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First	insights	on	Tuber	borchii	distribution	in	both	natural	forests	and	experimental	truffle	fields	in	Portugal
Conference	Paper	·	October	2014
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9	authors,	including:
Beatrice	Belfiori
Italian	National	Research	Council
23	PUBLICATIONS			302	CITATIONS			
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Francesco	Paolocci
Italian	National	Research	Council
87	PUBLICATIONS			1,926	CITATIONS			
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Available	from:	Marcos	Morcillo
Retrieved	on:	08	July	2016
First	
  insights	
  on	
  Tuber	
  borchii	
  distribu.on	
  	
  
in	
  both	
  natural	
  forests	
  and	
  experimental	
  truffle	
  fields	
  in	
  Portugal	
  
Anabela	
  Marisa	
  Azul1,	
  João	
  Trovão1,	
  Marcos	
  Morcillo2,	
  Andrea	
  Rubini3,	
  Alessandro	
  
Trusso3,	
  Beatrice	
  Belfiori3,	
  Francesco	
  Paolocci3,	
  Helena	
  Freitas1,	
  and	
  Claudia	
  Riccioni3	
  
1Centre	
  for	
  FuncAonal	
  Ecology,	
  Department	
  of	
  Life	
  Sciences.	
  University	
  of	
  Coimbra,	
  3001-­‐401	
  Coimbra,	
  Portugal.	
  E-­‐mail:	
  amjrazul@ci.uc.pt	
  
2Micologia	
  Forestal	
  &	
  Aplicada,	
  Rbla.	
  Arnau	
  6	
  local	
  D,	
  Vilanova	
  i	
  la	
  Geltrú,	
  08800	
  Barcelona,	
  Spain	
  
3	
  CNR	
  (NaAonal	
  Research	
  Council)	
  -­‐	
  InsAtute	
  of	
  Biosciences	
  and	
  BioResources	
  (IBBR),	
  Via	
  della	
  Madonna	
  Alta,	
  130	
  -­‐	
  06128	
  Perugia,	
  Italy	
  
	
  
AIM	
  To	
  monitor	
  the	
  distribuAon	
  and	
  
dynamics	
  of	
  T.	
  borchii	
  in	
  naAve	
  forests	
  and	
  
experimental	
  truffle	
  trials	
  established	
  in	
  
Portugal	
  (Alentejo	
  region).	
  
	
  
BACKGROUND	
  Among	
  the	
  most	
  valuable	
  ectomycorrhizal	
  fungi	
  belonging	
  to	
  Tuber	
  spp.,	
  Tuber	
  borchii,	
  is	
  becoming	
  an	
  
increasingly	
  popular	
  fungus	
  in	
  the	
  marketplace.	
  Thanks	
  to	
  a	
  good	
  adaptaAon	
  to	
  mulAple	
  environments,	
  T.	
  borchii	
  is	
  also	
  
culAvated,	
  in	
  both	
  naAve	
  and	
  non	
  endemic	
  areas.	
  As	
  all	
  symbioAc	
  species,	
  this	
  fungus	
  can	
  improve	
  mineral	
  nutriAon	
  and	
  
stress-­‐tolerance	
   of	
   the	
   host	
   plant.	
   The	
   scienAfic	
   relevance	
   of	
   T.	
   borchii	
   as	
   a	
   model	
   species	
   for	
   studying	
   plant-­‐fungus	
  
symbioses	
   is	
   also	
   recognized.	
   MaAng	
   type	
   (MAT)	
   genes	
   governing	
   the	
   sexual	
   reproducAon	
   (i.e.	
   frucAficaAon)	
   have	
   been	
  
recently	
  idenAfied	
  unveiling	
  that	
  this	
  is	
  a	
  heterothallic	
  fungus	
  (MarAn	
  et	
  al	
  2012	
  European	
  Patent	
  	
  EP2426215).	
  	
