19

INFLUENTA UNOR PARAMETRI ASUPRA PCREfectul dilutiei probei de ADN in DMSO (dimetil-sulfoxid)

0 game de concentratii au fost testate peprobe din prima extractie, in functie de concentratiainitiala determinate pe spectrofotometrul Nanodrop.De exemplu, trei concentratii au fost testate pe probanumarul 1 (34090A): Pura (11.3ng.pL-1); Jumatate(5.65ng.pL-1); Sfert (2.83ng.pL-1)

MT 1 1 1 2 2 4 3 5 6 6 6pur 1/2 1/4 pur 1/2 1/40 1/10 1/2 pur 112 1/4

INFLUENCE OF SEVERAL PARAMETERS ONPCREffect of DNA sample dilution in DMSO (DimethylSulphoxide)

A range of concentrations was tested on thesamples of the first extraction depending on theinitial concentration determined by the Nanodrop.For example, three concentrations were tested onsample number one (34090A): Neat (11.3ng.p1=1);Half (5.65ng.p1=1); Quarter (2.83ng.p1=1).

MT 7 X77 888 8 9 9 101-010 i+pur 1/2 1/4 pur 1/2 114 pur 1/2 pur 1/2 114

MOD

IB

Fig. 1Gelurile electroforezei ilustrand efectul cantitatii de ADN asupra amplificarii/

Electrophoresis gels illustrating the effect of the quantity of DNA on amplificationA - 1, 34090A; 2, 5473; 3, 18571; 4, 23730; 5, 34090B; 6, 30235; B - 7, 18865; 8, 16476; 9, 6070; 10,

18122; +, positive control.

In general s-a constatat ca amplificarea estemai bung in teste pure decat in teste diluate pentruprobele cu concentratie scazuta (in jur de 1 Ong.p1=1),ca in Fig. 1, unde probele 1, 2, 6 §i 9 afi§eaza obanda de intensitate mai mare pentru concentratiimai marl. Cu toate acestea, diluarea a fost necesarapentru probe cu o concentratie initiala mai mare.

Generally speaking, we found thatamplification seemed better in the neat assays thanin the diluted assays for the low-concentrationsamples (around 10ng.p1=1), as in Fig. 1 wheresamples 1, 2, 6 and 9 display a band of greaterintensity for the higher concentrations. However,dilution was necessary for the samples with a higherinitial concentration.

10



Efectul DMSODup5 testarea acestei game de concentratii,

s-a efectuat in continuare PCR utilizand trei probe,pentru care amplificarea a dat un rezultat pozitiv:Proba 1 (putt.); Proba 2 (putt.); Proba 6 (putt.).Totu§i, de data aceasta s-a adaugat DMSO laamestecul de reactie.

DMSO effectAfter testing this range of concentrations,

further PCR were carried out using three samples forwhich amplification gave a positive result: Sample 1(neat); Sample 2 (neat); Sample 6 (neat). However,this time, we did not add DMSO to the reactionmixture.

Fig. 2.Gelul electroforezei ilustrand influenta DMSO asupra PCR /Electrophoresis gel showing the influence of DMSO on PCR

(1, 34090A; 2, 5473; 6, 30235; +, positive control; -, negative control).

Fig. 2 arata ca DMSO poate avea un efectpozitiv asupra amplificarii. Intr-adevar, intensitateabenzii pare a fi mai mare pentru amplificari efectuatecu DMSO (Fig. 2), ceea ce dovede§te ca au fostprezenti cu siguranta cativa inhibitori.

Efectul volumului amestecului de reactieUn alt parametru care poate afecta, de

asemenea, rezultatele PCR, este volumul final dinamestecul de reactie. De fapt, s-au realizat dou5tipuri de PCR, unul cu un volum final de 50pL (1) saucu un volum final de 15pL (2).

Fig. 2 shows that DMSO can have a positiveeffect on amplification. Indeed, the band intensityseems greater for the amplifications carried out withDMSO (Fig. 2), which is proof that some inhibitorswere definitely present.

Effect of the reaction mixture volumeAnother parameter which can also affect

PCR results is the final volume of the reactionmixture. In fact, we carried out two types of PCReither with a final volume of 50pL (1) or with a finalvolume of 15pL (2).

