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A morphological and cytological study of Audouinella porphyrae and A. vaga (Rhodophyta) Tam, Carol Elizabeth 1985

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A MORPHOLOGICAL  AND CYTOLOGICAL  STUDY OF AUDOUINELLA AND A. VAGA  PORPHYRAE  (RHODOPHYTA) By  CAROL ELIZABETH TAM B.  SC., The U n i v e r s i t y  of B r i t i s h  Columbia,  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE. in THE  FACULTY OF GRADUATE (Department  We a c c e p t t h i s to  THE  STUDIES  of Botany)  thesis  the required  as conforming standard.  UNIVERSITY OF BRITISH COLUMBIA August, © Carol  1985  Elizabeth  Tarn, 1985  1982  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree a t the  the  University  o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may department o r by h i s o r her  be granted by the head o f representatives.  my  It i s  understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be  allowed without my  permission.  Department o f The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T  1Y3  written  ABSTRACT  A comparative  Audouinella  endophytes, vaga  s t u d y was  porphyrae  (Drew) G a r b a r y ,  A c r o c h a e t i a l e s ) and  in  stellate  their  host  their  "free"  may  from  features  cell not  their  and  forms.  monospores was either  A.  on  a  vaga  and  dimensions,  extensive prostrate portions  in  forms  do  branching i n the  r e p r o d u c t i o n was  The  were n o t  b l a d e s of Porphyra  field  the f r e e - l i v i n g  forms  of t h e  for re-infection  growth.  Pterosiphonia  cultured  two  r e p r o d u c t i o n w i t h r e g e n e r a t i o n by  Sexual  were u s e d  o u t s i d e the  significantly  and  Under a l l e x p e r i m e n t a l c o n d i t i o n s ,  and tort  differ  reside  cytological  p a t t e r n s are i d e n t i c a l  b o t h of the endophytes.  porphyrae  growing  Cell  and have  were  were compared w i t h  endophytes  Asexual  showed o n l y e p i p h y t i c on  portions  endophytes  branching patterns,  observed.  forms  experiments.  Porphyra  forms  endophytic or f r e e - l i v i n g  Free-living  effect  The  spp.  p y r e n o i d and  m o r p h o l o g i c a l and  t h e two  p a t t e r n s and d e v e l o p m e n t a l  in  producing erect  these d i f f e r e n c e s .  free-living  algal  Audouinella  Both endophytes  with a central  free-living  dimensions retain  walls,  h o s t s and  Although  red  and  h o s t s Porphyra  respectively.  bear monosporangia.  of t h e  material.  (Drew) G a r b a r y  red a l g a l  chloroplasts  host's c e l l  that  similar  Hansen e t S c a g e l , ( A c r o c h a e t i a c e a e ,  Pt er os i phoni a bipinna:a, axial,  made between two  not  observed  endophytes. cross-infection the  endophytes  h o s t s seem t o have some Audouinella  Epiphytes,  selective bipinnata.  tended  and  grew on b o t h h o s t s , Both  t o b r a n c h and  (3-5 c e l l s )  epiphtyes  have more  whereas b o t h e p i p h y t e s  -i i i -  on Pt erosiphoni cells  a bi pi nnat a t e n d n o t t o b r a n c h and have o n l y  i n the p r o s t r a t e  portions.  both endophytes  showed t y p i c a l  Ultrastructural  features  were s i m i l a r a n d f r e e - l i v i n g , to  field  the this  material.  field  material  U l t r a s t r u c t u r a l s t u d i e s of  florideophycean  of f i e l d  material  cultured  A l a r g e v a c u o l e was of both p r o s t r a t e  of t h i s  conspecific.  endophytes  Audouinella  porphyrae  vaga  endophytes  were s i m i l a r  o b s e r v e d i n s e c t i o n s of  and e r e c t  study i t i s proposed that  Audouinella  features.  of t h e two  was n o t o b s e r v e d i n c u l t u r e d m a t e r i a l .  results  1-2  portions  Based  whereas  on t h e  t h e two e n d o p h y t e s a r e  i s r e f e r r e d t o synonymy i n  (Drew) G a r b a r y , Hansen e t  Scagel.  - i v-  Table  of C o n t e n t s : Page  Abstract  ii  List  of Tables  vi  List  of F i g u r e s  v i i  Acknowledgements  ix  Dedication  x  Introduction  1  Materials  8  a n d Methods  Field  material  8  Cultures  10  Establishing  Cultures  D e v e l o p m e n t a l and L i f e Re-infection Light  of F r e e - l i v i n g History  E n d o p h y t e s . . . . 10  Studies  and C r o s s - i n f e c t i o n S t u d i e s  and E l e c t r o n M i c r o s c o p y  Studies  16 material  16  Audouinella  porphyrae  16  Audouinella  vaga  17  Development  o f E n d o p h y t e s : Audouinella Audouinella  Cultured  porphyrae  20  vaga  22  material  Establishment Infection Light  12 13  Results Field  11  23 of F r e e - l i v i n g  Experiments  and E l e c t r o n M i c r o s c o p y  Endophytes  23 26 28  Discussion  33  References  44  -  Figures  V-  52  - vi-  List  of Tables  Table I.  Page Similarities porphyrae  and d i f f e r e n c e s between  a n d Audouinella  vaga  Audouinella  according  to  G a r b a r y e t a l . (1982) II.  III.  Monthly  field  species  o f Porphyra  Measurements  bipinnata V.  Cell and  VI.  collections  o f A. porphyrae  o f Porphyra, o f A.  from  in situ,  and g e n e r a l  vaga  field  growing  i n i t s host  host  field  f e a t u r e s . . . 19  Pi erosi  phoni  collections  d i m e n s i o n s and b r a n c h i n g A. vaga  in various 18  D i m e n s i o n s o f A. porphyrae collections  IV.  7  from P o i n t  a  21  patterns  o f A.  porphyrae  No P o i n t  24  Summary o f d i f f e r e n c e s and s i m i l a r i t i e s o f morphological, of  field  A.  vaga  reproductive  and c y t o l o g i c a l  and c u l t u r e d A u d o u i n e l l a obtained  i n the current  porphyrae  study  characters and 32  — vii —  List  of F i g u r e s  Figures  Page  1.  Diagrammatic drawings of h o s t s  52  2-5.  Light micrographs of f i e l d  54  6-9.  Komarski c o n t r a s t  10-14.  Development  15-17.  L i g h t micrographs of basal of  micrographs of f i e l d  of spores  material  from c u l t u r e d m a t e r i a l and e r e c t  Nomarski c o n t r a s t  22-25.  Host m a t e r i a l  26-34.  Infected  filaments  Audouinella  58  micrographs of c u l t u r e d  uninfected  material  material..58  by e n d o p h y t e s  o f Pt erosi  60  phoni a bipinnata  by  porphyrae  Infected material  62  o f Porphyra  torta  Audouinella  by  vaga  38-4-1.  42-46.  sections  o f A.  porphyrae/Porphyra  col a  Methacrylate fuci  47-50.  64  Methacrylate fuci  66  sections  o f A.  porphyrae/Porphyra  col a  Methacrylate  68  sections  o f A.  vaga/Pl  erosiphonia  bi pi nnat a  51-53.  Methacrylate  70  sections  o f A.  vaga/Pt  er os i phoni a  bi pi nnat a  54-56.  U l t r a s t r u c t u r e o f A. fuci  57-60.  72  porphyrae  in  Porphyra  col a  U l t r a s t r u c t u r e of uninfected fuci  56 56  cultured material  18-21.  35-37.  material  col a  74  Porphyra 76  -viii -  61-64.  Ultrastructure fuci  65-68.  o f A. porphyrae  in  Porphyra  col a  Ultrastructure  78 o f A.  vaga  i n Pt erophoni  a  bi pi nnat a  80  69-71.  Ultrastructure  of f r e e - l i v i n g  A. porphyrae  82  72-75.  Ultrastructure  of f r e e - l i v i n g  A.  84  vaga  i X -  -  Acknowledgements I am i n d e b t e d t o D r . K. C o l e f o r a l l o f h e r wisdom, guidance  and f i n a n c i a l  support throughout  constant  encouragement a n d e n t h u s i a s m  t h r o u g h my y e a r s a s a g r a d u a t e thank  D r . D. G a r b a r y  introducing  were g r a t e f u l l y  thesis.  student.  I would a l s o of t h i s  of Phycology.  Editorial  for  a l l o f h e r h e l p and a d v i c e i n t h e l a b o r a t o r y .  Warm g r a t i t u d e  a l s o expressed to Kermit  knowledge t h r o u g h o u t Morin  comments  my t h e s i s .  f o r drawing  i s e x p r e s s e d t o Bev. Hymes  Ritland  figure  V a n d e r m e u l e n , Bev. Hymes, E l l e n  d u r i n g many c o l l e c t i n g a c k n o w l e d g e Mr. L . V e t o  trips  Appreciation  f o r h i s s u p p o r t a n d computer  A special one.  Rosenberg,  to Point  for technical  thanks  Thanks a l s o  Borgmann, Sandy L i n d s t r o m a n d L a r r y G o l d e n  laboratory.  and f o r  a c c e p t e d f r o m D r . K. C o l e , D r . D. G a r b a r y a n d  P. G a b r i e l s o n .  Karen  like to  project,  Dr.  is  Her  were g r e a t l y a p p r e c i a t e d  f o r the i n i t i a t i o n  me t o t h e f i e l d  this  i s given to go t o H e r b .  Dawn R e n f r e w , I r a for their  company  No P o i n t .  I would  like to  assistance  i n t h e E.M.  -  Dedicated Mom,  with  Dad,  X  -  l o v e t o my  Stella  and  family: Glory.  -  -1  INTRODUCTION Red  a l g a e may  g r o w i n g on inside  the  found  g r o w i n g on  s u r f a c e s of a n i m a l s  the c e l l  non-organic  be  parasitically,  (epizoic),  w a l l s of o t h e r a l g a e  substrata. or  just  and  a l g a e may  or w i t h i n t h e h o s t  as w e l l a s  cell  walls  t h e p h y s i o l o g y or m o r p h o l o g y of t h e h o s t  symbiotic  a s s o c i a t i o n s between p a r a s i t i c  the  relationship  algal  h o s t s has  between r e d a l g a l not  Traditionally, classified  "obligate"  many r e d a l g a l  as d i s t i n c t  a particular  host  entities  (White  interactions  and  m a t e r i a l s and  to  analysis.  Audouinella  (Bory)  cross-infection host-specific 1979b).  as  (Batters) Dixon,  (Ellis)  from  Boney  and  A.  (White ( 1969,  1970)  as paragopsis  Bonnemai  Boney,  observations  these  not  1969,  subjected  and be  1970;  as Garbary,  endophyt  (Chemin) D i x o n  dimensions  of  some s p e c i e s o f  s t u d i e d A.  soni a hamifera  with  These  h o s t s , Het erosiphonia  They measured c e l l compared  been  using r e - i n f e c t i o n  and  red  1983).  recently  C u l t u r e s t u d i e s of  but  association  1970).  inferred  et I r v i n e ,  presumed  B a t t e r s , and  h o s t s and  1969,  have been o n l y  Naegeli), in their  respectively. their  Dixon  (Goff,  their  their  their  s t u d i e s , have shown t h a t t h e y may  W h i t e and  Acrochaetium  Boney,  on  Many  1982),  e n d o p h y t e s have  based  have been  field-collected experimental  see G o f f ,  e n d o p h y t e s and  without  cells.  r e d a l g a e and  r e c e i v e d much a t t e n t i o n  on  grow  affecting  h o s t s have been w e l l s t u d i e d ( f o r r e v i e w  (epiphytic), partially  (endophytic)  Epi/endophytic on  other algae  i ca  (both  as  plumosa  Hariot, of e n d o p h y t e s i n  with c u l t u r e d ,  "free-living"  -2-  endophytes c o n c l u d i n g  that  They a l s o  the  not  showed t h a t  show any  substrate  Garbary Audouinella,  A.  tetraspora  as  an  A.  A.  endophyt  i ca,  species  larger  the  host  of  branch l e s s f r e q u e n t l y .  and  He  endophytically  i t s m o r p h o l o g y was  field  material  emphasised before  e n d o z o i c members of To  contribute  relationships, species  of  the  Audouinella,  that  of  the  one  site  Garbary  forms were  cell  importance  forms  tended  d i a m e t e r , and  A.  endophyt  He  i ca  to  grew  speculated  m o r p h o l o g y of  of  that the  comparing c u l t u r e  of  endophytic  and  red a l g a l  cultured material A.  porphyrae Scagel  of  and  and  (Rhodophyta,  w i t h Porphyra  in B r i t i s h  endophyte/host  two  endophytic  (Drew) G a r b a r y and  spp.  ( P o s t e l s et Ruprecht) Falkenberg, at  shape,  when g r o w i n g  altered.  knowledge of  Acrochaetiaceae) associated  studied  only  of alternate  free-living in c e l l  He  Acrochaetiaceae.  t o our  field  The  well  and  free-living  delimiting species  (Drew) G a r b a r y , Hansen e t  bipinnata  and  h o s t s and  hosts caused m o d i f i c a t i o n s  original  s i z e and  found that  i n t o given  e n d o p h y t e and  the  Dixon,  h o s t s as  some d e g r e e  smaller  endophytically  the  do  of  (Batters)  as par agopsis.  cell  endophytic  length  species  soniae  A.  (both  different species.  in c e l l  species.  h o s t s and  from r e d a l g a l  from h y d r o i d s  observations  that  other  endophytic  a l l isolates established  B a s e d on  morphologically  infect  bonnemai  Garbary et Rueness,  that  were d i s t i n c t  preference. several  (1979b) n o t e d  t o be  taxa  endophytes  r e l a t i o n s h i p with a given hosts).  two  (1979b) s t u d i e d  endozoic  concluded  the  Columbia.  A.  vaga  Acrochaetiales, and  Pt erosi  phoni a  r e s p e c t i v e l y , were  -3-  Members o f t h e A c r o c h a e t i a c e a e branched 20  filaments.  urn t o 10 mm  and t h e i r  g r e e n and v i o l e t distinguished carpogonial that  The a l g a e  taxa  marine.  