  
PRELIMINARY	
  RESULTS	
  
REFERENCES	
  
Azul	
  et	
  al.	
  2010	
  Mycorrhiza	
  20,	
  73-­‐88	
  
MarAn	
  F	
  et	
  al.	
  2012	
  European	
  Patent	
  	
  EP2426215	
  
Murat	
  et	
  al.	
  2013	
  New	
  Phytol	
  199:176-­‐187	
  
Rubini	
  et	
  al.	
  2011	
  New	
  Phytol	
  189:710-­‐722	
  
Rubini	
  et	
  al.	
  2014	
  Mycorrhiza	
  24,	
  S19-­‐S27	
  
ACKNOWLEDGEMENTS	
  
Bilateral	
  Agreement	
  between	
  FCT-­‐MCTES	
  
(Portuguese	
  FoundaAon	
  for	
  Science	
  and	
  
Technology)	
  and	
  CNR	
  (Italian	
  NaAonal	
  Research	
  
Council),	
  and	
  VIDT	
  19118	
  Co-­‐funded	
  by	
  COMPETE-­‐
FEDER-­‐EU,	
  for	
  financial	
  support.	
  All	
  faciliAes	
  at	
  the	
  
farm	
  units,	
  Freixo,	
  Breijinho,	
  and	
  Quinta	
  de	
  Sousa.	
  
FUTURE	
  RESEARCH	
  
During	
  Summer	
  2014	
  a	
  consistent	
  number	
  of	
  new	
  T.	
  borchii	
  
ECMs	
  has	
  been	
  found	
  and	
  collected	
  in	
  the	
  same	
  natural	
  areas.	
  
This	
  material	
  is	
  now	
  under	
  analysis	
  to	
  assess	
  whether	
  the	
  
distribuAon	
  pajern	
  of	
  T.	
  borchii	
  ECMs	
  with	
  different	
  maAng	
  
type	
  is	
  biased	
  on	
  their	
  hosts	
  as	
  it	
  has	
  been	
  recently	
  
demonstrated	
  to	
  occur	
  on	
  host	
  plants	
  colonized	
  by	
  T.	
  
melanosporum	
  (Rubini	
  et	
  al.	
  2011,	
  2014;	
  Murat	
  et	
  al.	
  2013).	
  
CONCLUSIONS	
  
Our	
  data	
  show	
  that,	
  at	
  least	
  in	
  experimental	
  truffle	
  grounds,	
  T.	
  borchii	
  ECMs	
  of	
  
different	
  maAng	
  type	
  can	
  coexist	
  under	
  the	
  same	
  host-­‐plant.	
  This	
  pajern,	
  if	
  
supported	
  by	
  further	
  data,	
  disAnguishes	
  this	
  species	
  from	
  the	
  valuable	
  black	
  truffle	
  
T.	
  melanosporum,	
  where	
  a	
  biased	
  distribuAon	
  of	
  the	
  two	
  maAng	
  types	
  on	
  single	
  
host	
  plants	
  was	
  evidenced,	
  suggesAng	
  intraspecific	
  compeAAon	
  (Rubini	
  et	
  al.	
  2011;	
  
Murat	
  et	
  al.	
  2013).	
  It	
  could	
  be	
  very	
  interesAng	
  to	
  invesAgate	
  whether	
  or	
  not	
  such	
  a	
  
dissimilarity	
  in	
  the	
  strain	
  dynamics	
  of	
  the	
  two	
  species	
  might	
  be	
  at	
  the	
  basis	
  of	
  their	
  
very	
  different	
  ecological	
  requirements	
  and	
  pajern	
  of	
  geographic	
  distribuAon.	
  
Tuber	
  borchii	
  	
  
SAMPLING	
  SITES	
  	
  
1	
  Natural	
  forests	
  (NF),	
  corresponding	
  to	
  
naAve	
  Mediterranean	
  oak	
  woodlands	
  
dominated	
  by	
  Quercus	
  suber	
  L.	
  (cork	
  oak,	
  Qs).	
  