3101b3b9b 13b + 2b6b7b8b10b13b+

(2)

ae

Fig. 3.Gelul electroforezei indicand efectul volumului amestecului de reactie /Electrophoresis gel indicating the effect of the reaction mixture volume

(3, 18571; 10, 18122; lb, 34090 bis; 3b, 18571 bis; 9b, 6070 bis; 13b, 23730 B; 2b, 5473 bis; 6b, 30235bis; 7b, 18865 bis; 8b, 16476 bis; 10b, 18122 bis; +, positive control; -, negative control).

11



PCR efectuat intr-un volum de 15pL arelevat o amplificare mai bung decat in 50pL. Fig. 3confirma acest lucru, dupa cum cele doua probe ingeneral (13b §i control pozitiv) arata intensitateabenzii mai mare pentru PCR in 15pL.

PROFILELE COMUNITATII FUNGICEToate profilele pentru aceea§i extractie au

fost cumulate pe acela§i grafic pentru a determinanumarul de varfuri decisive sau OTUs (Unitatea deTaxonomie Operationala). OTUs au fostcaracterizate de pozitia lor pe profil §i de intensitateafluorescentei lor.

12

c 8

6

4L7-

2

0

O

PCR carried out in a volume of 15pL revealedbetter amplification than in 50pL. Fig. 3 confirms this,as the two samples in common (13b and positivecontrol) show greater band intensity for PCR in 15pL.

FUNGAL COMMUNITY PROFILESAll the profiles for the same extraction were

cumulated on the same graph to determine thenumber of decisive peaks or OTUs (OperationalTaxonomy Unit). The OTUs were characterized bytheir position on the profile and the intensity of theirfluorescence.

MINIM 11M1 A11111=1111111111111. Illff 1111.1111. WOW

rs1 O-1 h- m m VI I-1 I",

N.) L.0 00 0 rn LD 00rN rN rJ rJ

Cn 1 r-kSD

cr, Li)0)

u")r-121

CS) N 1.11

Fig. 4.Exemplul profilului SSCP (proba 5473 bis) /

Example of a SSCP profile (sample 5473 bis).

ANALIZA COMPONENTILOR PRINCIPAL! (PCA)Datele obtinute dupa separarea ampliconului

in CE-SSCP au fost prelucrate utilizand o abordaremultifactoriala pentru a releva orice corelatii dintreOTUs (variabile) §i dintre indivizi (cheresteaanalizata).

Investigarea corelatiei dintre indivizi §i OTUs.

rnr-.

PRINCIPAL COMPONENTS ANALYSIS (PCA)The data obtained after amplicon separation

in CE-SSCP were processed taking a multifactorapproach to bring out any correlations between theOTUs (variables) and between individuals (timbersanalysed).

Search for correlations between individuals andOTUs.

TCi

6

4

2L RCA

LCo

T PolL Ben L Ci

Li RCA 2

-2Li Ci

Li RCA1Li Ca L La

-4-4 -2 2 4

axe F1 (27 %)

3 10

Fig. 5.Reprezentarea indivizilor in conformitate cu axele Fl si F2 (49%) /Representation of individuals according to axes Fl and F2 (49%).

12



1,5

0,5

-0,5

-1,5

17TO 13

0 ,,TO 29UTO 17

UTQ 12

UTO 31UTO 20

UT0 2UTO 23 UTC 14 UTO 7

UTOUTO 3b

1TO 11 +11 U 0 5

s uro 9 UTO 15UTO 10 UTO 24

UTO 6 : 0UTO O UTO 22 UTO 2

UTO 26

018UTO 27u-ro

U,3(N 5

UTOT6) 25

-1,5 -0,5 0,5

axe Fl (27 %)Fig. 6.

Cercul corelatiei variabilelor in conformitate cu axele Fl si F2 (49%) /Correlation circle for the variables according to axes Fl and F2 (49%).

Fig. 5 nu aduce nici o informatie importantacu privire la corelarea Intre diferiti indivizi, in ciudaunei u§oare tendinte care poate fi vazuta Intre celedoua specii, Afara §i Limbali. Intr-adevar, aceasta dinurma pare a fi in continuare lasata pe axa Fl decatAfara, sugerand ca axa Fl ar putea reprezentaspecia. In plus, Fig. 6 arata ca anumite OTUs aucontribuit foarte mult la aceasta axa, cum ar fi de ex.OTU 15, 18, 25 §i 26.