to generic  incorporating 20th century  absence of  growth of u n i s e r i a t e f i l a m e n t s  t o many d i f f e r e n t  Although  niches.  and e i t h e r  epibiotic  classification,  with  over  1983).  a dozen Criteria  complete  sexual  life  history  o f t h e 390 d e s c r i b e d collected  at least  24 d i f f e r e n t  schemes employed f o r generic histories  and c e l l  large  morphological  about  1969) a n d / o r 1980).  Four -  g a m e t o p h y t e and t e t r a s p o r o p h y t e ;  2)  a diminutive  g a m e t o p h y t e and a  3) D i p h a s i c , d i m o r p h i c  and no c a r p o s p o r o p y t e  a diminutive  A  a r e known: 1) T r i p h a s i c , d i m o r p h i c  trimorphic - with  tetrasporophyte - with  histories  tetrasporophyte;  morphology.  Many s p e c i e s a r e known o n l y t o  1979; G a r b a r y and R u e n e s s ,  Triphasic,  include  from o n l y  tetrasporangia  similar  s i n c e the e a r l y  segregation  has been r e p o r t e d  schemes  s i n c e 1970  ( W h i t e and Boney,  sexual  With  s p e c i e s o f w h i c h 7 s p e c i e s a r e from  populations.  (West,  most  or e n d o b i o t i c .  produce monosporangia  with  Different  some s p e c i e s a r e e p i l i t h i c ,  of c h l o r o p l a s t s , l i f e  distinct  thalli.  h a s e x p l o i t e d a wide r a n g e of  features  field  Acrochaetiales i s  one t o e i g h t g e n e r a have been u s e d  (Woelkerling,  23  blue t o  i n f r e s h a n d m a r i n e h a b i t a t s ; however, t h e y a r e  are symbiotic  regard  The o r d e r  approximately  steel  i n t o pseudoparenchymatous  e n v i r o n m e n t s and a d a p t e d s p e c i e s occur  from  r a n g e s from d a r k  branches, and a p i c a l  g r o u p of a l g a e  primarily  i n height  by t h e two l a y e r e d p i t p l u g s ,  a r e not e l a b o r a t e d This  vary  colour  red to rose.  are small, uniseriate  - with  a large  and 4) D i p h a s i c ,  carpotetrasporophyte  and no  dimorphic  tetrasporophyte,  - 4-  (for  more d e t a i l s ,  life  history  characters  see W o e l k e r i n g , 1983).  s t u d i e s and  B e c a u s e of t h e  inadequate d i s t i n c t  i t is difficult  to u t i l i z e  these  few  morphological f e a t u r e s as  generic  criteria. Drew be  (1928) p r o p o s e d t h a t  included  i n t h e genus Rhodochorton  that  chloroplast  used  to d i s t i n g u i s h  and  sporangia genera.  (1979a) a l l c o n c u r r e d should  be u s e d .  Audouinella  only  ( 1 9 4 5 ) , Feldmann,  patterns:  germination  proposed the thesis  I also  These  also continue  of c h a r a c t e r s  include:  1) b a s a l  t o be  used.  i s used t o systems: uni-or  structure: parietal cell:  (1979), Bold  vs  stellate,  d i a m e t e r and  length,  irregular,  secund or o p p o s i t e ,  or e p i l i t h i c ,  7) m o r p h o l o g y ,  6)  9) m o r p h o l o g y  size,  o f g a m e t a n g i a and  and G a r b a r y ( 1 9 8 4 ) .  5)  habitat: shape  8) s p o r e  10)  development. For r e v i e w of c h a r a c t e r s n i n e  see Hansen  genus  (Garbary et a l . ,  of monosporangia and/or t e t r a s p o r a n g i a ,  pattern,  fertilization  system  o r a b s e n t , a n d one o r more i n number,  endobiotic  and c l u s t e r i n g  ten,  variants  2) c h l o r o p l a s t  4) p y r e n o i d : p r e s e n t  epibiotic,  genus  A l t h o u g h many  t o many i n number, 3) m a t u r e  branching  a one  In t h i s  classification  a variety  species.  multicellular,  name.  (1977) and G a r b a r y  ( 1 9 6 2 ) , S t e g e n g a and van W i s s e n  Audouinella  differentiate  however,  t o be  1983), t h e o p p o s i n g schemes o f P a p e n f u s s  (1985) and t h e i r  Within  and one  this  Irvine  (1928) t h a t  genus Audouinella.  Woelkerling,  and Wynne  D i x o n and  i t i s the o l d e r  one  She s u g g e s t e d  f e a t u r e s were t o o v a r i a b l e  w i t h Drew  p h y c o l o g i s t s agree with 1982;  (Naegeli).  D i x o n and I r v i n e ,  since  recognize  a l l members of t h e A c r o c h a e t i c e a e  post and  -  The thesis  two  species  of  endophytic  Audouinella  have been p l a c e d  in several  d i f f e r e n t genera.  (1944) t r a n s f e r r e d i n t o Acrochaetiurn Similarly.,  Jao  Rhodochorton Rhodochor  because  p r e s e n c e of  Acr ochaet  porphyrae  a  Acrochaetiurn  material  classification  of  Acrochaet  i urn vagum  stellate  chloroplast  species  characters  concept  and  both  S t e g e n g a and t o Chromastrum  species  (1945)  porphyrae,  a number of  found p r i m a r i l y  urn b a s e d i n the  Mexico  from  made  the  on  (1947) a g a i n replaced Mulder  the  supposed  described  revised  Chromastrum  (1979)  b a s e d on  the  However, due  now  referred  the with  transferred p r e s e n c e of  to a  chosen  the  to  the  retained  original  species  i n the  Audouinella  an  a  similar  lack one  of  to  stable  genus  Audouinella  mid-  e n d o p h y t e w h i c h grows i n t h e  of Porphyra to  lower  porphyrae  (Bangiophyceae),  intertidal  in species  of  w i d e l y d i s t r i b u t e d , i n western North America Alaska,  Drew  transferred  However, he  (1979a) has  are  vagum  h i s t o r y presumed t o be  rum.  Garbary  on  a l . , 1982).  Audouinella  Endophytic  and  life  monosporangia.  (Drew) P a p e n f u s s , b a s e d on  Papenfuss  and  Rhodochorton  P a p e n f u s s , and  i n Acrochaeti  of Chromast  generic  produced  in t h i s Smith  Drew f r o m  Papenfuss  t h i s family  studied  Rhodochort  chloroplast.  1928).  Rosenvinge.  of  i urn.  porphyrae  Jao  Kylinia  walls  plants  parietal chloroplast  (Drew,  (Garbary et  the  porphyrae  t o Chromastrum  stellate vagum  p r e s e n c e of  on  (1937) t r a n s f e r r e d  to t on  Rhodochort  c o m b i n a t i o n Chromastrum  other  5-  U.S.A.  (Hansen e t  (Garbary et  1976), A u s t r a l i a  a l . , 1981)  a l . , 1982), New  (Woelkerling,  1971)  cell  is  regions. Porphyra  are  from Yakutat  Bay,  to Baja, C a l i f o r n i a ,  Zealand and  ( S o u t h and  T r i s t a n da  Adams,  Cuhna  -6-  (Baardseth,  1941).  Audouinella genera,  vaga,  Pt er osi phoni a,  to C a l i f o r n i a  similar  similar  stellate  a n d A.  (Garbary  might  species  (Table  be c o n s p e c i f i c ,  being a r e f l e c t i o n  i s r e p o r t e d from  (Garbary  Alaska  e t a l . , 1982).  are morphologically  e t a l . , 1982).  and  B o t h have  cells  t h a t c o n t a i n s one p y r e n o i d ,  germination.  p a t t e r n s , presence  t e t r a s p o r a n g i a , Garbary  different  a,  vaga  chloroplast  p a t t e r n s of spore  in hosts, branching and  a n d Pol ysiphoni  porphyrae  ontogenetically with a s i n g l e  i n two f l o r i d e o p h y c e a n  b u t i s unknown e l s e w h e r e  Audouinella  and  an e n d o p h y t e  B a s e d on d i f f e r e n c e s  or absence of h a i r  e t a l . (1982) m a i n t a i n e d  1), but suggested  the morphological  cells  them a s  t h a t t h e two t a x a  differences  between  them  of t h e i n f l u e n c e o f t h e h o s t s on t h e i r  development. Using  culture  comparative  study  Audouinella  vaga.  morphological  s t u d i e s and l i g h t was i n i t i a t e d  on Audouinella  microscopy,  porphyrae  and r e p r o d u c t i v e f e a t u r e s of both  m a t e r i a l , 2) l i f e  unialgal  culture,  histories  a  and  The f o l l o w i n g f e a t u r e s were i n v e s t i g a t e d :  cultured  their  and e l e c t r o n  1)  f i e l d and  of the endophytes i n  3) t h e r e l a t i o n s h i p between t h e e n d o p h y t e s and  h o s t s a n d 4) t h e q u e s t i o n o f c o n s p e c i f i c i t y .  Table I :  Similarities Audoui  net I a  and D i f f e r e n c e s between Audouinella porphyrae a c c o r d i n g t o G a r b a r y e t a l . ( 1982).  and  vaga  A.  porphyrae  vaga  Similarities: Growth p a t t e r n Chloroplast Pyrenoid Spore g e r m i n a t i o n  I n s i d e host c e l l w a l l Single, axial, stellate One Internal division  I n s i d e host c e l l w a l l Single, axial, stellate One Internal division  Differences: Host C e l l length Tetrasporangia Hair c e l l s Branching pattern  Porphyra  Spp.  18-25pm Unknown R e p o r t e d t o be r a r e Irregular  Pi er ai  phoni  a  sp.  L e s s than 16pm R e p o r t e d t o be r a r e Unknown At r i g h t a n g l e s  -8-  MATERIALS AND Field  Material  Monthly Point  field  No P o i n t  t r i p s May  (48° 23'N,  Audouinella  vaga  collected  on  123° 59'W).  v a r . vaga  (48° 28'N, 123° O T W )  San J u a n  Island,  sites  Audouinella of Porphyra to  bipinnata),  vaga  porphyrae  the  field.  this  for light  i n the  (UBC) c o n t a i n i n g previous  infected  i n the h o l d f a s t  by r e d p a t c h e s .  region  C a r e was t a k e n  s p p . when t h e s e were c o l l e c t e d . s p p . a n d Pt  t o be d e s i c c a t e d  and e l e c t r o n  of f i e l d  requires  Columbia  t e n d s t o grow  Some were r e h y d r a t e d  contrast,  nnat a  usually  (48° 27'N 2 2 ° 58'W)  Collections  i n large  erosiphonia  following  i n t o the laboratory  were k e p t d r y a n d c o l d  bipi  were  were examined t o d e t e r m i n e  (Porphyra  specimens  t i d e , were b r o u g h t  In  of B r i t i s h  o f Porphyra  low  examination  vaga)  and  and d a t e s o f c o l l e c t i o n .  which tended  overnight  A.  British  C o l l e c t i o n s were a l s o made a t E a g l e  s p p . and i s d e t e c t e d  host  nelI a porphyrae  from d i f f e r e n t s i t e s .  remove h o l d f a s t s  Infected  Island,  W a s h i n g t o n , U.S.A., i n May 1984, i n o r d e r t o  a n d A.  collection  Audoui  and C a t t l e P o i n t  herbarium of the U n i v e r s i t y porphyrae  1985, were made t o  of Vancouver  (henceforth  a t t h e same t i m e .  compare m a t e r i a l  A.  1984 - A p r i l  on t h e west c o a s t  Columbia  Cove  METHODS  exposure a t  on i c e d i r e c t l y quantities  microscopic  studies,  from  of seawater and o t h e r s  i n t h e r e f r i g e r a t o r (4°C) f o r f u r t h e r  characters.  i t i s not possible by Audouinella  microscopic  t o i d e n t i f y Pterosiphoni  vaga  examination  with  a  the u n a i d e d eye;  (Fig. 4).  Consequently,  -9-  several sites  h a n d f u l s o f P.  i n the mid-  t o lower  Audouinella  species  chloroplast  shapes  of  A.  vaga.  arcuata  bi pi nnat a were c o l l e c t e d  Such  intertidal on P.  epiphytic and  growth  bipi  A.  a  bipinnata  but  Audouinella  Porphyra  included:  (cell  of  maximal e x t e n t o f the endophyte outside and  fully  t h e h o s t and  the endophyte  a n a l y s i s was  square t e s t  to v e r i f y  in  t h e two  ( S o k a l and  used  i t s signifance  A.  porphyrae  distance  of  growth  A linear  statistical  regression  analysis  t o t h e a r e a of p r i m a r y 1973).  Secondary Another  of independence  and a C h i  ( S o k a l and R o h l f ,  and A.  vaga  of c e l l from  1973).  A  dimensions  field  material  1973).  F o r permanent c o l l e c t i o n s , dried  in  filament;  e x t e n t of  in this analysis. test  of a  t y p e s of  t o compare t h e s i g n i f i c a n c e  species, Rohlf,  a 2X2  the  endophytes  cells  The  by  n a t u r e of both the h o s t  o f Porphyra  were n o t c o n s i d e r e d  was  and  infection  w i d t h ) ; and  Three  ( S o k a l and R o h l f ,  statistical  "t-test"  noted.  to the d a t a .  the s i z e  infection  infections  ( l e n g t h and  the r e p r o d u c t i v e  were a l s o  used t o r e l a t e  endophyte  elongated apical  from the h o s t ' s h o l d f a s t .  a n a l y s e s were a p p l i e d was  of  with d i s s e c t i n g  Measurements o f t h e  infection  A.  1982).  d i m e n s i o n s ) l e n g t h and d i a m e t e r o f c e l l s  p r i m a r y a x e s and  those  thuretii  s p e c i e s and  were examined  species.  from  (Drew) G a r b a r y ,  compound m i c r o s c o p e s t o d e t e r m i n e t h e e x t e n t of two  their  S c a g e l and A.  (Bornet) W o e l k e r l i n g (Garbary et a l . ,  Pt er os i phoni  nnata,  densa  (Drew) G a r b a r y , Hansen and  specimens  There are other  patterns are d i f f e r e n t  taxa include:  Some i n f e c t e d  zone.  f r o m a number of  on h e r b a r i u m s h e e t s .  i n f e c t e d Porphyra  P o r t i o n s of  b l a d e s were  i n f e c t e d Pterosiphoni  a  bipi  nnat  a  plants  were mounted on s l i d e s i n 30% K a r o .  material  was p r e s e r v e d  possible  the  Porphyra  species  specimens of f i e l d British  Columbia  i n 5% f o r m a l i n / s e a w a t e r  material  were  phycological  a n d where  identified.  