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Detoxification of thujaplicins in living western redcedar (Thuja plicata Donn.) trees by microorganisms Jin, Lehong 1987

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DETOXIFICATION OF THUJAPLICINS IN LIVING WESTERN REDCEDAR (Thuja  piicat  a Donn.)  TREES BY MICROORGANISMS by LEHONG JIN B. Eng. Nanjing F o r e s t r y U n i v e r s i t y 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the FACULTY OF GRADUATE STUDIES (Department of F o r e s t r y ) We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA February, 1987 ©Lehong J i n , 1987  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  Department  V6T  DE-6(3/81)  1Y3  Columbia  I  further  purposes  gain  shall  that  agree  may  representatives.  financial  permission.  T h e U n i v e r s i t y o f British 1956 M a i n M a l l Vancouver, Canada  study.  requirements  It not  be is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  head  make  it  extensive of  my  copying  or  my  written  ABSTRACT  Thujaplicins volatile  are  the major components i n {Thuja  f r a c t i o n of western redcedar  (WRC) heartwood e x t r a c t i v e s . t o x i c to fungi and are c h i e f l y  T h i s study proves that t h i s  concept of t o x i c i t y  i s not completely c o r r e c t .  toxic  wood  in  al bipel as  to common decay fungi living  Sporothrix  sp.  thujaplicins altered fungi  by  do  heartwood  traditional Thujaplicins  i s o l a t e d from decayed  t r e e s or wood i n  I uci da Baxter.  to be h i g h l y  f o r WRC  decay r e s i s t a n c e .  are  steam-  pi i cat a Donn.)  They are consided responsible  the  service,  On the other  such  WRC Poria  as  hand, when a fungus such  invades sound heartwood of l i v i n g  not  provide  that fungus,  resistance  but  WRC,  instead  so that t h e i r t o x i c i t y  to  are decay  i s destroyed. Evidence  mechanism involves  of the  thujaplicin blocked  by  obtained  i n t h i s study i n d i c a t e s  t h u j a p l i c i n t o x i c i t y to reactive  toxicity  keto-enolic  methylation. hri x sp.  As  fungi  example,  living  trees  and i s o m e r i z a t i o n of the  t o a new lactone compound.  ii  In  is  i s demonstrated t o i n v o l v e  a process of o x i d a t i v e d i m e r i z a t i o n thujaplicins  group.  decay  the  disappears i f t h i s r e a c t i v e group  laboratory  d e t o x i f i c a t i o n by Sporot  common  that  T h i s compound  is  proven  to  al bipel  have no t o x i c i t y to decay f u n g i ,  I uci da  destroy  Baxter.  the  thujaplicin  The d i m e r i z a t i o n  reactivity  and  of the k e t o - e n o l i c  Porta  such as  isomerization  group  and  thus  toxicity.  Isolation,  purification,  and  determination  of  the  chemical s t r u c t u r e of the new lactone compound produced from t h u j a p l i c i n s during by  chemical,  This  sp. i n f e c t i o n was c a r r i e d out  chromatographic  naturally  previously  Sporothrix  and  spectroscopic  o c c u r r i n g compound has  and  there  are  no  not  previous  been  rules,  the  compound  is  named  as  hexamethyl-2,6-dioxa-1,5-anthracene-dione, trivial  name  show  experiments that  in  involves a succession attacking WRC  Phialophora  Following 3,3,4,7,7,8-  and  given  carried  living  WRC  out  in  trees,  of microorganisms.  