INVESTIGATII PRIVIND CORELARILE UTILIZANDDATE PRIMARE

Cum definitia OTUs este subiectiva, s-adecis sa se repete analiza folosind datele primare(una din 10 valori a fost utilizata ca variabila).

15

10

0 50

-10-30 -20 -10

1,5

Fig. 5 does not bring out any majorinformation on the correlation between the differentindividuals, despite a slight tendency that can beseen between the two species, Afara and Limbali.Indeed, the latter seems further left on axis Fl thanAfara, suggesting that axis Fl might represent thespecies. In addition, Fig. 6 shows that certain OTUsgreatly contributed to this axis, such as OTU 15, 18,25 and 26, for example.

SEARCH FOR CORRELATIONS USING RAWDATA

As the definition of OTUs is subjective, wedecided to repeat the analysis using raw data (oneout of 10 values was used as a variable).

()L Be

T Ci

L RCA

L Co

L Ci (A)

Li

Li Ca L CaT POI Li RCA

RCA 2°Li Ci1

0 10 20

axe Fl (57 %)

Fig. 7.Reprezentarea indivizilor in conformitate cu axele Fl si F2 (70%) /

Representation of the individuals according to axes Fl and F2 (70%).

13



1,5

CI0,5

0

LL

xtC

-1,5

175174 - .10 23

W1 14i1 1

173 177*1961 W.1800113

_ 4'351

-1,5 -0,5 0

axe Fl (57 % )

0,5

Fig. 8.Cercul corelatiei variabilelor in conformitate cu axele Fl si F2 (70%) /Correlation circle for the variables according to axes Fl and F2 (70%).

S-a 0-sit din nou tendinta deja mentionata,cu separarea celor dou5 specii Afara §i Limbali (Fig.7). Specia Afara (L) a fost gasita preferential pepartea stang5 a axei F1 §i Limbali pe dreapta,exceptand Afara (L) din Camerun, care s-a regasit inacela§i grup ca Limbali.

Cercul de corelatie (Fig. 8) indica Inca o datafaptul ca variabilele au avut o contributie importantala construirea axei F1, cum acestea au aparut maiales la marginea cercului pe aceasta ax5.

DISCUTIIExtractia ADN

Cuantificarea pe Nanodrop a furnizatinformatii privind calitatea ADN-ul extras. Dintre toateextractiile, doar o proba a avut un raport de 260:280intre 1,6 §i 1,8, rezultand existenta certa aproteinelor, in toate celelalte probe. Faza deprecipitare a proteinelor in timpul extractiei, prinurmare, ar putea fi considerata insuficienta.

Sub aspect cantitativ, cele mai multe probedin prima extractie au avut o concentratie deaproximativ 1Ong.pL-1, in afara de patru probe acaror concentratie a fost de peste 10Ong.pL-1.Distributia a fost mai uniforma pentru a douaextractie, cu 10 probe care arata o concentratie intre10 §i 30ng.pl: Cantitatile de ADN extrase parsuficiente. Totu§i, in timpul extractiei, ADN-ul dincherestea a fost izolat in acela§i mod ca §i ADN-ulfungic. In timpul cuantificarii, deosebirea dintre celedou5 tipuri de ADN nu a fost dezvaluita in modevident prin spectrofotometrie. Nu s-a putut stabili,prin urmare, cantitatea de ADN fungic, comparativ cucantitatea de ADN din cherestea. In cazul in careultimul ar fi in majoritate, amplificarea materialuluifungic ar fi mult mai dificila.

Inhibitori in cheresteaExistenta inhibitorilor depinde de matricea

folosita in timpul extractiei. De fapt, in probele

1,5

We found again the tendency alreadymentioned, with a separation of the two speciesAfara and Limbali (Fig. 7). Afara (L) was foundpreferentially on the left of axis F1 and Limbali on theright, though with an exception for Afara (L) fromCameroon, which fell into the same group asLimbali.

The correlation circle (Fig. 8) indicates onceagain that the variables strongly contributed to theconstruction of axis F1, as they mostly occurred atthe edge of the circle on this axis.

DISCUSSIONDNA extraction

Quantification on the Nanodrop providedinformation on the quality of the extracted DNA. Overall our extractions, only one sample had a 260:280ratio between 1.6 and 1.8, resulting in a definiteexistence of proteins in all the other samples. Theprotein precipitation phase during extraction couldtherefore be considered insufficient.