were d e p o s i t e d herbarium  Other  Voucher  i n the U n i v e r s i t y of  (UBC).  Cultures Establishing  cultures  Some h e a v i l y free-living millimeter blades of  of f r e e - l i v i n g  i n f e c t e d host  endophytic  s p e c i m e n s were u s e d t o e s t a b l i s h  c u l t u r e s of the endophytes  (Table  d i s c s of n o n - r e p r o d u c t i v e , h e a v i l y  from c l o s e  PES medium  t o the h o l d f a s t  region  ( P r a v o s o l i , 1968) w i t h  then  II).  (5-10 mg/1) G e 0  5-6 m i c r o e i n s t e i n Conditions  m~  2  were c h o s e n  (L:D),  s e c " i n PES medium 1  to simulate  field  Porphyra  infected  1966).  t o e n s u r e no e p i p h y t e s were p r e s e n t .  k e p t a t 6°C 16:8 h L i g h t : D a r k  Ten  were p l a c e d  d i a t o m g r o w t h on t h e h o s t s t h a l l u s ( L e w i n , screened  species.  i n 30-40 ml to stop  2  Discs  were  The m a t e r i a l  photon  f l u x d e n s i t y of  t o induce  sporulation.  conditions.  Some  c u l t u r e s were a l s o m a i n t a i n e d a t 10°C 8:16 h (L:D) p h o t o n density  o f 4-5 m i c r o e i n s t e i n s  m"  2  s e c " i n PES medium. 1  s p o r e s were o b s e r v e d on t h e b o t t o m o f c u l t u r e d i s h e s , material plastic  was removed. dishes,  containing Audouinella  unialgal  S p o r e s were t h e n grown  with or without  75 ml o f medium. porphyrae  stock flux  When host  i n 60 X 20 mm  c o v e r s l i p s , a n d i n 125 ml f l a s k s  R e - i s o l a t i o n o f s p o r e l i n g s of  i n t o new v e s s e l s  c u l t u r e s , and p e n i c i l l i n  (Hoshaw a n d R o s o w s k i ,  was  was n e c e s s a r y  l e v e l s of about  1973) were sometimes u s e d  to achieve  150 mg/1  i n cultures  that  -11-  were o v e r l y  i n f e c t e d by b a c t e r i a a n d f u n g i .  changed e v e r y cheesecloth a high to  three  flux  t h e amount  density  reduce p i g m e n t a t i o n  Pieces  similarly  i f t h e medium  compare t h e i r  spore,  width),  and branching  filament  sec"  porphyrae  types  a  were  histories.  cell  were  History  and photon  i n PES medium.  spore germination,  A.  Various  6°C  conditions m"  2  8:16 h ( L : D ) ,  16:8 h  (L:D).  weekly.  vaga.  and spore  of b o t h  with  sec" ). 1  patterns,  Dimensions of the  filaments  ( l e n g t h and  of free  living  were grown on c o v e r s l i p s a t  density stages  o f 4-5 m i c r o e i n s t e i n s of t h e l i f e  initiation  branching  microscopy.  species  were  temperature and  flux  density  (2  The f o l l o w i n g c o n d i t i o n s were  6°C 16:8 h ( L : D ) ,  using  phase of t h e l i f e  Audouinella  t h e same p h o t o n  2  pattern,  contrast  under v a r i o u s  m"  c y c l e , such as  r e l e a s e were p h o t o g r a p h e d  free-living  grown on c o v e r s l i p s a n d p l a c e d  microeinstein  1  recorded.  as w e l l as Nomarski modulation  Filaments  daylength  s e c " tended  treated  dimensions  A t t e m p t s were made t o i n d u c e t h e s e x u a l history.  that  Studies.  vaga  flux  filament  monospore p r o d u c t i o n brightfield  nnat  developmental patterns,  16:8 h ( L : D ) , 1  2  c u l t u r e s o f Audouinella  and l i f e  lengths,  D e v e l o p m e n t a l and L i f e  Audouinella  irr  morphologies, developmental  abilities  initial  study  I t was o b s e r v e d  was n o t r e p l a c e d  bipi  with  s p e c i m e n s o f b o t h e n d o p h y t e s were t h e n u s e d t o  general  cross-infection  6°C  a  to establish free-living  Free-living  To  of l i g h t .  o f 25 m i c r o e i n s t e i n s  Pt er os i phoni  of i n f e c t e d  was  weeks, a n d t h e c u l t u r e s were c o v e r e d  t o reduce  photon  The medium  tried:  15°C 8:16 h (L:D) a n d 15°C  12-  Re-infection  and C r o s s - i n f e c t i o n  T e t r a s p o r e s o f Pi erosiphoni 1984) were grown  Studies. a bi pi nnat a  i n the laboratory  medium i n 60 X 20 mm  plastic  f i l a m e n t s were p l a c e d medium a t a p h o t o n  flux  density  l o n g were u s e d  P.  abbottae  re-  Krishnamurthy  b l a d e s were c l e a n ,  seconds  repeated  gently  K r i s h n a m u r t h y and  F e b . 1985) were used To e n s u r e t h a t  then r i n s e d  in sterile  in a solution  in sterile  were v i s i b l e . and R o s o w s k i ,  and G i b o r  (3-8 cm i n  d i s t i l l e d water  sterile  epiphytes,  f o r 15  until  B l a d e s were a l s o Several  species.  Antibiotics  The p r o c e d u r e  seawater.  rinsed  soaked f o r  isolates  with a n t i b i o t i c s of A.  Infection  three  involved  porphyrae  a l l isolates  cultures.  i n which  eliminate  no t r a c e s o f t h e brown B e t a d i n e  f o r one week when e s t a b l i s h i n g  flasks  was  (TM): 100 s e a w a t e r and  (Hoshaw and R o s o w o s k i ,  experiments  To  b l a d e s were n e x t  of 1 B e t a d i n e  seawater  1973).  seawater.  I I ) were made t o e n s u r e t h a t  control  i n the  these  from P o l n e - F u l l e r  in sterile  t h r e e more t i m e s u s i n g  7 minutes  used  (collected  torta  experiments.  p r o t o z o a n s and b a c t e r i a l  Table  1  i n an A r t e k S o n i c D i s m e m b r a t o r Model 300 a t t h e 30  position,  rinsed  100 ml o f PES  studies.  Young b l a d e s of Porphyra  l e n g t h ) were s o n i c a t e d  individual  2  a method m o d i f i e d  (1984) was e m p l o y e d .  rhizoids  o f 55 m i c r o e i n s t e i n rn" s e c "  b l a d e s of Porphyra  and c r o s s - i n f e c t i o n  (L:D) w i t h PES  i n l e n g t h and then containing  June,  Clumps o f m a t e r i a l a p p r o x i m a t e l y 4-5  f o r subsequent  Young u n i n f e c t e d  h  Filaments with  i n 250 ml f l a s k s  with continuous a e r a t i o n . cm  a t 10°C 8:16  dishes.  were grown t o a p p r o x i m a t e l y 2-3 cm  (collected  (Hoshaw (see  were t h e same  1973) were  series  p i e c e s of the host p l a n t s  also  of f l a s k s : were  1)  cultured  -13-  without  any e n d o p h y t e s ,  Audouinella  porphyrae  in  the f l a s k s  placed  in flasks  a 250ml f l a s k  Pt erosiphoni  experiments  Porphyra.  P.  bipi  2  Porphyra  nnat  a  and  and  bi pi nnat A.  i n which  a  vaga  was  was p l a c e d i n  i n a 250ml  flask  flux  d e n s i t y o f 55 m i c r o e i n s t e i n s  s e c " a t 10°C 8:16 h (L:D) a n d a e r a t e d u s i n g an a i r pump. 1  Observations  were made weekly  f o r s i g n s of i n f e c t i o n .  S i n c e e p i p h y t i c g r o w t h o n l y was o b s e r v e d experiments,  an a t t e m p t a  were p l a c e d  i n 250 ml e r l e n m e y e r  daily  bipi  nnat  a  was made t o i n d u c e  Pt erosiphoni  out  A.vaga  a n d 3)  porphyrae  A.  was p l a c e d  A l l c o n d i t i o n s were e s t a b l i s h e d i n t r i p l i c a t e .  F l a s k s were p l a c e d a t p h o t o n m"  a  i n which  coverslips  2  with  with  flasks  grown on 18 X 18 mm  with cleaned p i e c e s of  cross-infection  with  2) r e - i n f e c t i o n  together  with  with d e s i c c a t i o n replicates  times  increasing  of t h i s experiment  were n o t d e s i c c a t e d . 16:8 h ( L : D ) , p h o t o n  over  growth.  endophytes  The medium was  t o d e s i c c a t e from  poured  2-48 h o u r s  a f o u r week p e r i o d .  were p e r f o r m e d .  The e x p e r i m e n t a l flux  endophytic  free-living  flasks.  a n d p l a n t s were a l l o w e d  i n the i n f e c t i o n  Control  Two  flasks  c o n d i t i o n s were 15°C,  d e n s i t y o f 55 m i c r o e i n s t e i n s m"  2  sec" . 1  Light  and E l e c t r o n M i c r o s c o p y For  Porphyra  fixed  light fucicola  with  microscopic  s t u d i e s , r e h y d r a t e d and  Krishnamurthy  and  Pt erosiphoni  2-3% g l u t a r a l d e h y d e / p h o s p h a t e  7.2) o r 5% f o r m a l i n / p h o s p h a t e S p e c i m e n s were d e h y d r a t e d propylene  Studies  oxide  series,  buffer  a  infected  bipi  nnata  were  (0.067M; pH =  b u f f e r (0.067M; pH = 7 . 2 ) .  i n a methanol  and t h e n  series,  embedded  f o l l o w e d by a  in glycol  methacrylate  -14-  plastic  from P o l y s c i e n c e s , W a r r i n g t o n ,  Wa. ( F e d e r  and O'Brien,  1968). Serial using  sections, approximately  g l a s s k n i v e s on a S o r v a l l  2-2.5 urn t h i c k  Porter-Blum  microtome.  s e c t i o n s were o b t a i n e d  by a p p l y i n g a s m a l l amount  cement a l o n g  of the blocks  were p i c k e d  the width up, d r i e d  histochemical for  stains:  alcian  various polysaccharides  proteins  (McCully  McCully,  1981), p e r i o d i c  and  starch  lipids B-1,3  stain  and M c C u l l y ,  and M c C u l l y ,  blue-0  (O'Brien For  porphyrae  material  a n d Pt erosiphoni  free-living  1983a).  unsatisfactory sucrose  i n various schiff  fast  green  (AB/PAS) (FG)  (SB)  1981), C a l c o f l u o r W h i t e (Hughes a n d M c C u l l y ,  for  (CW) ST f o r 1975) a n d  1981).  o f Porphyra  a bi pi nnat a/Audoui  A. porphyrae  Several other on f i e l d  (T.E.M.) s t u d i e s o f  fuci  a n d A.  vaga  cola/Audoui nelI  nelI a  a vaga,  were f i x e d  as w e l l  i n 2-3%  (0.067 M; pH = 7.2)(Hymes a n d  fixatives  were f o u n d  t o be  m a t e r i a l : phosphate-0.1M cacodylate-0.2M  (pH = 7 . 2 ) / 2-3% g l u t a r a l d e h y d e  (Pueschel  and C o l e ,  1985), s e a w a t e r - N a C l / 2-3% g l u t a r a l d e h y d e  (Pueschel,  comm.).  w i t h PIPES/2-3%  Free-living  glutaraldehyde  for  (PAS) f o r p o l y s a c c h a r i d e s  1981), sudan b l a c k  g l u t a r a l d e h y d e / phosophate b u f f e r Cole,  1981),  Sections  (0.05%, pH = 4.4) (TBO) was u s e d a s a g e n e r a l  and M c C u l l y ,  field  1977).  and p l a c e d  transmission e l e c t r o n micorscopy  rehydrated  as  acid/schiff  o r B-1,4 p o l y s a c c h a r i d e s  toluidine  of rubber  blue/periodic acid  (Kiernan,  Serial  e t a l . , 1980), IKI f o r s t a r c h ( O ' B r i e n and  (O'Brien  (O'Brien  (Henry,  on g l a s s s l i d e s ,  were c u t  s p e c i m e n s were f i x e d  (Hayat,  pers.  1981), b u t t h e phosphate b u f f e r  pH = 7 . 2 ) / 2-3% g l u t a r a l d e h y d e  (Hymes a n d C o l e ,  1983a)  (0.067M; fixation  -15-  p r o v e d most s a t i s f a c t o r y m a t e r i a l s was p o s t f i x e d  for free-living  vaga,  contrast graded  which  were p o s t f i x e d  of m e t h a n o l  o x i d e and embedded replaced for  every  7-8 h o u r s  glass  24 h o u r s  uranyl  a c e t a t e (Watson,  porphyrae  a t 4°C t o r e d u c e  f o l l o w e d by g r a d e d  f o r 7 days)  ( S p u r r , 1969).  up on u n c o a t e d  EM-10  f o r 1 hour  A.  (0.067M; pH  S p e c i m e n s were d e h y d r a t e d  k n i v e s on a R e i c h e r t  citrate  free-living  i n Spurr's p l a s t i c  picked  lead  except  i n the cytoplasm.  series  All  i n 2% 0 s 0 « / p h o s p h a t e b u f f e r  = 7.2) a t 4°C f o r 2 h o u r s , A.  specimens.  series  was h a r d e n e d  in a  was a t 70°C  S e c t i o n s of 60-90nm were c u t u s i n g  0mU3 u l t r a m i c r o t o m e .  copper  f o r 45 m i n s w i t h  1958) a n d r e s t a i n e d  f o r 15 m i n s w i t h  transmission electron  grids,  S e c t i o n s were  stained  (Reynold,1963).  dark  of p r o p y l e n e  (fresh p l a s t i c  that  and  S e c t i o n s were v i e w e d microscope.  in a  Zeiss  -16 -  RESULTS Field  material: Monthly  collections  Porphyra  plants,  at Point Pi  spp. and  from February to October. Audouinella  and  their host  initial plants  present in  vaga  UBC  during  The  spring.  that  and  July.  t h e s e months may current the  fall  Triple  Island,  morphologically  the  collections  occurred in their frequently  hosts  reported  - October.  to c o l l e c t  in July  common i n t h e  f r o m San  (collected  effort,  p l a n t s a r e abundant  September  became l e s s  identical  and  h o s t p l a n t s were n o t  of c o l l e c t i o n  that  and  collected  B.C.  and  were most  were more d i f f i c u l t  H o s t / endophytes  to October,  a b u n d a n c e of h e r b a r i u m c o l l e c t i o n s  - June  when h o s t p l a n t s  porphyrae  A study of the h e r b a r i u m  o b s e r v a t i o n s suggest  endophytes  were p r e s e n t  w i t h the m a t u r a t i o n of  Endophytes  be a r e f l e c t i o n  p e r i o d s o f May  host  Audouinella  f r o m May  both endophytes  The  showed t h a t  bipinnala,  endophytes  f r o m F e b r u a r y t o September and from June  a  coincided  the w i n t e r .  indicated  Point  erosiphoni  were o b s e r v e d  appearance  in late  No  Juan  Island  by L . G o l d e n )  to those c o l l e c t e d  and throughout  The and  from  two  in late  intertidal  zone.  (Washington)  and  were from P o i n t  No  Point.  Audouinella  porphyrae:  Audouinella  in  porphyrae  the host c e l l  randomly  and  wall,  is a  just  filamentous  under  p e n e t r a t e s the w a l l  endophyte,  the s u r f a c e .  It  between t h e c e l l s  growing  branches of the h o s t .  -17-  of A.  Cells of  the  porphyrae  f i l a m e n t s and  project  outward  reproductive  porphyrae  of  the  vegetative  endophyte  first  area.  September many o t h e r  noted that the  reproductive  cm  2  later  areas  III).  of  No  the  primary  areas.  blades  areas  of  were r e p r o d u c t i v e outside  infection  i s greater  AudouinelI  a  the  the  the  i n the  infection  of  1).  main  when  holdfast  were a l s o  blade.  e n d o p h y t e s were s e e n  host  blades.  the than  The  ranged  It  host  The  infection  was  (Table host  cell  3.0  cm .  the  cm  to  2  = 0.006)  the  size  is restricted  Most  III).  and  2  The  of  than  occupied  from 0.1  blades  spp.  in  area  (b = 0.032; r  the  p o r t i o n s of Porphyra  monosporangia  of  parts  correlation  size  vegetative  In A p r i l  Fig.  i n f e c t e d , but  than  observed only  porphyrae  f o u n d between t h e infection  a l s o be  I I ) , most  i n f e c t i o n s were g e n e r a l l y s m a l l e r  of A. No  i t was  vegetative  infection.  infection  (Table  holdfast area.  appeared,  these  original  primary  i n the  I t grows i n  (Table  I I I and  usually less extensive  site  more d i s t a l  may  (Fig. 2).  specific.  (Table  filaments  asexual  formed  present  portions  Erect  where t h e  are  blade  in older  apex.  species  when t h e y  infection  n o t e d on  surface  holdfast regions  i n f e c t i o n s are  By  towards the  i s not  species  i n the  Other p a r t s  host  cylindrical  s t r u c t u r e s , monosporangia, are  s e v e r a l Porphyra frequently  t o be  to taper  from t h e  Audouinella  these  tend  the  22.5 was  of  the  to  infected  endophyte  w a l l when t h e  by  produces  area  of  2  vaga  Audouinella endophyte that  vaga  i s an  grows j u s t  abundantly  beneath the  branching surface  of  filamentous the  host,  the  -18 -  Table I I :  Monthly f i e l d c o l l e c t i o n s of v a r i o u s s p e c i e s of Porphyra.  Audouinella  Date o f collection  Location of c o l l e c t i o n  May 16, 1 984 May 16, 1984 June 14, 19B4 June 29, 1 984 June 29, 1984 June 29, 1984 June 29, 1984 June 29, 1984 June 29, 1984 June 29, 1 984 June 29, 1984 June 29, 1 984 June 29, 1984 June 29, 1984 June 29, 1984 June 29, 1 984 June 29, 1 964 J u l y 15, 1984  E a g l e ' S Cove , S . J . E a g l e ' s Cove, s . J . T r i p l e I s . , B .c # * T r i p l e I s . , B .c * P.N.P. B.C. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P.  p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p.  s c hi 2 o ph yl la I or l a I or l a s c hi zophylI a fucicola Sc hizophyl1ia sanj uanes is abbot t ae s c hizophylI a t or i a fuci col a fuci col a fuci col a fuci cola fucicola fucicola fucicol a  Aug. Oct. Oct. Oct. Oct. Oct. Dec. Feb. Mar. Apr .  P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P. P.N.P.  p. p. p. p. p. p.  Porphyra I or l a kanakaensis 1 or l a t or l a I or l a I or t a NO Porphyra  15, 1 984 12, 1 984 1 2 , 1 984 12, 1 964 12, 1 984 12, 1984 1 9 , 1984 4, 1984 4 , 1 964 10, 1 984  S.J.= San Juan I s l a n d , Washington B r i t i s h Columbia * = c o l l e c t e d by L. Golden  S p e c i e s of  Porphyra  p.  very  in  porphyrae  P r e s e n c e of Isolates endophyte in culture  little  Juven i l e Juvenile l or i a  P.N.P.= P o i n t No P o i n t ,  + + + + + +  + + + + + + +  _  + + + + +  +  -19 -  Table  III:  Dimensions of Audouinella porphyrae g r o w i n g in situ, f i e l d c o l l e c t i o n s of Porphyra, and g e n e r a l host f e a t u r e s .  Endophytes  Host  Length  Width  Area  (cm) 7.5 B 3.8 3.5 2.5 3.5 3.5 2.0 1.8 2.5 2.0 1 .6 1.8 1.5 1.5 1 0.5 2 0.1  (cm) 3.0 21 .5 4 3.5 2.3 1.5 1.5 2.4 2.5 1 .8 2.0 1.8 1.8 1.5 1.5 1 0.5 0.1 0.1  (cm ) 22.5 20 16.2 12.25 5.75 5.25 5.25 4.6 4.5 4.5 4 3.24 3.24 2.25 2.25 1 0.25 0.2 0.1  Reprod.  2  + + + + + + + + + + + +  -  Length (cm) 12.5 (a) 14(a) 11.5(a) 11 (a) 6(a) 7(a) 1.5(a) ' 6(a) 1.8(d) 9.5(c) 4.5(a) LB(b ) 11 (e) 4(a) 9.5(e) 10(a) 4(a) 4.5(b) 15.5(c)  Width (cm) 13 7 8 7 5.5 B 3.5 8 2.5 7.0 5.5 1 .8 6 2.8 7 8 6 5.5 12.5  Area (cm ) 162.5 98 92 77 33 56 5.25 48 4.5 66.5 24.75 3.24 66 11.2 66.580 32 24.75 193.75  Repi  2  + •+  + + + + + +  -  + + +  -+ + +  • • r e p r o d u c t i v e s t r u c t u r e s were p r e s e n t - • r e p r o d u c t i v e s t r u c t u r e s were absent Note: a l l i n f e c t i o n s b e g i n a t t h e h o l d f a s t of t h e h o s t and o t h e r subsequent s m a l l e r i n f e c t i o n s b e g i n f u r t h e r up t h e host b l a d e , a b c d e  » * « «= •  Porphyra fuci col a Porphyra abbot Iae Porphyra s anj uanes i s Par phyr a t or l a Por phyr a schi zophylI a  -20-  mainly  i n the p e r i c e n t r a l  grows p a r a l l e l the host portion Erect  cell  t o the primary  (Fig. 5).  Cells  walls  (Fig. 4).  The e n d o p h y t e  a x i s of the l o n g i t u n d i n a l  tend  t o be c y l i n d r i c a l  a x i s of  i n the older  of t h e endophyte and t a p e r toward the growing p o i n t .  filaments are e x t e r i o r  abundant, a r i s i n g filaments.  from  t o the host w a l l and u s u a l l y  almost  any c e l l  M o n o s p o r a n g i a a r e formed  of t h e e n d o p h y t i c  from  the e x t e r n a l , erect  f ilaments. Audouinella  vaga  grows most  main a x i s and i n l a r g e r young a p i c a l areas  tips  o f A. vaga  two  adjacent  individual  ranged  host  portions  (greater  tube  Infection  (Table I V ) .  correlated  factor  between  t h e s i z e of (x  2  = 3.03;  Thus, t h e age o f  for infection  by t h e  by t h e o b s e r v a t i o n t h a t o l d e r  t o become h e a v i l y i n f e c t e d  occur.  rarely in  wall i n the area  reproductive.  The l a r g e r  2  before  the i n f e c t i o n  area  the endophyte w i l l  be  (Table I V ) . o f e n d o p h y t e s : Audouinella  same way.  of both  porphyrae  h o s t s by t h e two e n d o p h y t e s commences i n  Monospores a t t a c h t o o u t e r w a l l s of t h e i r  h o s t s and d i v i d e  i s then  2  250 ;um ), t h e more l i k e l y  Infection  respective  being  tend  infections  Development  t o 7.5 mm  As f o r A. vaga,  a determining  of the host  than  2  i s positively  This i s supported  reproductive  the  the host  i s probably  secondary  180 urn  cells.  areas  and o n l y  ( T a b l e IV and F i g . 1 ) .  a t t a c h t o the host  infection  endophyte.  from  pericentral  p = 0.05) w i t h the  branches of i t s host  and r h i z o i d s  Monospores u s u a l l y  f r e q u e n t l y i n t h e w a l l of the  produced  from  internally  (Fig. 6).  one o f t h e two d a u g h t e r  A  germination cells  (Fig.  -2 1-  Table  Length  (pm) 151.5 138  165 129 49.5 198 90 75 77.1 69 54  43 45 42 7.5 30 39 52.5 37.5 34.5  13.5 90  IV:  Measurements of Audouinella vaga i n i t s host bipi nnata from f i e l d c o l l e c t i o n s .  Endophyte Width Area (urn)  50 42 28.5 25.5 60 13.5 27 21 IB 18 20 24 18 15 63 15 9 4.5 6 6 9 20  Reprod.  (pm ) 7575 5796 4702.5 3289.5 2970 2673 2430 1575  Erect portions outside host  Pterosiphonia  Reprod. of host  Location on host 1 2 3  1  1387.8  1242 1080 1032 810 630 472.5 450 351 236.25 225 207 121.5 180  -+  -+ +  +  -+ -  / /  +  /  + + +  + +  -  +  +  -+ -—  + + +  -  -+ -  -  /  + + + + +  /  -+ -  / / / /  / / /  +  -  -  Total  2  of l o c a t i o n  bi pi nnat a  on h o s t . )  ( F i g . 1)  (see F i g . 1 f o r e x p l a n a t i o n  / / / /  / / / / 13 6  + = r e p r o d u c t i v e s t r u c t u r e s ( c a r p o g o n i a , or spermatangia or t e t r s p o r a n g i a ) are present - = r e p r o d u c t i v e s t r u c t u r e s a r e absent / = l o c a t i o n of endophyte on host a c c o r d i n g t o a schematic drawing of Pterosiphonia  /  -2 2-  7) a n d t h e r e s u l t i n g f i l a m e n t cell  wall.  walls  The i n i t i a l  throughout  urn ±0.3 i n l e n g t h  were All  cell  pyrenoid it  formed.  division  infection  the  surface,  each e r e c t  axis.  examined  Development Settled  (n=50).  c h l o r o p l a s t a n d one p r o m i n e n t  reddish  be s e e n e a s i l y  the host  pink  using  wall,  point of area i s  Hoffman  host  cells.  wall.  diameter  Erect  filaments  a r e formed  These d e v e l o p e d p e r p e n d i c u l a r l y t o t o about  25 pm, a n d h a d two t o s i x  one t o two m o n o s p o r a n g i a were p r e s e n t  No t e t r a s p o r a n g i a in this  of endophytes: monospores  filaments  g i v i n g the blade  T h e r e were no i n d i c a t i o n s o f t h e  or h a i r c e l l s  were n o t e d i n  Audouinella  Audouinella  vaga  vaga  were  12 pm ± 0 . 3 i n  (n = 5 0 ) . The a p i c a l c e l l o f  measured 21 pm ±0.4 i n l e n g t h  a n d 3.0 pm ±0.3  (n = 5 0 ) . The narrow d i a m e t e r o f a p i c a l  to older  on  study.  a n d 8 pm ± 0 . 3 i n d i a m e t e r  compared  filament  and 7.5 pm ±0.8 i n d i a m e t e r  could  extended  In g e n e r a l ,  growing  i n the primary  r a d i a t i n g o u t from t h e i n i t i a l  appearance.  t o the host  length  (n=50) a f t e r t h e  become l a r g e r a n d l a r g e r , e v e n t u a l l y  external  material  Cells  a somewhat c i r c u l a r  features  endophyte p e n e t r a t i n g  cells.  V).  12.0  ( F i g s . 6 - 9 ) . As t h e e n d o p h y t e grows a r e a s o f  red blotchy  host  cell  measured  As t h e e n d o p h y t e grows w i t h i n  eventually  microscopy  in  (Table  irregularly,  These  the hosts'  porphyrae  a n d 9.0 urn ±0.3 i n w i d t h  (Fig. 8).  branches  the host  development.  have a s i n g l e , s t e l l a t e  infection;  a  spores remain o u t s i d e  13.5 pm ±2.3 i n l e n g t h cells  as i t p e n e t r a t e s  m o n o s p o r e s o f Audouinella  Settled  initial  tapers  vegetative  cells  was a c o n s i s t e n t  cells f e a t u r e of  -23-  both endophytes. lengths vaga  of c e l l s  on p r i m a r y  had s i g n i f i c a n t l y  differences the  T h e r e was a s i g n i f i c a n t  diameters  (Table  (Table  V).  Filaments  No  were n o t e d  A.  of  V).  vaga  tend  between t o grow  t o t h e main a x i s a n d t o t h e l o n g i t u d i n a l d i m e n s i o n o f  pericentral  cells  of t h e h o s t .  endophyte branching Erect  smaller  and A ,  o f t h e two s p e c i e s  i n the c h l o r o p l a s t and p y r e n o i d  two s p e c i e s  parallel  filaments  d i f f e r e n c e i n the  portions  tends  a r i s e from  In t h e p r o s t r a t e p o r t i o n s  t o occur  at right  the prostrate  and  protrude  cell  one  a n d one t o two m o n o s p o r a n g i a a r e p r o d u c e d on  these erect p o r t i o n s . patterns close  or b l o t c h e s  examination  growth c o u l d  5). field  Neither  with  of  d i d n o t show m a c r o s c o p i c  filaments,  a n d i t was o n l y by  a compound m i c r o s c o p e  that  the endophytic  M i c r o s c o p i c a l l y , the endophyte  filaments  A.  axes g e n e r a l l y c o n s i s t of  that  branch at r i g h t  nor h a i r c e l l s  angles  forms  (Fig.  were o b s e r v e d i n  vaga.  material  Establishment Audouinella  free-living material  i n the host  tetrasporangia  material  Culture  The i n f e c t i o n  be r e s o l v e d .  numerous p a r a l l e l  Erect  filaments  (Fig. 5).  through the host t o two c e l l s  walls.  angles  of the  of f r e e - l i v i n g porphyrae  endophytes  and t h e r e f o r e  Commencing w i t h developmental pattern free-living  endophytes  and  A.  vaga  grew r e a d i l y a s  i n c u l t u r e without a r e not o b l i g a t e  the presence  endophytes.  monospores r e l e a s e d  from mature p l a n t s , the  o f b o t h e n d o p h y t e s was s i m i l a r ,  forms were m o r p h o l o g i c a l l y  of host  identical.  and the  Upon  -24 -  T a b l e V:  Cell  d i m e n s i o n s and b r a n c h i n g p a t t e r n s of  porphyrae  and Audouinella  vaga  in their  Audouinella  respective  h o s t s from P o i n t No P o i n t . (* p = <0.05; a l l c a s e s n = 50)  A.  C e l l dimension: Settled spore**: Main  filament:  Mature  apical cell:  porphyrae  12pm ( 1 ) 9pm ( d ) *  A.  vaga  12pm ( 1 ) Bpm ( d ) *  I3.5jjm ( 1 ) * 7.5pm ( d ) *  15pm ( 1 ) * 6pm ( d ) *  15pm ( 1 ) * 3pm (d)  21pm ( 1 ) * 3pm (d)  Host:  Porphyra  Branching p a t t e r n :  Irregular  spp.  (1) = length (d) = d i a m e t e r * i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s a t p = <0.05. ** measurements a f t e r i n t e r n a l d i v i s o n .  Pt erosiphonia  At r i g h t  sp.  angles  -2  attaching  to a v a r i e t y of  nylon  thread,  10).  Attachment  hours of  5-  substrata  s u c h as p l a s t i c ,  monospores underwent an  spore  and  internal  release.  division  This  Commonly, a  second g e r m i n a t i o n  second c e l l  of  filamentous  plant  was  (Fig.  At  time d i d e i t h e r of  inside were  the  14.0  pm  the  in  diameter  in length  primary  from a  frequently;  (Figs.  and  Most  i n the of  the the  produced.  (Figs.  9.0  to  of  the  the  very  of  a  12). from  to erect  the  filaments  initial  cells Basal  cells  urn i n d i a m e t e r , whereas t h e  cells  were  16.5  four  erect  filaments.  urn i n l e n g t h axes c o u l d  than  and be  Apical cells Erect  t h o s e of lateral  the  9.0  urn  formed  were  filaments  18.0  branch  Cells  in  primary  axis  branches,  branches always d e v e l o p e d a d a x i a l l y i s comprised  basal  cells  are  of  erect  formed  f l a s k s became q u i t e d e n s e and  portions  (5-9  plants  cells).  grew on  the  vessels. p l a n t s matured, monosporangia  ( F i g s . 16,17) were  M o n o s p o r a n g i a were e i t h e r t e r m i n a l  16,17). a  formation  later  primarily unilateral.  plant  few  (Fig.  prostrate  two  to erect  urn i n d i a m e t e r . was  small  or  24-48  ( F i g s . 11,  gave r i s e  o c c a s i o n a l l y , the  b r a n c h e s , and  f o r m e d on  A  the  formed  ( F i g s . 13).  These secondary  (50-100 c e l l s ) ;  erect  rise  b r a n c h e s were s m a l l e r  18).  As  Up  system  branching  rebranched.  sides  that  by  cells  t u b e was  filament  9.0  15,18) and  Growth  and  ( F i g . 14).  in length  (Fig.  erect  s i n g l e basal  lateral  give  two  spore.  formed  spore wall  in  um  no  original  the  division  occured within  followed  tube  the  of  was  germination  13).  from one  internal  glass  either sessile  In most c a s e s o n l y  supporting  cell;  or  on  or  lateral  a one-celled  on  pedicell  a s i n g l e monosporangium  i n f r e q u e n t l y sporangia  were  was  formed  -26-  in  pairs  ( F i g . 21).  Similar  t o the formation of l a t e r a l  branches, monosporangia  were formed  s p o r a n g i a may be formed  i n succession  sporangium  wall  (Figs.  19,20).  walls could  be e a s i l y  (Fig.  Monosporangial  20).  and a t low p h o t o n  microeinsteinsm" produced  under  inoculation  the  Infection In  flux  the c o n t r o l  the  cycle  took  after  t h r e e t o s i x weeks. s t r u c t u r e s by  in field  series,  Pt erosiphonia  This corroborates  material.  re-infection  bi pi nnat  enough t o be u s e d  t h r e e weeks.  and grew l a r g e r  (Figs.  22-24).  Porphyra  and  tort  r e - and c r o s s - i n f e c t i o n growth.  a,  time  on t h e o t h e r field  deterioration  bipinnata  grown  conditions  the e n t i r e  than the o r i g i n a l  Pt erosiphonia  plants  for infection  during  experiments, both endophytes  Porphyra  a  Under t h e c u l t u r e  t h e r e was no e v i d e n c e o f t h a l l u s  endophytic  were  was c o v e r e d w i t h p l a n t s and  observed  hand, d i d n o t grow much l a r g e r  on  (6°C v s  experiments  the experiment  growth  days  F o u r months  c o n d i t i o n s were u n s u c c e s s f u l .  p r o v i d e d , they branched  In  i n long  (4-5 v s 22 v s 55  t o induce s e x u a l r e p r o d u c t i v e  experiments w i t h i n  and  microscopy  a few m o n o s p o r a n g i a  of a d i s h  f r o m t e t r a s p o r e s were l a r g e  of  densities  Only  2  o f gametangia  sporangium  a t a low t e m p e r a t u r e  other experimental conditions.  culture  absence  the o r i g i n a l  Remnants o f empty  The m o n o s p o r a n g i a l  Attempts  Two t o s i x  p r o d u c t i o n was g r e a t e r  sec" ).  t h e bottom  monospores.  varying  2  within  seen w i t h Hoffman c o n t r a s t  16:8 h (L:D) v s 8:16 h ( L : D ) ) , 15°C)  unilaterally.  showed  material  (Fig.  25).  epiphytic  b u t i n none o f  e x p e r i m e n t s was t h e r e e v i d e n c e o f  Monospores appeared  t o be p a r t i a l l y  embedded  -2 7-  in  the host  bipi  nnat  a  cell  wall  were i n f e c t e d  cross-infection re-infection free-living internal  plants  division  versus  cells).  species  bipi  nnat  Porphyra  tort  system  (three  a  cultures  or  plants  there  to s i x vs. five  was l e s s to nine  a x i s was  f r o m one o f t h e two c e l l s  f o r the production  evident  i n the  a x e s were  of l a t e r a l  monosporangia  31, 3 2 - 3 3 ) .  Audouinella  porphyrae  Numerous p l a n t s  from  and  within  portions.  A.  vaga  covered  18 d a y s e x c e p t  After  i t was t o t a l l y  When g r o w i n g on  much l o n g e r Pt er os pi phoni  t o develop a  bipinnata.  Porphyra  to host  covering the  cell  the host  Pt er os i phoni  the  Pt er osi  phoni  walls.  plants.  a  a  bipinnata  apical bipinnata  s l o w e d g r o w t h compared t o c o n t r o l  65 d a y s , t h e h o s t  s h o o t s even t h o u g h  attached  cultured  f o r the a c t i v e l y growing  Endophytes d i d not k i l l  i n c u l t u r e , but r a t h e r  cultures.  the f r e e - l i v i n g  were soon o b s e r v e d  Both endophytes completely  34).  on  the f r e e - l i v i n g  ( F i g s . 30, 3 2 - 3 3 ) , a n d most e r e c t  Monospores r e l e a s e d  plants  between  d i f f e r e n c e s were  In t h e e p i p h y t i c  always arose  unbranched except  plants  initial  T y p i c a l l y , one ( r a r e l y two t o t h r e e ) e r e c t  spore  (Figs.  a.  of the basal  produced that inital  g r o w t h was s i m i l a r t o  showed t h e same  However, s e v e r a l  growing e p i p h y t i c  Pterosiphonia  development  spores  t o those of  ( F i g s . 26,27) a n d a s i m i l a r m o n o s p o r a n g i a l  cycle.  those  The e p i p h y t i c  i n that  Pterosiphonia  of  The r e s u l t s o f  e x p e r i m e n t s were i d e n t i c a l  Audouinella  both  ( F i g s . 29-33).  experiments.  reproductive in  (Fig. 28). A l l parts  tort  still  grew a n d p r o d u c e d new  covered a,  by e p i p h y t e s  epiphytic  (two weeks t o t h r e e In a d d i t i o n ,  (Fig.  populations  to five  took  d a y s ) t h a n on  the population  was l e s s  -2  Porphyra  d e n s e on  torta.  Auduoinella  their  basal  Porphyra bipi  nnat  within  a  portions  basal  A.  and  vaga  a  Pt er os i phoni  On  s y s t e m was l i m i t e d t o t h e i n i t i a l  from e p i p h y t e s  erect  when g r o w i n g on  bipinnata.  t h e s p o r e and o c c a s i o n a l l y  cells  showed d i f f e r e n c e s i n  and b r a n c h i n g p a t t e r n s  Pt e r o s i phoni  and  the basal  different  the  porphyrae  torta  8-  on  t o one o t h e r c e l l .  Porphyra  might be p r o d u c e d  two  tort  a  Porphyra  conditions  including  intensity  torta  where up t o f i v e  ( F i g s . 28, 35, 3 7 ) .  (compare F i g s .  lack  and d e s i c c a t i o n  induce e p i p h y t i c  plants  In a d d i t i o n  of host  32 and 3 6 ) .  reduction  were u s e d  The o n l y  in  of e p i p h y t i c  were Various  in light  i n an a t t e m p t t o  t o become e n d o p h y t i c  bipinnata.  the rate  of aeration,  cells  This i s  a x e s were more commonly b r a n c h e d when p l a n t s  g r o w i n g on  a  in  Pt er osi  phoni  e f f e c t o f t h e s e e x p e r i m e n t s was a  a  reduction  g r o w t h w h i c h was n o t e d when p l a n t s  were  desiccated. Light  and t r a n s m i s s i o n Using  various  sections  electron  from m e t h a c r y l a t e  observations  Audouinella Porphyra  prostrate to  filaments  were seen  porphyrae fucicola  of  walls  the  host  curved,  arc  ( F i g s . 47, 4 9 - 5 0 ) .  of  Many  i n the hosts'  material,  endophytic  cell  walls.  irregularly  i n the  ( F i g s . 38-39, 4 3 ) , whereas t h e  Audouinella  the l o n g i t u d i n a l a x i s wall  hosts.  tended t o branch cell  portions  embedded f i e l d  were made on t h e c y t o l o g i c a l r e l a t i o n s h i p s  between t h e e n d o p h y t e s a n d t h e i r prostrate  microscopy  Pt er osi  vaga  tended  phoni  t h e endophyte a l s o  a  t o grow  parallel  bipinnata.  Thus,  appeared  t o grow i n an  Internally divided  when  s p o r e s were o b s e r v e d  -29-  on  t o p of t h e w a l l o f b o t h  germinating wall.  I t was n o t e d  f i l a m e n t but not the spore  (Figs.  portions these  Porphyra.  42,43) o f  outside their  to penetrate  nnata  Filaments  the cytoplasm  were p e n e t r a t e d  A.  by  Audouinella  vaga  in this  in their  study.  cytoplasm  of t h e  vaga  to indicate  green  fucicola  (Figs.  producing emerging (Figs.  e r e c t axes,  Audouinella  with T o l u i d i n e blue-0  reaction  with  the presence  the c e l l  of  When e n d o p h y t i c cell  In a d d i t i o n  f i l a m e n t s were  reaction areas  with  t h a t some p r o t e i n a c e o u s  released  into  wall prior  present host  showed a s i m i l a r  endophytic  t h a t s t a i n e d d a r k l y on t h e (Figs.  52,56).  fast  cells  green were  ( F i g . 44-46,  m a t e r i a l may  to upright axis reaction  to the  of s t a i n i n g  This suggests  vaga  1981).  Porphyra  of  walls adjacent  localized  above c e r t a i n  This i s  f o r p r o t e i n s i n the  52-53).  bipinnata  w a l l s and  polyphosphates,  immediately  cuticle  gave a  (O'Brien and M c C u l l y ,  reaction  the host  the host  a  ( F i g . 48).  observed  Audouinella  cells.  stains  f i l a m e n t showed a p o s i t i v e  45-46).  were  Pterosiphoni  of  as w e l l as i n the c u t i c l e  44-46).  from  r e a c t i o n s t o the h i s t o c h e m i c a l  showed a p o s i t i v e of a l l c e l l s  erect  porphyrae  Porphyra  cells  beween  p o l y s u l f a t e s and p o l y c a r b o x y l a c i d s  cytoplasm  A.  of  o f t h e two h o s t s and t h e two e n d o p h y t e s .  interpreted  wall  and  Staining  red-purple metachromatic  cell  porphyrae  T h e r e were no d i f f e r e n c e s  Fast  the host  h o s t s , a n d m o n o s p o r e s were formed  erect f i l a m e n t s ( F i g . 41).  seen  used  penetrated  Both endophytes produced  However, on two o c c a s i o n s p e r i c e n t r a l bipi  that the  T h e r e were many f i l a m e n t s w i t h i n t h e o u t e r c e l l  layer  not  hosts.  be  initiation.  except  t h e r e was  Pt er osi  phoni  no  a  IKI a n d Sudan b l a c k d i d n o t show  -30-  any  reaction.  No d i f f e r e n c e s  between t h e c e l l  e n d o p h y t e s were shown by t h e A l c i a n showed p u r p l i s h c o l o r a t i o n polysaccharides differences  (Kiernan,  pink-pinkish stained  (Cole  e t a l . , 1985).  test  1981).  indicated  polysaccharides  The d i f f e r e n c e s  t h e two e n d o p h y t e s  (Figs.  69-71, 7 2 - 7 5 ) .  (Hara a n d C h i h a r a , w i t h one p y r e n o i d within two  starch  central  material  free-living discussed algae.  microscopy  Bangi  a  showed a  polysaccharides  with C a l c o f l u o r  W h i t e ST  B-1,3 o r B-1,4  studies  hosts.  showed t h a t  axile  has an i r r e g u l a r p a t t e r n ( F i g s . 54, 56, 6 2 ) .  cells  of  cells  chloroplast chloroplast  thylakoids  P i t plugs  i s surrounded with  Mitochondria  i s often  in  are u l t r a s t r u c t u r a l l y i d e n t i c a l  and t h e n u c l e u s  have  abundant  and d i c t y o s o m e s a r e n o t  ( F i g s . 69, 7 1 - 7 3 ) ; when cytoplasm of c e l l s .  observed within  A  present, large  endophytic c e l l s i n  ( F i g s . 54, 56) w h i c h was n o t o b s e r v e d i n  material.  later  walls  They have t h e " N e m a l i o n " t y p e  i n the p e r i p h e r a l  vacuole  observed  of t h e e n d o p h y t e s and t h e i r  vivo  matrix  i n vegetative  they occur  field  that  ( F i g s . 69-71).  abundant  in  stained  whereas t h e o u t e r  1973) - a s i n g l e , s t e l l a t e ,  the pyrenoid  cap l a y e r s  bipinnata  the absence of d e t e c t a b l e i n the walls  showed  i n d i c a t e d i f f e r e n t types  i n d i c a t i n g a mixture of  electron  Both  however,  fucicola  was a l s o  Lack o f f l u o r e s c e n c e  Transmission of  Porphyra  Pt erosiphonia  coloration  (Kiernan,  wall.  The two h o s t s ,  bluish, this reaction  of p o l y s a c c h a r i d e s . purplish  blue/PAS r e a c t i o n .  r e d i n the c y t o p l a s m and c u t i c l e  walls  o f t h e two  i n d i c a t i n g a m i x t u r e of 1981).  