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Physiological  and  molecular  responses  of  lodgepole  and  jack  pine  trees  under   environmental  stress  to  the  blue  stain  fungus,  Grosmannia  clavigera   Adriana  ARANGO-­‐VELEZ,  Jill  Hamilton1,  Miranda  Meents1,  Dominik  Royko1,     L.  Irina  Zaharia2,  Janice  E.K.  Cooke1   1University  of  Alberta,  Department  of  Biological  Sciences,  Edmonton  AB  Canada  T6G  2E9  

2  NRC  Plant  Biotechnology  InsGtute  (NRC-­‐PBI)  /  CNRC-­‐InsGtut  de  biotechnologie  des  plantes  (CNRC-­‐IBP),  Saskatoon,  Saskatchewan  S7N  0W9  

Results  &  Discussion    

Our  objecNve  was  to  evaluate  the  influence  of  water  deficit  in  tree  responses  to  G.   clavigera,  and  idenNfy  differences  in  consNtuNve  and  induced  defenses  between   lodgepole  and  jack  pine.    

Experiments  

20  

10  

10  

0  

14  dpi   35  dpi   56  dpi   68  dpi   200  

150  

150  

100  

100  

20  dpi  

50  dpi  

InoculaNon  

Seedlings:  Second  year  seedlings  of  lodgepole,  and  jack  pine  were  grown  in  Sunshine   growing  media  4,  and  maintained  under  controlled  condiGons  as  follows:  19°C  (day/ night),  20-­‐35%  RH,  and  photoperiod  15h  day  light  (200  µmol  PAR)  and  9h  night.  A  total   of    4  independent  experiments  with  8  biological  replicates  per  experiment  were   sampled  across  treatment  combinaGons.     Mature  trees:  Natural  stands  of  jack  and  planted  lodgepole  pines  trees  located  in   Smoky  lake  Alberta  were  used.  Ten  trees  of  similar  diameter  breast  height  (dbh)  were   randomly  selected  for  each  treatment  combinaGon.   A  two  by  three  factorial  design  with  two  treatment  factors,  water  level  and  fungi   inoculaNon  with  G.  clavigera  was  performed.    

Physiological, biochemical & transcriptional analyses: Seedling  collecNon:  1,  7,  14,  28,  35,  56  and  68  days  post  inoculaNon  (dpi)   Mature  tree  collecNon:  20,  28,  50  and  70  dpi  

A  

B  

10  mm  

Well-­‐watered   Water  deficit  

C  

Fig.  2.  (A)  InoculaGon  and,  (B)  Lesion  length  of  mature  trees  aeer  five  weeks  of   inoculaGon.  (C)  Lesion  developed  in  pine  seedlings  aeer  28  days  post  inoculaGon.    

Acknowledgements  

We  thank  Leonard    Barnhardt  for  providing  the  experimental  field  site  at  Smoky  lake  (AB),  and  also  for  his   valuable  help  during  the  experiment.  We  also  want  to  thank  Dr.  Walik  El  KLeayal  and  Charles  Copeland  for   their  help  with  this  project,  and  Marlena  Muskens  for  her  help  with  the  microscopy  analyses.  

Control  

Inoculated  

Control  

Inoculated  

Control  

Inoculated  

Control  

Inoculated  

Jack  pine  

70  dpi  

Well  watered   Water  deficit  

Fig.  4.  Decreases  in  photosynthesis  under  water  deficit  treatment  indicates   a  reduced  in  carbon  gain,  therefore  we  hypothesize  that  this  will  influence   carbon  allocaNon  to  defense.    

Lodgepole  pine  

Jack  pine  

Jack  pine  

PB   Xylem  

S   PP  

Xylem  

C  

TD  

R  

PP   S   C  

10X  

10X  

R=rays,  C=cambial  zone,  TD=  traumaGc  resin,  S=sieve  cells,  PP=polyphenolic  parenchyma  cells,  PB=phenolic  bands.    

Fig.  5.  Anatomical  changes  of  inoculated  pine  seedling  under  water  deficit   condiGons.  InoculaGon  resulted  in  swollen  polyphenolic  parenchyma  (PP)   cells  in  lodgepole  pine,  and  development  of  addiGonal  phenolic  bands  (PB)   in  both  species,  indicaNng  an  induced  defense  against  G.  clavigera.    

Fig.  6.  Hydraulic  conducGvity  (Kstem)  as  a  measure  of  water  transport,  was   significantly  decreased  in  lodgepole  pine,  possibly  due  to  fungal  growth,   fungal  secondary  metabolites,  or  tree  secondary  metabolites  occluding   tracheids.     Lodgepole  pine  

JA  (ng/g  FW)  

• Water  deficit    (WD)    (<18  RWC)  

Fig.  1.  Experimental  set  up  for  lodgepole  (A)  and  jack  mature  pines  (B),  and  seedlings   (C).  Water  deficit  was  induced  by  placing  woven  laminated  polyethylene  tarps.  