In terms of quantity, most of the samples ofthe first extraction had a concentration of around10ng.pL-1, apart from four samples for which theconcentration was over 100ng.pL-1. The distributionwas more uniform for the second extraction, with 10samples showing a concentration of between 10 and30ng.pL-1. The quantities of DNA extracted appearedsufficient. However, during extraction, the timberDNA was isolated in the same manner as the fungalDNA. During quantification, the distinction betweenthe two types of DNA was obviously not revealed byspectrophotometry. We could not therefore ascertainthe amount of fungal DNA compared to the amountof timber DNA. If the latter were in the majority,amplification of the fungal material would be moredifficult.

Timber inhibitorsThe existence of inhibitors depends on the

14



prelevate de la plante, s-au 0-sit compu§i precumfenoli §i polizaharide, in time ce in probele de sol,acidul humic a fost majoritar. In cherestea, compu§iifenolici sunt printre cei mai frecventi inhibitoriprezenti (ex. taninuri). Ultimii sunt antioxidantii §i

asociatiile lor, prin legaturile covalente cu proteinele§i acizii nucleici, reduc puritatea ADN -ului deextractie §i astfel reduc randamentul. In plus,aceasta poate provoca negative false in cazuldetectarii ciupercilor prin PCR.

Cu toate acestea, exista componente carepot fi adaugate in timpul extractiei sau in amesteculde reactie, pentru a elimina actiunea inhibitorilor.Acidul humic poate fi precipitat in timpul extractiei deCaC12§i ulterior eliminat de etanol. Polizaharidele potreactiona cu CTAB (bromura dehexadeciltrimetilamoniu), prin legatura ionica. In celedin urma, DMSO (dimetil-sulfoxid) §i albumina aucapacitatea de a inhiba compu§i fenolici. DMSO, deasemenea, reduce formarea de hibridizari non-specifice, crescand rigurozitatea reactiei. Cu toateacestea, in cazul in care concentratia in amesteceste de peste 10%, prezenta sa poate reduceactivitatea de polimerizare a ADN-ului.

In scopul de a evidentia prezenta inhibitorilorde amplificare, s-a efectuat un PCR pe probe cu ocantitate identica de ADN tulpina putt- in toatetuburile, adaugand, de asemenea, proba ADN-uluide mediu, pentru a fi amplificate, Ia concentratii maimarl. De fapt, dou5 probe: (29151 150.63ng.pL-1 §i5256: 33.1ng.pL-1) au fost testate intr-un amestec dereactie de 15pL cu 1pL de ADN §i 1pL de ADNtulpina pura. Prima proba nu a dat nici un rezultat §is-ar putea prin urmare imagina ca au fost prezentiinhibitori in solutia ADN-ului recuperate dup5extractie, cum tulpina putt- nu a fost amplificate. Pede alta parte, cealalta proba a prezentat o bungamplificare pentru toate concentratiile de tulpina putt-testate.

0 game de concentratii a fost apoi testate peacelea§i dou5 probe. Proba 29151 a afi§at o bandapornind de Ia o concentratie de aproximativ 25ng.pL-1 'Dana Ia 1.30ng.pL-1, in timp ce proba 5256 a afi§ato banda pentru toate concentratiile. Am puteaconsidera, prin urmare ca, amplificarea s-a realizatmai bine atunci and probele, cu o concentratieinitiala mare, au fost diluate. Acest lucru a fost posibilpoate din cauza faptului ca, prin diluarea ADN-ului,am diluat de asemenea orice inhibitori. Prezentainhibitorilor poate fi, de asemenea, demonstrate cuajutorul DSMO. Fig. 4 arata intr-adevar ca, atunciand compusul a fost folosit, benzile au fost maiintense. Aceasta poate datorita faptului ca DSMOface ADN-ul mai accesibil pentru polimerizare princompu§ii fenolici inhibitori.

In cele din urma, o singura serie deamplificare a fost adoptata pentru analiza SSPC,care a fost efectuata pe a doua extractie, intr-unvolum de 15pL.

matrix used during extraction. In fact, for samplestaken from plants, compounds such as phenols andpolysaccharides are found, whereas for soilsamples, it is humic acid that will be in the majority.In timber, phenolic compounds are among theinhibitors most frequently present (such as tannins,for example). The latter are antioxidants, and theirassociations by covalent bonding with proteins andnucleic acids reduce the purity of DNA extractionand thereby reduce the yield. In addition, it cancause false negatives in the case of fungus detectionby PCR.