in their cell  walls  Further  when c o m p a r i n g  ultrastructural details will these  taxa  to other  be  acrochaetioid  -31  Preliminary in  situ  plants walls  ultrastructural  were c a r r i e d (Figs.  by growth recorded  on e n d o p h y t e s  T h e r e were no major  became d i s o r g a n i z e d  porphyrae  organization  out.  studies  effects  growing on  5 7 - 6 0 ) , however, some o f t h e f i b e r s w i t h i n  o f Porphyra  Audouinella  -  of w a l l s  o f A.  vaga  in Table  VI.  (Figs.  61,  63-64).  o f Pterosiphonia (Figs.  during  65-68).  The  infection  host the  by  structural  bi pi nnat a was  unaffected  A summary of a l l r e s u l t s  is  -32-  T a b l e V I : Summary of d i f f e r e n c e s and s i m i l a r i t i e s of m o r p h o l o g i c a l , r e p r o d u c t i v e and c y t o l o g i c a l c h a r a c t e r s of f i e l d and cultured  Audouinella  obtained  i n the c u r r e n t  porphyrae  Field porphyrae  Morphological Cell  Audouinella  vaga  material A.  vaga  C u l t u r e d ma t e r i a 1 porphyrae  A.  vaga  features:  dimensions:  12-15um(l) 3-7.5um(d)  Hosts:  Porphyra  Branching p a t t e r n : Infection cycle:  spp. i rregular monospore  Reproductive features: Life history: asexual via monospore Cytological  and  study.  l2-2lum(l) 3-6pm(d)  14-l8um(l) 9pm(d)  14-lBjjm(l) 9um(d)  right angular monospore  irregular monospore  i rregular monospore  asexual via monospore  asexual via monospore  asexual via monospore  Pi er os iphonia bipinnat a  features: s i m i l a r u l t r a s t r u c t u r a l f e a t u r e s f o r f i e l d and c u l t u r e d m a t e r i a l s (see t e x t )  Re-infection exper iments: Cross-infection exper iments:  epiphyt i c growth  epiphytic growth  epiphytic growth  epiphyt ic growth  -3 3-  DISCUSSION  A  study  of  similarities Audouinella  field  and  porphyrae  been c o r r o b o r a t e d of  the  structures,  showed t h a t  d i f f e r e n c e s between t h e A.  and  endophytes reported  aspects  material  vaga.  previously  in this  m o r p h o l o g y and  were n o t e d  relationship  between  endophyte/host size  of  the  e n d o p h y t e grows as the  oldest  A.  porphyrae  Initially, Porphyra  spp.  rhizoids. a  suggested any  the  basal  i n f e c t i o n s by Unlike  hosts  (Goff,  their  the  noted.  in  s i z e of  the This  hosts mature.  field  In  the  neither  could  their  vaga  i n f e c t s the  older  endozoic  Acrochaetiurn  of  alga  will  A  when  indicate  that  appear  hosts. region  regions  c l o s e s t to noted  the  hosts.  Both endophytes  of  bipinnata,  the  e n d o p h y t e and  holdfast  a red  taxa.  c o r r e l a t i o n between  i n f e c t s the  and  two  hosts.  first  region  this  of  of the  the  when  examining  species, usually  and  contain  endophytes.  parasites 1982)  i n the  r e l a t e p r i m a r i l y to  s i z e of  (1970) a l s o  include  reproductive  portions  Boney  endophytic  Such f e a t u r e s  They  f  was  the  a l . (1982) have  older  whereas A.  W h i t e and  s e r i e s of  the  or  et  of  endophytes  there a  the  of Pt erosiphonia  axis  the  i n f e c t i o n a r e a s and  h o s t s were r e p r o d u c t i v e  primary  study.  c o r r e l a t i o n between  infect  of  e n d o p h y t e s and  the  to  Garbary  spore g e r m i n a t i o n  r e l a t i o n s h i p was  endophytic  positive  the  endophytes,  i n f e c t i o n process,  in this  are  Many f e a t u r e s  by  Some a d d i t i o n a l c h a r a c t e r s material  two  investigation.  seasonality,  there  and  which r e q u i r e epiphytes  nutritional  w h i c h may  needs from  require  their  a certain  -3 4-  colonizable appear these  substrata (Harlin,  t o have any o f t h e s e symbionts  1975),  these endophytes  requirements.  t o become e n d o p h y t i c  stresses  wave a c t i o n .  T h i s has y e t t o be s t u d i e d .  such a s d e s i c c a t i o n ,  wounding by g r a z e r s p l a y s an i m p o r t a n t parasites  (Goff,  A possible  1982).  sand  s c o u r i n g and  The phenomenon o f  role  i n the p e n e t r a t i o n  T h i s was n o t o b s e r v e d  f o r the  endophyte  in this  was  i n t h e a r e a of s e t t l e m e n t and p e n e t r a t i o n .  noted  Secondary hosts probably primary  reason f o r  i s t o "escape"  environmental  of  do n o t  s t u d y and no o b v i o u s damage o f h o s t c e l l  infections from  infection.  i n f e c t i o n s a r e seen  occur  i n v e g e t a t i v e r e g i o n s of both  g e r m i n a t i o n of monospores p r o d u c e d In s u p p o r t  walls  o f t h i s argument,  o n l y when t h e p r i m a r y  by t h e  secondary  infections  are large  .and r e p r o d u c t i v e . D i f f e r e n c e s w h i c h were p r e v i o u s l y separate also  t h e two s p e c i e s ( G a r b a r y  observed  in this  the endophytes and  current c e l l  those  by G a r b a r y  vaga.  different,  e t a l . (1982) ( T a b l e I a n d T a b l e V ) . l o n g e r than  The b r a n c h i n g  i n the host m a t e r i a l  main g r o w i n g  filament  A.  vaga  just  e t a l . (1982) n o t e d  porphyrae  d i m e n s i o n s of  hosts are s t a t i s t i c a l l y  l e n g t h s were s l i g h t l y  whereas  A.  F o r example, c e l l  I ) were  measurements f o r t h e two t a x a o v e r l a p w i t h  irregularly,  Garbary  and used t o  e t a l . , 1982 a n d T a b l e  e t a l . (1982) t h u s e x t e n d i n g  Audouinella  endophytes  of  their  r e p o r t e d by G a r b a r y  However, c e l l  for  inside  study.  noted  and h a i r  branches inside  t h e range  pattern  differs;  A.  porphyrae  at right  length  branches  angles t o the  the host w a l l .  from  of c e l l  o f t h e two  t e t r a s p o r a n g i a from  cells  those reported  Although one c o l l e c t i o n  one c o l l e c t i o n  o f A.  vaga  -35 -  (Table  I ) , neither  of these  or c u l t u r e m a t e r i a l cells are  from t h i s  and t e t r a s p o r a n g i a  unsuitable  two c h a r a c t e r s study.  Since  a r e so s p o r a d i c  characters  was o b s e r v e d  t h e presence of h a i r  in field  investigation.  Occasionally,  vaga  were o b s e r v e d p e n e t r a t i n g  This  i s the f i r s t  report  the  rare penetration  was  not observed  of  A.  this  study. their  (Garbary  are three  i n separate  Porphyra  whereas  2)  A.  than  porphyrae A.  whereas  vaga A.  Other  hosts,  A.  hosts'  cell  vaga  A.  in their  are  t h e same.  could  hosts:  be u s e d  1) They  occurs i n bipinnata,  filmentous  cells  branches  angles  t o the primary  the d i f f e r e n c e s discussed  irregularly axis.  a b o v e , t h e two  similar morphologically  and the p a t t e r n  i s not a  porphyrae  than  field  The r a r i t y o f  i n Pterosiphonia  branches a t r i g h t  the  to distinguish  o f t h e two t a x a .  vaga  In a d d i t i o n , t h e m o n o s p o r a n g i a l  infrequently,  penetration  a p i c a l and main  ( T a b l e V ) , a n d 3)  this  endophytes  cells  porphyrae  i s present  has s h o r t e r  endophytes a r e q u i t e  both  major d i f f e r e n c e s t h a t  Audouinella  bipinnata.  cells,  e t a l . , 1982).  c h a r a c t e r i z i n g t h e two e n d o p h y t e s  reside  Since  a  (1928) n o t e d  Porphyra  i s not a u s e f u l c h a r a c t e r  of t h e growth s t r a t e g y  Thus, t h e r e  Drew  i n the Audouinella  of  Pt er os i phoni  into  t h i s phenomenon s u g g e s t s t h a t h o s t normal a s p e c t  filaments of  porphyrae  i n the present  between t h e s e two s p e c i e s  in  i n addition to  o f t h i s phenomenon.  have been o b s e r v e d t o p e n e t r a t e appears that  cells  they  species.  i n d i c a t e d by G a r b a r y e t a l . (1982) were o b s e r v e d  current  it  material,  f o r d i s t i n g u i s h i n g these  Some d i f f e r e n c e s between t h e e n d o p h y t e s those  in field  reproductive  and c y t o l o g i c a l l y . c y c l e s observed i n  o f p r i m a r y and s e c o n d a r y i n f e c t i o n s  Chloroplasts  were c o n s i s t e n t  with  t h e "Nemalion"  -36-  - t y p e a s d e s c r i b e d by H a r a and C h i h a r a chloroplasts as  Kyi  i ni a  Chihara,  sp.  in ultrastructural  than  chloroplast describing  the l i m i t e d  and p i t p l u g s  was  (1983a) and  "Batrochospermum" - t y p e  (1982);  Audouinella  have  with the  Lee (1971);  hermanni  (I983a,b).  A.  Like  mitochondria  periphery  structure  (Roth)  Hymes and  and d i c t y o s o m e s ;  A large  cells Unlike  central  of  A.  Audouinella  prominent  thylakoids.  hermanni  Furthermore,  pyrenoid with  i,  Audouinella,  72-73). A.  1983a). showed a  traversing  e n d o p h y t e s were commonly  T h i s h a s been o b s e r v e d parvula  similar to  endophytes  in erect  ( K y l i n ) Dixon  30% o f t h e  in field material  in culture,  both  at the  w a l l s of the  r e p r e s e n t i n g over  four to f i v e  the base of v e g e t a t i v e c e l l s  (Figs.  the c e l l  (Hymes and C o l e ,  hermanni  N u c l e i of both  alga  e n d o p h y t e s showed  were l o c a t e d  v a c u o l e was o b s e r v e d  i  papers  o f an a c r o c h a e t i o i d  these  i n o l d e r f i l a m e n t s of endophytes  older  the only  the f r e e - l i v i n g  e n d o p h y t e s were t h i c k ,  diameter.  s t u d i e s of  Duby) a r e by Hymes and C o l e  hermannii,  of the c e l l s .  free-living cell  i  ultrastructural  (see above),  the general f i n e  (Audouinella  near  types; t h i s  This i s consistent  and C o l e  Other  (1983a). Other  and  chloroplast  The two s p e c i e s o f e n d o p h y t i c  o b s e r v a t i o n s of Pueschel  few  s p . (Hara and  s t u d i e s by Hymes and C o l e ,  p l u g s w i t h two c a p l a y e r s .  Cole  s p e c i e s , such  s p . ( M i t r a k o s , 1960).  (1973) who r e p o r t e d a m o d i f i e d  chloroplast.  pit  Rhodochorion  s p p . have d i f f e r e n t  Lichtle, of  Acrochaetium  1970),  Similar  Audouinella  shown i n o t h e r  (Gibbs,  1974) and  Audouinella  shown  have been  (1973).  observed  (free-living)  i n one o t h e r (Garbary,  axes  s p e c i e s of  1978).  In  -37-  contrast, located  the nuclear  position  (Hymes and C o l e ,  Culture endophytic  studies  characters  free-living  endophytes.  similar  cycle  the  the  characters The d a t a  free-living  forms  free-living Het  i n the  were n o t m a i n t a i n e d revealed  porphyrae  dimensions,  in field  er s i phoni  not f i n d  and e n d o p h y t i c a  plumosa.  A.  m o r p h o l o g y when c o m p a r i n g  in cell  forms of  in  length  Garbary  vivo  are  a p p e a r s t o be However, t h e t h a t of  (1979b) a l s o  endophyt  i ca  between isolated  White and Boney in cell  and  than  dimensions  A.  differences  the  vaga  that  reproductive  i s greater  In c o n t r a s t ,  any s i g n i f i c a n t  clearly  and  material.  forms  differences  i n the  b r a n c h i n g and g r o w t h  forms, and c e l l  (9 v s 3-7.5 urn).  some s i g n i f i c a n t  centrally  i f the  persisted  obtained  Audouinella  forms o f  t h e range e n c o u n t e r e d  endophytic  from did  material  d i a m e t e r s of the f r e e - l i v i n g  noted  t o determine  They b o t h d e m o n s t r a t e a m o n o s p o r a n g i a l  in their  within cell  Field  i n morphology, c e l l  patterns.  i s more  1983a).  in field  state.  free-living  hermanii  were c o n d u c t e d  free-living  the  A.  in  in  situ  ( 1970)  dimensions and forms o f  A.  endophytica.  It the  i s possible  differences  field  materials  represented thicker this  in cell  endophytes.  t h e above o b s e r v a t i o n s  by t h e h o s t  cell  influence  walls.  Porphyra  Pt er os i phoni  the branching  a  of the w a l l s  in habitats a r e much w a l l s and  a n d growth p a t t e r n s  The o r i e n t a t i o n o f m i c r o b i l s (Young,  walls  bipinnata  i n the c e l l  of the wall  1980) and t h e c h e m i c a l  of both h o s t s c o u l d  that  patterns i n  a r e a consequence of the d i f f e r e n c e  of f l o r i d e o p h y c e a e  constituent  from  dimensions and b r a n c h i n g  (50pm v s 20pm) t h a n  could  matrix  t o conclude  influence the  -38-  observed d i f f e r e n c e s The  walls  mannan  i n the branching  of m a c r o t h a l l i  (Frei  of  and P r e s t o n ,  Porphyra  pattern  a r e composed o f x y l a n and  1964) whereas members o f  Florideophyceae  have s u l f a t e d p o l y s a c c h a r i d e  the  ( M a c k i e and P r e s t o n ,  wall  matrix  Re-  and c r o s s - i n f e c t i o n s t u d i e s  Audouinella  porphyrae  A.  and  vaga  studied  demonstrated  were n o t h o s t  They  found A.  and  B o t h W h i t e and Boney  various  bonnemai  red algae  shells  endophytes  from  A.  only  red algae  studied simple  g r o w t h on g i v e n that  However, t h e y  grew e n d o p h y t i c a l l y  and o t h e r  ranging  grew on v a r i o u s  and h y d r o i d s .  