50  dpi  

TD  

30  

30  

Lodgepole  pine  

3008  Genes  

Jack  pine  

2385  Genes  

2851  Genes  

2574  Genes  

20  

20  

10  

10   n.d.  

0  

Control  

SA  (ng/g  DW)  

• Well  watered  (WW)  (>40  RWC)  

20  dpi  

70  dpi  

Fig.  3.  Lesion  length  is  generally  associated  with  fungal  growth  and/or  the   response  to  the  fungi.  Under  well-­‐watered  condiNons  lesions  were  longer   in  lodgepole  pine  relaNve  to  jack  pine,  and  its  development  is  slowed   under  water  deficit.  However,  aeer  a  longer  exposure  to  the  fungi,  water   deficit    trees  developed  longer  lesions,  which  could  be  a  higher  fungal   development  and  a  delayed  tree  response.  

R  

•     Wound  (W)  –Seedlings-­‐   •     Wound/G.  clavigera  (F)   •     Control  (C)  

Mature  trees  

0  

0  

C  

Water  availability  

14  dpi   35  dpi   56  dpi   68  dpi  

50  

50  

PB  

B  

7  dpi   250  

200  

Lodgepole  pine  

Seedlings  

0  

7  dpi   250  

Lodgepole  pine  

A  

Jack  pine  

30  

20  

A  (μmol  m-­‐2s-­‐1)  

Lodgepole  pine  

30  

Lesion  length  (mm)  

Background   Conifers  exhibit  both  consGtuGve  and  induced  defense  mechanisms  in  response  to   aSack  by  pests  and  pathogens,  such  as  mountain  pine  beetle  (Dendroctonus   ponderosa  Hopkins)  and  their  fungal  associates.  However,  this  defense  can  be   modulated  by  environment,  where  abioGc  stress  may  reduce  the  species  ability  to   respond  to  bioGc  stress.  In  this  study,  we  examined  the  relaGve  influence  of  water   limitaGon  on  consGtuGve  and  induced  defenses  of  lodgepole  pine  (Pinus  contorta   Dougl.  ex  Loud.  var.  la1folia)  and  the  naïve  host  jack  pine  (P.  banksiana  Lamb.)  to   inoculaGon  with  the  mountain  pine  beetle  fungal  associate  Grosmannia  clavigera.   Molecular  and  biochemical  assays  evaluated  inducible  defense  responses  of  jack  and   lodgepole  pine,  in  response  to  inoculaGon  with  G.  clavigera  at  1,  7,  and  28  days  post-­‐ inoculaGon  (dpi).    

0  

Wound  

Inoculated  

400  

400  

300  

300  

200  

200  

n.d.  

n.d.  

Control  

Wound  

WD+F  vs  WD+C  

WW+F  vs  WW+C   Inoculated   Jack  pine  

*  

 4299  Genes  

4014  Genes  

4017  Genes  

4296  Genes  

100  

100   0  

0   Control  

Wound  

Inoculated  

Control  

Wound  

Inoculated  

*Significantly  different  at  p<0.05  

Fig.  7.  Jasmonic  acid  (JA)  and  salicylic  acid  (SA)  are  important  for  defense   signaling  in  response  to  insects  and  pathogen  ahack.  Fungal  inoculaGon   results  in  increased  JA  accumulaGon  in  both,  jack  and  lodgepole  pine.  SA   increased  significantly  in  jack  pine  under  fungal  inoculaGon  in  water  deficit   treatment,  probably  as  an  acNve  defense  mechanism.  

WW+F  vs  WW+C   1  28  1 7

7

WD+F  vs  WD+C   7 28  1  28  1

days  post  inoculaNon  

7

28  

Fig.  8.  IdenGficaGon  of  differenNally  expressed  genes  showed  an  early  (1   and  7  dpi)  inducGon  of  genes  associated  with  defense-­‐related  processes   such  as  terpenoid,  pehenylpropanoid,  and  phenolic  compound   biosynthesis,  cell  wall  modificaNon  and  hormone  signaling.    

Conclusions   Fine-­‐scale  differenNaNon  among  funcNonal  categories  of  differenNally  expressed  genes  point  towards  species-­‐specific  strategies  associated  with   defense.  These  data  suggest  that  water  limitaNon  modulates  defense  response,  and  abioNc  and  bioNc  factors  may  interact  to  influence  the  transiNon  of   MPB  and  associated  pathogens  from  lodgepole  pine  to  jack  pine.      


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