However, constituents exist that can beadded during extraction or in the reaction mixture, toeliminate the action of inhibitors. Humic acid can beprecipitated during extraction by CaC12 andsubsequently removed by ethanol. Polysaccharidescan chelate with CTAB(hexadecyltrimethylammonium bromide) by ionicbonding. Lastly, DMSO (dimethyl sulphoxide) andalbumin have the ability to inhibit phenoliccompounds. DMSO also reduces the formation ofnon-specific hybridization, increasing reactionstringency. However, if its concentration in the mix isover 10%, its presence can reduce DNA polymeraseactivity.

In order to reveal the presence ofamplification inhibitors, we carried out a PCR on oursamples with an identical quantity of pure strain DNAin all the tubes, also adding the environmental DNAsample to be amplified, at increasing concentrations.In fact, two samples (29151: 150.63ng.pL-1 and5256: 33.1ng.pL-1) were tested in a 15pL reactionmixture with 11_1L of sample DNA and 14 of purestrain DNA. The first sample did not give any result,and it could therefore be imagined that inhibitorswere present in the DNA solution recovered afterextraction, as the pure strain was not amplified. Onthe other hand, the other sample exhibited goodamplification for all the pure strain concentrationstested.

A range of concentrations was then tested onthe same two samples. Sample 29151 displayed aband starting from a concentration of around25ng.pL-1 up to 1.30ng.pL-1, whereas sample 5256displayed a band for all the concentrations. We couldtherefore consider that amplification worked betterwhen samples with a high initial concentration werediluted. This may have been due to the fact that bydiluting the DNA, we also diluted any inhibitors. Thepresence of inhibitors could also be demonstratedusing DSMO. Indeed, Fig. 4 shows that when thecompound was used, the bands were more intense.This may be because DSMO made the DNA moreaccessible to the polymerase by inhibiting phenoliccompounds.

Lastly, a single amplification series wasadopted for SSCP analysis, which was carried outon the second extraction, in a volume of 15pL.

15



Analiza SSPCProfilul obtinut a condus la detectarea a 31

de OTUs (unitati operationale taxonomice) pe setulde probe, de ex. in jurul a 2,8 OTUs (sau phylotypes)pe proba. S-ar putea considera ca probele au fostlipsite de diversitatea materials pentru analiza SSCP.

La observarea profilului, nu a fost deasemenea, posibil sa se vada nici o confuzie intreprobe, care ar fi putut modifica rezultatele statisticeobtinute ulterior.

Analiza statistics: analiza componentilorprincipali (PCA)

Nici o concluzie semnificativa statistic nupoate fi trasa din analiza pe baza de OTU (Fig. 5).Intr-adevar, a fost imposibil sa se grupeze originilegeografice ale fiecaruia. In plus, in aceasta figura,diferenta intre specii, nu este la fel de clar vizibila cain Fig. 7. Efectiv, in aceasta figura, faptul de a firealizat un PCA cu date brute luate la intamplare afacut posibil sa se scoata in evidenta mai bine celedoua grupuri (Afara §i Limbali). Totu§i, proba Afaradin Camerun a fost localizata in grupul Limbali. Cutoate acestea, exists o modalitate de a verifica dacapozitia sa in plan poate fi considerata corecta. Pentrua face acest lucru, s-a observat valoarea patratuluicosinusului §i daca este peste 0.4 se poateconsidera ca punctul de referinta al probei a fost inlocul potrivit. Valoarea pentru aceasta proba a fostde 0,39, adica sub valoarea de prag, dar suficient deaproape pentru a exprima o indoiala cu privire lainterpretarea acestui rezultat. Prin urmare, a fostdificil sa se concluzioneze asupra pozitiei reale aacestei probe.

Ceea ce se poate spune este ca, in general,probele aflate la dreapta axei Fl au apartinut specieiLimbali, in timp ce, cele de pe cealalta parte a axei incea mai mare parte au apartinut speciei Afara.

In concluzie, s-a investigat astfel tehnicaSSCP pentru a determina originea geografica acherestelei din specii exotice. Dupa analizelestatistice ale datelor SSCP, am avut posibilitatea dea vedea o tendinta care a parut sa grupeze probelein functie de specie.