endophytica  substrata.  endophytica  porphyrae  on  the hosts,  also  and  factors.  lack  nonendophytic  Differences  vaga,  matrix  other  only A .  such as  The  other  relationships  (1969,  1970) n o t e d  i n a h y d r o i d and a v a r i e t y of  began s h o r t l y a f t e r  of the given  differences  of endophytic walls  including  formed growth p a t t e r n s  spore  that  were  substratum.  For  i n r e s p o n s e s when g r o w i n g  the nature of t h e host  t h e development  The c e l l  infestans  t o some f o r m o f e p i p h y t i c  W h i t e a n d Boney  i n d i c a t e that  influences The  A.  conditions.  substrata  r e s p o n s e s when g i v e n  by t h e o r g a n i c  (1979)  both found that  grew e n d o z o i c a l l y  substrata.  A.  substrata  They  culture  Audouinella  i n various  entanglement  g e r m i n a t i o n and t h e g e r m l i n g s determined  and c u l t u r e  (not i t s normal h o s t ) .  established  showed d i f f e r e n t g r o w t h calcareous  field  both  specific.  under v a r i o u s  many e n d o p h y t e s s u c h a s soniae  that  (1969) and G a r b a r y  e n d o p h y t e s , under  that  and c e l l u l o s e i n  1974).  b o t h grew e p i p h y t i c a l l y on e i t h e r h o s t conditions.  of the endophytes.  cell  walls  of t h e endophytes.  growth c o u l d  r e s u l t from  of the j u v e n i l e host  plants  several may n o t be  -39-  "thick"  enough f o r t h e e n d o p h y t e s  argument  i s p r e s e n t e d by G a r b a r y  species  he  as sand  scouring  walls  studied  (1979b).  by  the  grow.  This Audouinella  f o r t h e some o f t h e  There c o u l d  o r wave a c t i o n  for penetration  t o p e n e t r a t e and  which  be  some mechanism  could  prepare host  such  cell  endophytes.  M o n o s p o r e p r o d u c t i o n i s t h e most common form of reproduction  i n t h e genus  monosporangia observed  A.  f o r both  a typical 1970;  within  feature  Boney,  porphyrae  production  and  A.  vaga.  Audouinella  Hymes and  (1979) n o t e d c h a n g e s  R e g e n e r a t i o n of  remnants o f o l d m o n o s p o r a n g i a l  o f many  1967;  Audouinella.  in different  (White and  C o l e , 1983b).  culture  White  and  Boney,  monosporangial  conditions.  Differences  p r o d u c t i o n were a l s o n o t e d  current  out  morphological reproductive conditions  features  in culture  s u c h as h a i r  s t r u c t u r e s were n o t  used  did  not  find  the  endophytes,  infestans,  carried  under  any  sexual reproductive  structures  endophyt  i ca,  A.  as par  Other  and s e x u a l  White  A.  in  i n the  conditions.  i n the p r e s e n t study.  any  even  cells  seen  t o be  Boney,  abundance of m o n o s p o r a n g i a l experiments  was  This appears  spp.  i n t h e abundance of  walls  agops  culture  and  Boney  (1979)  or h a i r  i s  and  cells  in  A.  when e x p e r i m e n t i n g w i t h d i f f e r e n t  daylengths  Audouinella  (West)  and  temperatures. In  the asexual s p e c i e s ,  G a r b a r y Hansen e t S c a g e l , West production photoperiod been hairs  was  with hair  free-living  (1971) n o t e d t h a t  primarily  or temperature.  reported from  related  cells  proskaueri  to l i g h t  Field  intensity  material  of  (Garbary et a l . ,  cultures  hair  A.  rather  porphyrae  1982)  of the endophytes  cell  but  were  than has  no  observed  '  - 40-  under d i f f e r e n t photon m"  2  sec" ).  The  1  porphyrae  may  conditions, genetic  lack  flux densities of  hairs  r e s u l t from an  or  the  fact that  environmental  insufficient features  to  that  particular  absence of the  combinations  of  tetrasporangia,  hair c e l l s ,  such s t r u c t u r e s In  between  i n t e n s i t y are  are  their studies  Hansen e t  Scagel,  two  suggested  not  of  t h e y may  several  have  the  and  P e r h a p s more  photoperiod, induce It  these  is also  (Drew) G a r b a r y ,  (1984) n o t e d arcuata  similarities A. vaga  and  tetrasporophytic  s i n g l e sexual  possible  entities.  arcuata  A.  be  morphological  field.  needed t o  Garbary  may  varying  life  erect  filaments  triphasic,  trimorphic  t e t r a s p o r o p h y t e s are extensive  erect  S t e g e n g a and  life  1976;  history.  There  (Naegeli)  and  A.  mi croscopi  Woelkerling  gametophytic In  these  stages  in  life histories,  of m u l t i c e l l u l a r b a s e s and S t e g e n g a and  Mulder,  1979;  see  Woelkerling,  1983).  Hansen  A. ca  arcuata  with a  also with a  growing with  A.  vaga  few on  Pterosiphonia  bipinnata.  I have a l s o  observed  collections.  Audouinella  arcuata  A.  and  are  and  (eg.  (1984) have o b s e r v e d  filaments,  the  histories.  comprised  filaments  Vroman,  are  and  and  e x a m p l e s where e p i p h y t e s w i t h u n i c e l l u l a r b a s e s  diminutive  Garbary  of  f o r m e d by  represent  g a m e t o p h y t i c p h a s e s of a  not  gametangia.  Audouinella:  culture  laboratory  i n the  Audouinella  on  Hansen and  species  that  i n the  seen  and  A.  and  I t would a p p e a r t h a t  conditions  light  microeinstein  vaga  suitable  induce c e r t a i n reproductive  were i n f r e q u e n t l y  55  A.  of  e n t i t i e s do  factors  t e m p e r a t u r e and  that  25,  in cultures  p o t e n t i a l t o p r o d u c e them.  individual  (4-5,  few  filaments  same h o s t ,  t h i s i n my  microscopica  and  erect  erect the  more  are  field  -41-  diminutive plants with u n i c e l l u l a r axes and s i n g l e ,  stellate A.  (1984) s u g g e s t e d t h a t vaga.  Audouinella  chloroplasts. might  arcuata  from San Juan  A.  at  10°C 16:8 h (L:D) a t a p h o t o n 2  structures to  gametophytic  Other  culture  f o r A.  porphyrae  porphyrae  and  least life  results  of t h e i r  in their  originial  sexual l i f e  data  because  under  a s shown from t h e  could  histories,  then a t  be s y n o n y m i z e d  forms,  and b o t h showed  implicata  with  this  complex.  its'  host  (Halosaccion  experiments.  (Table V I ) , I propose  of l e s s (Drew,  Audouinella  var.  provisionally  i f A.  Thus,  when t h e  o b s e r v a t i o n s , the endophytes a r e  free-living  description  endophytes  1982).  and i f t h e y have t h e s u g g e s t e d  Audouinella  are conspecific.  vaga  (sensu  1979) maybe a p o s s i b l e  in culture.  i n t h e r e - and c r o s s - i n f e c t i o n  endophytes  ca  are resolved.  and c u l t u r e  A.  thesis  summarize c u l t u r e  identical  over  as part  histories  mi croscopi  a r e t h e same e n t i t y  three s p e c i e s of  To  field  vaga  s t u d i e s of t h i s  gametophytes  o f 4-5  (Garbary e t a l . ,  needs t o be i n v e s t i g a t e d  culture  I s l a n d was grown  c o n d i t i o n s a r e needed  et a l . ,  This also  A.  A.  Similarly,  1982 n o t S t e g e n g a  phase  density  phase  1  on t h i s p l a n t .  et a l . ,  be t h e g a m e t o p h y t i c  s e c " , a n d d i d n o t o b s e r v e any s e x u a l  induce gametogenesis.  Garbary  flux  erect  Hansen and G a r b a r y  arcuata  of  m i c r o e i n s t e i n s m"  b a s e s , one t o s e v e r a l  Audouinella  that  porphyrae  similar B a s e d on  t h e two i s chosen  taxonomical confusion i n i t s 1928).  I propose  porphyrae.  synonymizing the  Another  taxon  A.  vaga  (Drew) G a r b a r y , Hansen e t S c a g e l i s a s s o c i a t e d It differs  from  glandiforme  may be s y n o n y m i z e d  A.  vaga  var.  vaga  only i n  (Gmelin) R u p r e c h t ) , and with  A.  vaga  (Garbary e t a l . ,  -42 -  1982). A complete  listing  synonyms o f Audouinella  Audouinella  of the taxonomic porphyrae  porphyrae  1982 Basionym:  Rhodochor Chromastrum Kylinia Audouinella  In c o n c l u s i o n , information  on  porphyrae  Drew  1928: 188  history  ecological endophytes,  vagum vagum vagum  future  s t u d i e s a r e needed porphyrae  the current  would  life  S c a g e l 1982 [ 1 9 8 3 ] : 42 Drew 1928: 188 (Drew) J a o , 1937: 111 (Drew) S t e g e n g a a n d M u l d e r 1979: 305 i n Tarn t on implicatum Drew 1928: 190 implicatum (Drew) P a p e n f u s s 1945: 324 implicata (Drew) P a p e n f u s s 1 947: 438 vaga v a r . implicata (Drew) G a r b a r y Hansen e t S c a g e l 1982 [1983]:63 on  Audouinella  complement  alternate  well  (Drew) G a r b a r y , Hansen a n d S c a g e l , [ 1 9 8 3 ] : 42  Rhodochorton  Rhodochort Acrochaetium Chromastrum  life  follows:  Acrochaetium porphyrae (Drew) S m i t h 1944: 179 Chromastrum porphyrae (Drew) P a p e n f u s s 1945: 325 Kylinia porphyrae (Drew) P a p e n f u s s 1947: 438 Col aconema porphyrae (Drew) W o e l k e r l i n g 1971: 50 Audouinella porphyrae (Drew) G a r b a r y , Hansen e t  Synonyms:  would  i s as  and n o m e n c l a t u r a l  be u s e f u l  history  explored. crevices  A.  F o r example,  stages i n nature.  The e n d o p h y t e s of r o c k s s i n c e  that  a complete  More e x t e n s i v e  to determine  the l o c a t i o n  when t h e h o s t p l a n t s a r e a b s e n t d u r i n g  There are s e v e r a l  vaga)  i n understanding the possible  s t u d i e s a r e needed  the host p l a n t s . residing  (including  knowledge.  as the source of i n f e c t i o n  spring.  t o p r o v i d e more  f o r t h e young  possibilities could  infect  Another p o s s i b i l i t y  i n an a l t e r n a t e h o s t .  which  could form  the oldest  i s that  F o r example,  t h e w i n t e r as  hosts during  be i n a c r y p t i c  they both  of the  be i n the p o r t i o n s of  t h e y c o u l d be A.  vaga  early  var.  -43-  implicata,  i s d e s c r i b e d from  Halos  acci  on  a b u n d a n t d u r i n g w i n t e r months, however, is  a p o o r l y known t a x o n .  characterize s t u d i e s may  adequately add  relationships Audoui  nel  I a .  t o our  and  I t thus  glandiforme A.  vaga  g e n e r a l knowledge of  r e s o l v e t a x o n o m i c and  implicata  var.  becomes i m p o r t a n t  i t to resolve this  which i s  problem.  to Such  host-endophytic  ecological  problems i n  -44-  References: Baardseth, Res.  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Unusual c e l l  (Rhodophyta).  nell  Br.  wall  ultrastructure in  Phycol  J.  15: 119-124.  - 52-  FIGURE LEGENDS 1  Diagrammatic Pt erosiphoni  1a  Porphyra  representations a  bi pi nnat  blade:  3 = reproductive 1b  P.  bi pi nnat  a  a  of  showing  Porphyra  sp. and  a r e a s of  infection.  1 = holdfast; 2 = vegetative  region;  region.  thalli:  1 = h o l d f a s t ; 2 = main  3 = older branches; 4 = a p i c a l  growth.  filament;  -53-  -54-  Fig.  2  Cross-section with the  the endophyte, erect  portion Fig.  3  P.  4  filaments  heavily  endophyte  (e).  Pt erosiphoni  a  host Fig.  5  Close in  (e) g r o w i n g  from t h e p r o s t r a t e  up o f  A.  Scale  by A.  branching  Scale  b a r = 18pm  porphyrae.  pattern  of the  primary axis heavily A.  vaga  of the endophyte cells.  vaga  Note  b a r = 19pm  bipinnata  pericentral  (h),  porphyrae.  by t h e e n d o p h y t e  growth p a t t e r n  torta  Audouinella  infected  the i r r e g u l a r  infected  Porphyra  (p) o f t h e e n d o p h y t e .  torta  Note  Fig.  of l i v i n g  Scale  (en).  Note  i n the walls  of the  b a r = 75pm  ( a r r o w ) a s i t grows a n d b r a n c h e s  the wall of i t s host.  Note  the branching  Scale  b a r = 25pm  c  and growth p a t t e r n  of t h e e n d o p h y t e .  -56-  Fig.  6-9:  Micrographs Porphyra  fucicola  Contrast Fig.  Fig.  6  7  A spore  of l i v i n g  taken  Audoinella  porphyrae/  w i t h Nomarksi  Modulation  Microscopy. A.  of  field  porphyrae  w i t h an i n t e r n a l  Note g e r m i n a t i n g  cell  (arrow).  Note g e r m i n a t i n g  cell  ( a r r o w ) from  division.  S c a l e b a r = 15pm settled  spore.  S c a l e b a r = 11 urn Fig.  8  P r o s t r a t e f i l a m e n t s i n the host c e l l prominent bar  Fig.  9  Extensive  branching  internally  pattern.  S c a l e b a r = 14pm  A . porphyrae  s e t t l i n g and  ( a r r o w ) on a p l a s t i c  cell  from  settled  spore  12 G r o w i n g  filament.  (arrow).  13 P r o s t r a t e c e l l s erect  Cytoplasm  Scale  i s being  axial  (p) o f  A.  porphyrae  f i l m e n t s (e) ( a r r o w s ) .  chloroplast  large vacuole  expanding  S c a l e b a r = 8pm  14 E r e c t f i l a m e n t s o f  a  plate.  = 8pm  vacuole  Fig.  petri  (arrow).  " p u s h e d " t o w a r d s t h e g r o w i n g t i p by an  Fig.  Scale  b a r = 12pm  11 G e r m i n a t i n g bar  (arrow).  by p r o s t r a t e f i l a m e n t s i n t h e h o s t .  10 Monospore o f f r e e - l i v i n g  Scale  Fig.  infection  irregular  dividing  Fig.  i n the c e l l  Note  = 14urn  Note Fig.  pyrenoid  walls.  A.  porphyrae  with  initial  S c a l e b a r = 20pm showing a  (c) with a c e n t r a l  (v).  two  stellate,  pyrenoid  S c a l e b a r = 13pm  (py) and  -58-  Figs.  15-21:  15 E r e c t  both  endophytes a r e i d e n t i c a l ,  free-living  porphyrae  16 E r e c t  filaments with  filaments  17 M o n o s p o r a n g i a erect  Figs. Fig.  18 E r e c t  branchlets Fig.  Fig.  Fig.  arrow).  Contrast  and/or  terminal  Note  many l a t e r a l  filaments.  cell  Scale  growing  (arrow).  i n t o an empty  Scale  sporangium.  b a r = 17pm  c a n be p r o d u c e d f r o m one  o l d wall  a r e formed  on s e c o n d a r y  b a r = 8pm  layers  one t o two o r o c c a s i o n a l l y  b a r = 17pm  on an  monosporangium  (arrow).  b a r = 17pm  monosporangia  b a r =15pm  f i l a m e n t s c a n a l s o be f o r m e d  the vacuole  21 U s u a l l y  Scale  Scale  i n an  Modulation Microscopy  20 F o u r t o s i x m o n o s p o r a n g i a  Scale  o f a new c e l l  b a r = 15pm  (arrows).  sporangium.  form  discharged  Note p e d i c e l s below  19 A b u d d i n g a p i c a l Note  (dark  filaments with  Monosporangia  branches which  arrow) with  c a n be l a t e r a l  Scale  18-21: N o m a r s k i  lateral  Note p r o d u c t i o n  filament.  (arrow).  only  b a r = 14pm  (clear  empty monosporagium Fig.  short  Scale  monosporangia.  (since  i s shown).  monosporangia. Fig.  porphyrae  of l i v i n g  A.  Fig.  A.  Culture material  three  from one c e l l .  -60 -  Figs.  22-25: U n i n f e c t e d Porphyra  and Fig.  living  blades  Pt er os i phoni  used  cross-infection  22 A young b r a n c h  a  bipinnata  and  for re-infection experiments.  on t h e main a x i s  of  P.  bipinnata.  S c a l e b a r = 375pm Fig.  23 A p i c a l  t i p s of  (arrow). Fig.  P.  24 The main a x i s  25 C l e a n e d  tend  to curve  adaxially  S c a l e b a r = 375pm of c u l t u r e d  ( r ) and a b r a n c h . Fig.  bipinnata  Porphyra  P.  bipinnata  with a r h i z o i d  S c a l e b a r = 188pm blade.  S c a l e b a r = 90pm  - 6 1 -  -62-  Figs.  26-34: C r o s s - i n f e c t e d on on  Fig.  Pier  living  of  (arrow).  28 E r e c t  Fig.  29 E r e c t P.  Fig.  Fig.  filament  internal  divison  from s e t t l e d  spore  (arrow).  growing  penetration  f r o m a monospore.  by t h e b a s a l  cell  Note t h e  (arrow).  b a r = 12pm filaments  g r o w i n g on r h i z o i d Scale  Scale  31 O l d e r  apical  (r)  of  b a r = 45pm  g r o w i n g on a r h i z o i d  of i n f e c t e d  b a r = 45pm a x i s of host  covered  with  epiphytes.  b a r = 45pm  32 E p i p h y t e s  g r o w i n g on a b r a n c h i n g  point  of the host.  b a r = 50pm infection  host. Fig.  filament  host.  33 Heavy  Note  infecting a  b a r = 10pm  30 T u f t s o f e p i p h y t e s  Scale Fig.  bipinnata.  bipinnata.  bipinnata.  Scale Fig.  na  b a r = 10pm  partial Scale  P.  Scale  27 Note e r e c t Scale  Fig.  phi  porphyrae  26 G e r m l i n g s o f e p i p h y t i c A . porphyrae rhiziod  Fig.  osi  Audouinella  Scale  infected  A.  porphyrae  obscuring the  b a r = 50pm  34 A young a p i c a l heavily  by  shoot  of  i n f e c t e d host. (arrows).  P.  bipinnata  growing  from  The new s h o o t h a s a l r e a d y  Scale  b a r = 40pm  been  -64-  Figs.  35-37:  Audouinella Porphyra  Fig.  35 I n f e c t i o n  vaga lorta  cross-infecting  area  Few e p i p h y t e s  of the blade.  filaments of the epiphytes Fig.  36 E r e c t  f i l a m e n t s of  extensive Fig.  blades  A.  branching  37 B a s a l c e l l s  vaga  bipinnata.  Note the long  erect  S c a l e b a r = 30um  on h o s t .  Note t h e  of the e p i p h y t e .  (p) o f e p i p h y t e s  P.  a r e seen on t h e  (arrow).  (h) a r e more e x t e n s i v e  g r o w i n g on  living  blade.  i s sparse.  vegetative  a  (e)  S c a l e b a r = 30um  g r o w i n g on  (arrow) than  S c a l e b a r = 15um  Porphyra  epiphytes  -6  Figs.  38-41: M e t h a c r y l a t e Porphyra  sections  fucicola  6-  of  Audouinella  (2.5um) s t a i n e d  porphyrae/  with  Toluidine  Blue-O. Fig.  38 E x t e n s i v e Scale  Fig.  P.  within  Fig.  growing  Majority  the outer  wall  of the host.  (arrow). 41 An empty the  (p) i n h o s t  cell  wall.  between h o s t of e n d o p h y t i c  cells growth i s  of the host.  b a r = 36pm  40 C l o s e - u p o f an e r e c t wall  Fig.  filament  fucicola.  Scale  filaments  b a r = 40pm  39 P r o s t r a t e of  prostrate  host  Scale  filament  Note t h e i n n e r  wall  of the host  b a r = 11pm  monosporangium cell  a s i t grows t h r o u g h t h e  wall.  on an e r e c t  Scale  b a r = 8pm  filament,  outside  - 6 7 -  -68-  Figs  42-46: M e t h a c r y l a t e fucicola  42  Close  up  43  44  (Figs.  Close  up  cells  (arrow) w i t h i n the  of p r o t e i n o u s  the c u t i c l e  stained.  Scale Fig.  stain bar  (Figs.  =  44-46).  7pm  i n n e r w a l l of t h e h o s t .  of an  =  S c a l e bar  substance  (dark  i n the cytoplasm  the h o s t .  P i t plug  =  host Erect  9pm  s t a i n ) i s seen of b o t h  the  (arrow) i s a l s o  14pm  erect i n i t i a l  i n the host  cell  wall beside  (arrow).  the  Note  protuberance.  = 6jum  46 Note t h e d a r k the outer  (cu) and  S c a l e bar  Protuberance dark  S c a l e bar  (e) a r e a l s o p r e s e n t .  e n d o p h y t e and  45  F a s t Green  of p r o s t r a t e p o r t i o n s g r o w i n g between two  Suggestion on  Fig.  42-43) and  w a l l of t h e h o s t .  filaments Fig.  porphyrae/Porphyra  of p r o s t r a t e p o r t i o n s g r o w i n g e x t e n s i v e l y i n  the o u t e r Fig.  A.  (2.5um t h i c k ) s t a i n e d w i t h T o l u i d i n e  Blue-0 Fig.  s e c t i o n s of  stain  just  above the endophyte c l o s e to  edge of t h e h o s t  (arrow).  S c a l e bar  =  3pm  -7 0-  Figs  47-50: M e t h a c r y l a t e Pt er os i phoni  with Fig.  Erect  of a p e r i c e n t r a l  the host  49 E n d o p h y t i c  against  growing a l o n g the  with a large vacuole ( v ) . from p r o s t r a t e f i l a m e n t s  A.  vaga  into  cell.  the cytoplasm  S c a l e b a r = 6pm  growth l i m i t e d  by t h e h o s t  wall  (dark  ( v ) and c y t o p l a s m  the p e r i p h e r y of the host  50 F i l a m e n t s  of the host  (n) a n d l a r g e v a c u o l e s ( v )  Note t h e l a r g e v a c u o l e  arrow).  cells  cell  Note t h e n u c l e u s  arrow).  Fig.  Audouinella  S c a l e b a r = 8um  48 P e n e t r a t i o n of  of  (2.5um t h i c k ) s t a i n e d  f i l a m e n t s a r e produced  (arrow).  Fig.  bipinnata  vaga/  T o l u d i n i e Blue-O.  (arrow). Fig.  a  47 Note t h e f i l a m e n t s o f wall  Audouinella  s e c t i o n s of  cell  (clear  S c a l e b a r = 8pm o f t h e e n d o p h y t e g r o w i n g between two  of the host  (arrow).  S c a l e b a r = 10^im  -7 2-  Figs.  51-53: M e t h a c r y l a t e  s e c t i o n s of  A.  vaga/Pt  (2.5um t h i c k ) s t a i n e d w i t h A l c i a n and Fig.  51  F a s t Green  Endophyte branching (arrow).  52  Suggestion within  =  cytoplasm  of  both  of  darken area  the host  53 A  few  bar  =  of  Blue/PAS  host c e l l  branching  ( F i g . 51)  p a t t e r n of  substance  the  located  e n d o p h y t e and  the endophyte  host.  (arrows).  wall just  Note  Note  above t h e  i n host  S c a l e bar  =  8pm  wall beside  endophyte.  (arrow).  the  the  the  10pm  Note d a r k e n e d a r e a s  a  wall  e r e c t f i l a m e n t s (e) o u t s i d e of h o s t  endophyte.  phoni  10pm  of p r o t e i n a c e o u s  large vacuoles  Scale Fig.  w i t h i n the  S c a l e bar  osi  52-53).  Note the a n g u l a r  endophyte. Fig.  (Figs.  er  81  -74-  F i g s 54-56: U l t r a s t r u c t u r e of Porphyra  Fig.  fuci  col  Audouinella  a  54 Note the l a r g e vacuole (c) to  The host c e l l  be a f f e c t e d by the endophyte.  pit  plug, (arrow).  56 Note the nucleus of  the c e l l .  chloroplast  w a l l appears  Note the i r r e g u l a r  w a l l (hew).  S c a l e bar = 2pm  55 S e c t i o n through a growing f i l a m e n t of i n s i d e the host.  Fig.  (v) and the s t e l l a t e  i n the endophyte c e l l .  matrix of the host c e l l Fig.  porphyrae/  A.  Note the m i t o c h r o n d r i a  porphyrae  (m) and h a l f  S c a l e bar = 1pm (n) of the endophyte towards the end  Part of the s t e l l a t e c h l o r o p l a s t  seen w i t h i n the vacuole  ( v ) . Scale bar = 2pm  (c)  is  -76 -  Figs. Fig.  57 A c e n t r a l in  58 A  bar =  nucleus  Scale Fig.  59 C l o s e - u p  60 O v e r v i e w matrix.  (py) with  o f a P.  fucicola.  mitochondria  fucicola  (arrow)  cell.  4pm  (n) w i t h  bar =  regular Fig.  pyrenoid  the periphery  Scale Fig.  o f u n i n f e c t e d Porphyra  57-60: U l t r a s t r u c t u r e  mitochrondria  (m) a r o u n d i t .  1pm  of the c e l l fibrous  matrix.  o f P.  fucicola  Scale  bar =  o f P.  wall  Bar = inner  1.5pm  fuci  col  a.  Note t h e  0.2pm wall.  Note  the regular  -7 7-  -78 -  F i g s 61-64: U l t r a s t r u c t u r e of fuel  Fig.  61 S e c t i o n  Audouinella  porphyrae/Porphyra  cola.  of endophyte growing o u t s i d e  of the h o s t .  Note the i r r e g u l a r matrix of the host c e l l A mitochondrion  62 S e c t i o n  Scale  bar = 0.5pm  through the c h l o r o p l a s t of the endophyte  showing a c e n t r a l pyrenoid thylakoids Fig.  (py) with i r r e g u l a r  (arrow) t r a v e r s i n g i t .  Scale  63 Note the i r r e g u l a r matrix of the host the w a l l of the endophyte (ecw).  Fig.  64 Enlargement of endophytic c e l l wall  (hew).  (m) and p a r t s of the c h l o r o p l a s t (c)  of the endophyte. Fig.  wall  (hew).  Scale  bar = 0.2pm  (hew) compared to  Scale  wall  bar = 1pm  bar = 0.8_pm  (ecw) and host  cell  -80-  Figs.  65-68: U l t r a s t r u c t u r e o f  Audouinella  vaga/  Pt er os i phoni  a  bipinnata.  Fig.  65 S e c t i o n  of a part  regular  matrix  of u n i n f e c t e d  of t h e w a l l  (v) a n d m i t o c h r o n d r i o n Scale Fig.  66 S e c t i o n  Host c e l l  67 S e c t i o n showing  Scale  wall  at the periphery  (hew) d o e s  of a  vacuole  of the c e l l .  o r g a n e l l e s of the host  Scale  follows  Note  cell  w a l l (hew)  (h) w h i c h do n o t a p p e a r  large vacuole  of endophyte  b a r = 2pm of t h e endophyte (e)  t h e l o n g i t u d i n a l growth  b a r = 2pm  outside the  n o t a p p e a r t o be  i n s i d e the host  68 Note how t h e g r o w t h p a t t e r n  Scale  showing t h e  b a r = 0.2um  of endophyte  be a f f e c t e d .  (arrow). Fig.  (hew) and p a r t  of t h e endophyte p r o t r u d i n g  affected.  to  cell  b a r = 0.8um  host.  Fig.  host  of t h e h o s t .  - 8 2  Figs.  69-71: U l t r a s t r u c t u r e porphyr  Fig.  of  ae.  the c e l l .  arrow).  with  Note m i t o c h o n d r i a  of the c e l l  a c e n t r a l p y r e n o i d (py)  (double  N o t e t h e two c a p l a y e r s .  b a r = 0.5pm  frequently  Scale  wall  S c a l e b a r = 1pm  71 A p r o m i n e n t n u c l e u s  starch  (m) a l o n g t h e  and t h e t h i c k  70 C l o s e up o f a p i t p l u g . Scale  Fig.  Audouinella  the florideophycean s t a r c h (s) at the periphery  periphery  Fig.  of f r e e - l i v i n g  69 Note t h e l a r g e c h l o r o p l a s t and  -  (n) w i t h a n u c l e o l o u s  (nu)  l o c a t e d a t t h e base of t h e c e l l .  grains  (s) surrounding  b a r = 1pm  the nucleus.  is  Note t h e  -84 -  Figs. Fig.  72-75: U l t r a s t r u c t u r e o f f r e e - l i v i n g 72 Note t h e numerous l a y e r s of a g r o w i n g the  Fig.  filament.  Scale  wall  (n) t o w a r d  Note t h e n u c l e u s ( n ) , of a  stellate  t h e c y t o p l a s m of the c e l l .  74 C r o s s s e c t i o n o f a f i l a m e n t .  75 S e c t i o n  grains  (s) around  it.  Note t h e n u c l e u s Scale  through the p r o s t r a t e p o r t i o n  Note t h e c e n t r a l p y r e n o i d grains  the c e l l  b a r = 1pm  (mb) a n d p a r t s  (c) w i t h i n  vaga.  b a r = 0.8pm  starch Fig.  Scale  73 C l o s e - u p o f a p i c a l t i p .  chloroplast  Fig.  (arrow) w i t h i n Note t h e n u c l e u s  base of the c e l l s .  membranous b o d i e s  Audouinella  (s).  Scale  b a r = 0.8um of t h e endophyte.  ( p y ) a n d numerous  b a r = 2pm  (n) a n d  starch  

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