study  fungal  attack  Three e a r l y stage  ei ni eI I a sp.  They thuji  are na  identified  as  discolored  Sporothrix  sp.  (Peck) Pomerleau & Etheridge  Biological  roles  of  these  demonstrated based upon the r e s u l t s of wood block and  the  this  fungi were c o n s i s t a n t l y i s o l a t e d from  heartwood.  Ki rschst  i n the  'Thujin'.  Biological clearly  isolated  reports  l i t e r a t u r e about a compound with t h i s s t r u c t u r e . IUPAC  methods.  chemical a n a l y s i s of wood blocks  fungi  and are  bioassays  t r e a t e d with the three  fungal  isolates.  TABLE OF CONTENTS Page ABSTRACT  i i V  TABLE OF CONTENTS LIST  OF  TABLES  ix  LIST OF FIGURES  xi  ACKNOWLEDGEMENTS  xiv  INTRODUCTION  1  LITERATURE REVIEW  7  2.1. Importance of western B r i t i s h Columbia 2.2. The  redcedar to the  forest industry  chemical composition of WRC  7 wood  2.3. Fungi and decay i n WRC  11 21  2.4. Tropolones, tropolone d e r i v a t i v e s and t h e i r p r o p e r t i e s  27  2.5. B i o l o g i c a l a c t i v i t i e s of wood e x t r a c t i v e s  45  2.6. P o s s i b l e mechanisms of e x t r a c t i v e t o x i c i t y  58  MATERIALS AND METHODS  69  3.1. Sample s e l e c t i o n  69  3.2. I s o l a t i o n c f microorganisms  74  3.3. Determination of e x t r a c t i v e s  77  3.4. I d e n t i f i c a t i o n of WRC Heartwood extractive fractions 3.5. Wood block bioassay  83 85  v  3.6. I s o l a t i o n and i d e n t i f i c a t i o n of an unknown compound from d i s c o l o r e d heartwood e x t r a c t i v e s a) Solvent f r a c t i o n a t i o n b) Column chromatography c) Thin l a y e r chromatography d) C r y s t a l i z a t i o n and elemental a n a l y s i s of the unknown compound e) Mass spectrometry f) Nuclear magnetic resonance spectrometry (proton and carbon-13 NMR) g) V i s i b l e and u l t r a v i o l m a - t h u j a p l i c i n h) I n f r a r e d  Spectrometry  92 92 93 95 96 97 97 98  3.7. T o x i c i t y t e s t b i o a s s a y s  100  a) T o x i c i t y of t h u j a p l i c i n b) T o x i c i t y of the unknown compound  100 102  c) T o x i c i t y of methylated t h u j a p l i c i n  104  3.8. Examining o r i g i n of the new unknown compound 3.9. D e t e c t i o n  107  of metal c h e l a t e s from d i s c o l o r e d  WRC heartwood  toluene e x t r a c t i v e s  RESULTS AND DISCUSSION  110 112  4.1. Microorganisms i s o l a t e d and t h e i r d i s t r i b u t i o n in heartwood of WRC 112 4.2. E x t r a c t i v e d i f f e r e n c e s between sound and d i s c o l o r e d WRC heartwood 119 4.3. B i o l o g i c a l and chemical r o l e s of Sporothrix sp. K.  thujina  and Phialophora  sp.  in l i v i n g WRC t r e e s 4.4. E l u c i d a t i o n of the unknown compound structure  131 1 43  4.5. Mechanisms of thu j a p l i c in ' s t o x i c i t y 4.6. Future p e r s p e c t i v e s  177 183  CONCLUSIONS  185  LITERATURE CITED  188 vi  APPENDIXES  201  Appendix 1 . l i s t of Synonymi f o r the Fungi Used i n the Text.  201  Appendix 2. T h u j a p l i c i n Content from O l d Growth by C o l o r i m e t r i c Method  WRC  Appendix 3. T h u j a p l i c i n Content from O l d Growth by Gas Chromatography Method  WRC  Appendix 4. T h u j a p l i c i n Content from Second Growth by Gas Chromatography Method  202  203 WRC  Appendix 5. Check L