Se poate imagina cheresteaua colonizata dediferite comunitati, cum ar fi cele specifice speciei,locului de productie, sau de depozitare, in cazuldepozitarii pe termen lung. Prin urmare, ar fi dificil sase determine efectiv originea geografica folosindtoate comunitatile existente in cherestea. Ideal ar fisa se §tie taxonii prezenti in probe, pentru ca analizastatistics sa se bazeze exclusiv pe cei de interes §inu pe contaminanti. Pentru a realiza acest lucru, ar fide preferat sa efectueze clonarea ampliconuluiinainte de secventiere. 0 alts tehnica ar duceaparent la acela§i rezultat: D-HPLC. Aceasta tehnicaface posibila recuperarea fractiilor specifice unui \fartin functie de timpul de retentie (Troedsson §.a. 2008)facand astfel posibila secventa probei.

SSCP analysisThe profile obtained led to the detection of 31

OTUs on the set of samples, i.e. around 2.8 OTUs(or phylotypes) per sample. We could consider thatthe samples possessed not insubstantial diversity forSSCP analysis.

When observing the profile, we were alsoable to see any staggering between the samples,which might have modified the statistical resultsobtained thereafter.

Statistical analysis: principal componentsanalysis (PCA)

No statistically significant conclusions couldbe drawn from the OTU-based analysis (Fig. 5).Indeed, it was impossible to group the geographicalorigins with each other. In addition, in this figure, thedistinction between species is not as clearly visibleas in Fig. 7. In effect, in that figure, the fact of havingcarried out a PCA with raw data taken at randommade it possible to bring out the two groups moreeffectively (Afara and Limbali). However, the Afarasample from Cameroon was located in the Limbaligroup. Nevertheless, there was one way of checkingwhether its position in the plane could be consideredcorrect. To do this, we observed the value of thesquared cosines and if it was over 0.4 we couldconsider that the point referencing the sample was inthe right place. The value for this sample was 0.39,i.e. below the threshold value but close enough toexpress a doubt as to the interpretation of this result.It was therefore difficult to conclude on the trueposition of that sample.

What we can say is that, all in all, thesamples located to the right of axis Fl belonged tothe Limbali species, whereas those on the other sideof the axis mostly belonged to the Afara species.

In conclusion, we thus investigated the SSCPtechnique for determining the geographical origin ofexotic timbers. After statistical analyses of SSCPdata, we were only able to see a tendency thatseemed to group samples according to timber type.

It can be imagined that timber is colonized bydifferent communities, such as those specific to thespecies, production site or storage site in the eventof long-term storage. It may therefore be difficult tosuccessfully determine geographical origin using allthe communities existing in timber. The ideal thingwould be to know the taxa present in samples, inorder to base the statistical analysis solely on thoseof interest, and not on contaminants. To achievethat, it would be preferable to carry out ampliconcloning prior to sequencing. Another techniquewould apparently lead us to the same result: D-HPLC. This technique makes it possible to recoverthe specific fractions of a peak according to itsretention time (Troedsson et al. 2008) therebymaking it possible to sequence the sample.

16



AbrevieriCE-SSCP: Electroforeza Capilara- tehnica de analiza(utila in detectarea poliformismului determinat de celputin o nucleotide)D-H PLC : Cromatografie lichida de inaltaperformanta de denaturareDNA: acid dezoxiribonucleicdNTP: Deoxyribonucleotide TrifosfatOTU: Unitati taxonomice operationalePCA: Analiza Componentilor PrincipaliPCR: Reactie in lant a polimerazeiRT-PCR: Reactie de polimerizare in lant cu detectiein timp realUV: Ultra Violet

BIBLIOGRAFIE / REFERENCES

AbbreviationsCE-SSCP: Capillary Electrophoresis Single StrandConformation PolymorphismD-HPLC : Denaturing High Performance LiquidChromatographyDNA: Deoxyribonucleic AciddNTP: Deoxyribonucleotide TriphosphateOTU: Operational Taxonomic UnitPCA: Principal Components AnalysisPCR: Polymerase Chain ReactionRT-PCR: Real Time Polymerase Chain ReactionUV: Ultra Violet