2	
  Experimental	
  field	
  trial	
  1	
  (EFT1),	
  with	
  
plantlets	
  of	
  Pinus	
  pinea	
  L.	
  (stone	
  pine,	
  Pp)	
  
inoculated	
  with	
  T.	
  borchii,	
  introduced	
  in	
  areas	
  
dominated	
  by	
  Qs	
  and	
  Pp	
  (in	
  spring	
  2010).	
  	
  
3	
  Experimental	
  field	
  trial	
  2	
  (EFT2)	
  inoculaAon	
  
in	
  situ	
  of	
  Qs	
  and	
  Pp	
  young	
  trees	
  with	
  spores	
  
of	
  T.	
  borchii	
  in	
  oak	
  woodlands	
  (spring	
  2011).	
  	
  
METHODS	
  
Samplings	
  were	
  performed	
  in	
  summer	
  
2013.	
  Four	
  distant	
  plots,	
  two	
  in	
  the	
  NF,	
  
and	
  two	
  in	
  EFT1	
  and	
  EFT2,	
  and	
  5	
  trees	
  
per	
  plot,	
  were	
  selected.	
  Around	
  each	
  
tree,	
  roots	
  and	
  soil	
  samples	
  were	
  
collected	
  from	
  different	
  points.	
  
Ectomycorrhizae	
  (ECM)	
  and	
  soil	
  
samples	
  were	
  molecularly	
  analyzed	
  by	
  
PCR-­‐amplificaAon	
  of	
  the	
  ITS	
  region	
  and	
  
maAng	
  type	
  locus.	
  
T.	
  borchii	
  ECMs	
  were	
  present	
  in	
  both	
  NF	
  
and	
  EFTs	
  (Fig.	
  6),	
  with	
  higher	
  frequency,	
  
up	
  to	
  50%	
  mycorrhiza.on,	
  in	
  EFT1	
  (Table	
  
1),	
  and	
  ≤5%	
  in	
  EFT2.	
  In	
  NF,	
  T.	
  borchii	
  
ECMs	
  were	
  more	
  irregularly	
  found.	
  	
  
Moreover,	
  preliminary	
  data	
  revealed	
  
that	
  under	
  the	
  same	
  host	
  plant,	
  T.	
  
borchii	
  ECMs	
  of	
  opposite	
  ma.ng	
  type	
  
can	
  coexist	
  in	
  experimental	
  grounds.	
  	
  
EFT	
  2	
  Pp	
   aTbor	
   aECM	
   iECM	
   nECM	
  
Abundance	
   ≤5%	
   75-­‐85%	
   12-­‐17%	
   <5%	
  
EFT	
  2	
  Qs	
   aTbor	
   aECM	
   iECM	
   nECM	
  
Abundance	
   ≤5%	
   70-­‐80%	
   15-­‐20%	
   <5%	
  
EFT	
  1	
  Pp	
  
aTbor	
   aECM	
   iECM	
   nECM	
  
Abundance	
   ≤50%	
   45-­‐50%	
   40-­‐45%	
   ≤5%	
  
Interes.ngly,	
  although	
  T.	
  borchii	
  
ECMs	
  were	
  infrequently	
  found	
  in	
  
natural	
  forest,	
  almost	
  all	
  of	
  the	
  
soil	
  samples	
  revealed	
  the	
  
belowground	
  presence	
  of	
  
mycelium	
  of	
  T.	
  borchii	
  and	
  a	
  
dominance	
  of	
  one	
  ma.ng	
  type	
  
over	
  the	
  other.	
  	
  
Figure	
  2.	
  above	
  experimental	
  field	
  trial	
  1,	
  plantlets	
  of	
  Pinus	
  pinea	
  inoculated	
  with	
  
T.	
   borchii	
   (spring	
   2010);	
   below	
   experimental	
   field	
   trial	
   2,	
   inoculaAon	
   in	
   situ	
   of	
  
young	
  trees	
  of	
  Quercus	
  suber	
  and	
  P.	
  pinea	
  with	
  spores	
  of	
  T.	
  borchii	
  (spring	
  2011).	
  
Figure	
   3.	
   Natural	
   forest	
   –	
   Herdade	
   do	
   Freixo,	
   Montemor-­‐o-­‐Novo,	
   Portugal,	
  
represenAng	
  oak	
  woodlands	
  dominated	
  by	
  Quercus	
  suber.	
  
Figure	
  4.	
  EFT1	
  –	
  Herdade	
  do	
  Brejinho,	
  Grândola,	
  Portugal.	
   Figure	
  5.	
  EFT2	
  –	
  Quinta	
  de	
  Sousa,	
  Montemor-­‐o-­‐Novo,	
  Portugal.	
  	
  
Tabela	
  1.	
  Abundance	
  of	
  T.	
  borchii	
  ECM	
  in	
  EFT1	
  and	
  EFT2.	
  aTbor	
  =	
  acAve	
  T.	
  
borchii	
  ECM;	
  aECM	
  =	
  total	
  acAve	
  ECM,	
  iECM	
  =	
  total	
  inacAve	
  ECM,	
  nECM	
  =	
  total	
  
non	
  mycorrhizal	
  roots	
  (see	
  Azul	
  et	
  al.	
  2010)	
  
Natural	
  forests	
  	
  
cork	
  oak	
  woodlands	
  
Experimental	
  truffle	
  trials	
  
Figure	
  1.	
  DistribuAon	
  of	
  naAve	
  Mediterranean	
  oak	
  
woodlands	
  dominated	
  by	
  Quercus	
  suber	
  in	
  Portugal	
  
with	
  the	
  sampling	
  sites	
  located	
  in	
  the	
  Alentejo	
  region.	
  
	
  	
  1	
  	
  	
  	
  2	
  	
  	
  	
  3	
  	
  	
  	
  4	
  	
  	
  	
  5	
  	
  	
  	
  6	
  	
  	
  	
  	
  7	
  	
  	
  	
  	
  8	
  	
  	
  	
  	
  9	
  	
  	
  10	
  	
  	
  11	
  	
  12	
  13	
  
500bp	
  
1Kb	
  
Figure	
  6.	
  MulAplex	
  PCR	
  with	
  specific	
  primers	
  of	
  both	
  maAng	
  types	
  
on	
  ECMs	
  collected	
  under	
  the	
  same	
  host	
  plant	
  (site	
  EFT1).	
  Lane	
  (L)	
  
2:	
   posiAve	
   control,	
   MAT1-­‐2-­‐1.	
   L13:	
   posiAve	
   control,	
   MAT1-­‐1-­‐1.	
  
L3-­‐11:	
  each	
  lane	
  represent	
  a	
  single	
  ECM.	
  L12:	
  negaAve	
  control.	
  L1:	
  
Gene	
  Ruler	
  DNA	
  ladder	
  mix	
  (Fermentas).	
  

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Poster tuber borchii research in portugal iwemm morocco