CAO, C.P., GAILING, 0., SIREGAR, I., INDRIOKO, S., FINKELDEY, R. (2006). Genetic variation at AFLPsfor the Dipterocarpaceae and its relation to molecular phylogenies and taxonomic subdivisions. Journal ofPlant Research 119(5):553-558.CHASE, M.W., SALAMIN, N., WILKINSON, M., DUNWELL, J.M., KESANAKURTHI, R.P., HAIDAR, N.,SAVOLAINEN, V. (2005). Land plants and DNA barcodes: short-term and long-term goals. Philosophicaltransactions of the Royal Society B-Biological Sciences 360:1889-1895.EL SHEIKHA, A.F., CHALIER C., ZAREMSKI, A., MONTET, D. (2009). Determination of the geographicalorigin of timber by using an innovative molecular tool "PCR-DGGE": Preliminary application to tropicaltimber. In Conference "How to trace the origin of food?", 2009-12-02/2009-12-03, Brussels, Belgium.EL SHEIKHA, A.F., CONDUR, A., METAYER, I., LE NGUYEN, D.D., LOISEAU, G., MONTET, D. (2009).Determination of fruit origin by using 26S rDNA fingerprinting of yeast communities by PCR-DGGE:preliminary application to Physalis fruits from Egypt. Yeast 26: 567 - 573.LE NGUYEN, D.D., NGOC DIJOUX, D., LOISEAU,G., MONTET, D. (2006). Determination of fish originby using 16S rDNA fingerprinting of bacterial communities by PCR-DGGE: An application on Pangasius fishfrom Viet Nam. Food Control 19:454-460.FINKELDEY, R., LEINEMANN, L., GAILING, 0. (2009). Molecular genetic tools to infer the origin of forestplants and wood. Appl Microbiol Biotechnol. 85(5):1251-1258.HAMILTON, M.B. (1999). Four primers pairs for the amplification of chloroplast intergenic regions withintraspecific variation. Mol Ecol 8:513-525.HARVARD, M.L., DEFREMOND, E., ZAREMSKI, A., SALES, C. (2007). Using strontium isotopes todetermine the provenance of tropical timber Bois et forets des tropiques. n°294.HOFFMAN, M.T., ARNOLD, E. (2008). Geographic locality and host identity shape fungal endophytecommunities in cupressaceous trees. Mycological research, 112:331-344.HEBERT P.D.N., CYWINSKA, A., BALL, S.L., DE WAARD, J.R. (2003). Biological identifications throughDNA barcodes. Proceedings of the royal society of London series B-Biological Sciences 270: 313-321.HEBERT, P.D.N., GREGORY, T.R. (2005). The promise of DNA barcoding for taxonomy.Systematic Biology 54:852-859.JANZEN, D.H.: 2004. Now is the time. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 359:731-732.JANZEN, D.H. (2005). In Plant Conservation: A natural History Approach, eds. Krupnick, G.A., & Kress, W.J.(Univ. Chicago Press, Chicago), pp. IX-XIII.KRESS, W.J., WURDACK, K.J., ZIMMER, E.A., WEIGT , L.A., JANZEN, D.H. (2005). Use of DNA barcodesto identify flowering plants. PNAS 23:8369-8374.ORITA, M., SUZEKI ,Y., SEKIYA ,T., HAYASHI, K. (1989). Rapid and sensitive detection of point mutationsand DNA polymorphisms using the polymerase chain reaction. Genomics 5:874-879.TABERLET, P., COISSAC, E., POMPANON, F., GIELLY, L., MIQUEL, C., VALENTINI, A., VERMAT, T.,CORTHIER, G., BROCHMANN, C. ,WILLERSLEV, E. (2007). Power and limitations of the chloroplast trnL(UAA) intron for plant DNA barcoding. Nucleic Acids Research 35:e14; doi:10.1093/nar/gk1938.TROEDSSON, C., LEE, R., STOKES, V., WALTERS, T., SIMONELLI, P., FRISCHER, M. (2008).Development of a denaturing high-performance liquid chromatography method for detection of protistparasites of metazoans. Appl Environ Microbiol, 74:4336-4345.VAINIO, E.J., HANTULA, J. (2000). Direct analysis of wood-inhabiting fungi using denaturing gradient gel

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electrophoresis of amplified ribosomal DNA. Mycological research 104:927-936.WHITE, T.J., BRUNS, T., LEE, S., TAYLOR, J.W. (1990). Amplification and direct sequencing of fungalribosomal RNA genes for phylogenetics. 315-322 In: PCR protocols: a guide to methods and applications(Innis MA, Gelfand DH, Sninsky JJ & White TJ eds), Academic Press, New York.

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