  • 2. First  insights  on  Tuber  borchii  distribu.on     in  both  natural  forests  and  experimental  truffle  fields  in  Portugal   Anabela  Marisa  Azul1,  João  Trovão1,  Marcos  Morcillo2,  Andrea  Rubini3,  Alessandro   Trusso3,  Beatrice  Belfiori3,  Francesco  Paolocci3,  Helena  Freitas1,  and  Claudia  Riccioni3   1Centre  for  FuncAonal  Ecology,  Department  of  Life  Sciences.  University  of  Coimbra,  3001-­‐401  Coimbra,  Portugal.  E-­‐mail:  amjrazul@ci.uc.pt   2Micologia  Forestal  &  Aplicada,  Rbla.  Arnau  6  local  D,  Vilanova  i  la  Geltrú,  08800  Barcelona,  Spain   3  CNR  (NaAonal  Research  Council)  -­‐  InsAtute  of  Biosciences  and  BioResources  (IBBR),  Via  della  Madonna  Alta,  130  -­‐  06128  Perugia,  Italy     AIM  To  monitor  the  distribuAon  and   dynamics  of  T.  borchii  in  naAve  forests  and   experimental  truffle  trials  established  in   Portugal  (Alentejo  region).     BACKGROUND  Among  the  most  valuable  ectomycorrhizal  fungi  belonging  to  Tuber  spp.,  Tuber  borchii,  is  becoming  an   increasingly  popular  fungus  in  the  marketplace.  Thanks  to  a  good  adaptaAon  to  mulAple  environments,  T.  borchii  is  also   culAvated,  in  both  naAve  and  non  endemic  areas.  As  all  symbioAc  species,  this  fungus  can  improve  mineral  nutriAon  and   stress-­‐tolerance   of   the   host   plant.   The   scienAfic   relevance   of   T.   borchii   as   a   model   species   for   studying   plant-­‐fungus   symbioses   is   also   recognized.   MaAng   type   (MAT)   genes   governing   the   sexual   reproducAon   (i.e.   frucAficaAon)   have   been   recently  idenAfied  unveiling  that  this  is  a  heterothallic  fungus  (MarAn  et  al  2012  European  Patent    EP2426215).     PRELIMINARY  RESULTS   REFERENCES   Azul  et  al.  2010  Mycorrhiza  20,  73-­‐88   MarAn  F  et  al.  2012  European  Patent    EP2426215   Murat  et  al.  2013  New  Phytol  199:176-­‐187   Rubini  et  al.  2011  New  Phytol  189:710-­‐722   Rubini  et  al.  2014  Mycorrhiza  24,  S19-­‐S27   ACKNOWLEDGEMENTS   Bilateral  Agreement  between  FCT-­‐MCTES   (Portuguese  FoundaAon  for  Science  and   Technology)  and  CNR  (Italian  NaAonal  Research   Council),  and  VIDT  19118  Co-­‐funded  by  COMPETE-­‐ FEDER-­‐EU,  for  financial  support.  All  faciliAes  at  the   farm  units,  Freixo,  Breijinho,  and  Quinta  de  Sousa.   FUTURE  RESEARCH   During  Summer  2014  a  consistent  number  of  new  T.  borchii   ECMs  has  been  found  and  collected  in  the  same  natural  areas.   This  material  is  now  under  analysis  to  assess  whether  the   distribuAon  pajern  of  T.  borchii  ECMs  with  different  maAng   type  is  biased  on  their  hosts  as  it  has  been  recently   demonstrated  to  occur  on  host  plants  colonized  by  T.   melanosporum  (Rubini  et  al.  2011,  2014;  Murat  et  al.  2013).   CONCLUSIONS   Our  data  show  that,  at  least  in  experimental  truffle  grounds,  T.  borchii  ECMs  of   different  maAng  type  can  coexist  under  the  same  host-­‐plant.  This  pajern,  if   supported  by  further  data,  disAnguishes  this  species  from  the  valuable  black  truffle   T.  melanosporum,  where  a  biased  distribuAon  of  the  two  maAng  types  on  single   host  plants  was  evidenced,  suggesAng  intraspecific  compeAAon  (Rubini  et  al.  2011;   Murat  et  al.  2013).  It  could  be  very  interesAng  to  invesAgate  whether  or  not  such  a   dissimilarity  in  the  strain  dynamics  of  the  two  species  might  be  at  the  basis  of  their   very  different  ecological  requirements  and  pajern  of  geographic  distribuAon.   Tuber  borchii     SAMPLING  SITES     1  Natural  forests  (NF),  corresponding  to   naAve  Mediterranean  oak  woodlands   dominated  by  Quercus  suber  L.  (cork  oak,  Qs).   2  Experimental  field  trial  1  (EFT1),  with   plantlets  of  Pinus  pinea  L.  (stone  pine,  Pp)   inoculated  with  T.  borchii,  introduced  in  areas   dominated  by  Qs  and  Pp  (in  spring  2010).     3  Experimental  field  trial  2  (EFT2)  inoculaAon   in  situ  of  Qs  and  Pp  young  trees  with  spores   of  T.  borchii  in  oak  woodlands  (spring  2011).     METHODS   Samplings  were  performed  in  summer   2013.  Four  distant  plots,  two  in  the  NF,   and  two  in  EFT1  and  EFT2,  and  5  trees   per  plot,  were  selected.  Around  each   tree,  roots  and  soil  samples  were   collected  from  different  points.   Ectomycorrhizae  (ECM)  and  soil   samples  were  molecularly  analyzed  by   PCR-­‐amplificaAon  of  the  ITS  region  and   maAng  type  locus.   T.  borchii  ECMs  were  present  in  both  NF   and  EFTs  (Fig.  6),  with  higher  frequency,   up  to  50%  mycorrhiza.on,  in  EFT1  (Table   1),  and  ≤5%  in  EFT2.  In  NF,  T.  borchii   ECMs  were  more  irregularly  found.     Moreover,  preliminary  data  revealed   that  under  the  same  host  plant,  T.   borchii  ECMs  of  opposite  ma.ng  type   can  coexist  in  experimental  grounds.     EFT  2  Pp   aTbor   aECM   iECM   nECM   Abundance   ≤5%   75-­‐85%   12-­‐17%   <5%   EFT  2  Qs   aTbor   aECM   iECM   nECM   Abundance   ≤5%   70-­‐80%   15-­‐20%   <5%   EFT  1  Pp   aTbor   aECM   iECM   nECM   Abundance   ≤50%   45-­‐50%   40-­‐45%   ≤5%   Interes.ngly,  although  T.  borchii   ECMs  were  infrequently  found  in   natural  forest,  almost  all  of  the   soil  samples  revealed  the   belowground  presence  of   mycelium  of  T.  borchii  and  a   dominance  of  one  ma.ng  type   over  the  other.     Figure  2.  above  experimental  field  trial  1,  plantlets  of  Pinus  pinea  inoculated  with   T.   borchii   (spring   2010);   below   experimental   field   trial   2,   inoculaAon   in   situ   of   young  trees  of  Quercus  suber  and  P.  pinea  with  spores  of  T.  borchii  (spring  2011).   Figure   3.   Natural   forest   –   Herdade   do   Freixo,   Montemor-­‐o-­‐Novo,   Portugal,   represenAng  oak  woodlands  dominated  by  Quercus  suber.   Figure  4.  EFT1  –  Herdade  do  Brejinho,  Grândola,  Portugal.   Figure  5.  EFT2  –  Quinta  de  Sousa,  Montemor-­‐o-­‐Novo,  Portugal.     Tabela  1.  Abundance  of  T.  borchii  ECM  in  EFT1  and  EFT2.  aTbor  =  acAve  T.   borchii  ECM;  aECM  =  total  acAve  ECM,  iECM  =  total  inacAve  ECM,  nECM  =  total   non  mycorrhizal  roots  (see  Azul  et  al.  2010)   Natural  forests     cork  oak  woodlands   Experimental  truffle  trials   Figure  1.  DistribuAon  of  naAve  Mediterranean  oak   woodlands  dominated  by  Quercus  suber  in  Portugal   with  the  sampling  sites  located  in  the  Alentejo  region.      1        2        3        4        5        6          7          8          9      10      11    12  13   500bp   1Kb   Figure  6.  MulAplex  PCR  with  specific  primers  of  both  maAng  types   on  ECMs  collected  under  the  same  host  plant  (site  EFT1).  Lane  (L)   2:   posiAve   control,   MAT1-­‐2-­‐1.   L13:   posiAve   control,   MAT1-­‐1-­‐1.   L3-­‐11:  each  lane  represent  a  single  ECM.  L12:  negaAve  control.  L1:   Gene  Ruler  DNA  ladder  mix  (Fermentas).