i s t of Fungi C o l l e c t e d on Western in B r i t i s h Columbia from 1943 to 1945  204  Redcedar 205  Appendix 6. R e s u l t s of A n a l y s i s of V a r i a n c e f o r the WRC Heartwood Weight Loss  208  Appendix 7. R e s u l t s of M u l t i p l e Range Tests f o r Wood Block Bioassay  212  Appendix 8. Chemical S h i f t s of Some Protons  213  Appendix 9. Chemical S h i f t s of Some Carbon-13 Appendix 10. IR Data f o r S e l e c t e d Absorption Bands  Resonances  Carbon-Hydrogen  Appendix 11. IR Data f o r S e l e c t e d X-H A b s o r p t i o n Bands  vii  214  215  216  Appendix 12. IR Data f o r S e l e c t e d Carbon-Carbon Bands  Absorption  Appendix 13. IR Data f o r S e l e c t e d Carbon-Oxygen Bands  Absorption  viii  217  218  LIST OF TABLES Table 1. The S i g n i f i c a n c e of WRC i n B.C. as a F o r e s t Resource and Timber Cut i n 1980.  8  Table 2. The Volume of D i f f e r e n t Grades of WRC Log Export from B.C. i n 1980.  8  Table 3. Chemical Hemlock  Composition of Western Redcedar, Western and D o u g l a s - f i r Wood.  Table 4. Composition Heartwood.  of Steam V o l a t i l e O i l from WRC Butt  12  16  Table 5. List  of Some Trees C o n t a i n i n g Terpenoid T r o p o l o n e s —  29  Table 6. Q u a n t i t a t i v e Analyses of Tropolones i n Cupr es s aceae . Table 7. Carbon-13 Chemical S h i f t s of S e v e r a l D e r i v a t i v e s of 2-methoxytropone i n C D C l j .  43  Table 8. R e s u l t s of T o x i c i t y T e s t s f o r a Number of Heartwood Extractives (Decay as a percentage of decay i n control) .  47  Table 9. C o n c e n t r a t i o n S e r i e s f o r Standard Solutions.  81  Gamma-thujaplicin  Table 10. Frequency of Microorganism I s o l a t i o n on Malt Agar from WRC Heartwood v s . Sample Age.  ix  30  1 13  Table 11. BE E x t r a c t i v e s and T h u j a p l i c i n of the WRC Heartwood.  Content  120  Table 12. TLC R Values f o r Four WRC Heartwood E x t r a c t s Using BE (9:1) S o l v e n t .  127  Table 13. TLC R Values of Known WRC E x t r a c t i v e s Using BE (9:1) S o l v e n t .  129  f  f  Table 14. R e s u l t s of Weight Loss and E x t r a c t i v e A n a l y s i s of WRC Sound Heartwood Blocks Treated with Sporothrix  sp. , K.  t huj i na and  Phialophora  sp.  133  Table 15. Data of the Low R e s o l u t i o n Mass Spectrum of the Unknown Compound.  148  Table 16. Broad Band and Attached Proton Test Carbon-13 NMR Spectra Data of the Unknown Compound.  157  Table 17. Atomic Weight of Isotopes Used i n High R e s o l u t i o n Mass Spectroscopy.  162  Table 18. F o u r i e r Transform IR S p e c t r a l Data of the Unknown Compound.  171  x  LIST OF FIGURES F i g u r e 1. The V o l a t i l e Components of WRC Extractives.  Heartwood  F i g u r e 2. The N o n - v o l a t i l e Components of WRC Extractives.  14  Heartwood  F i g u r e 3. S t r u c t u r e s of Methyl Ether, Conjugated A c i d and Amine S a l t s of Tropolone. F i g u r e 4. Oxidation  18 Cations  of H i n o k i t i o l with Hydrogen Peroxide.  F i g u r e 5. A n i o n i c S u b s t i t u t i o n and Tropolone.  Rearrangement of  F i g u r e 6. The Complex S t r u c t u r e between Tropolone, and S-Adenosyl Methionine (COMT) . F i g u r e 7. The Chemical S t r u c t u r e s of the WRC F e r r i c Chelates.  35 38  39 Magnesium  Extractive  62  67  F i g u r e 8. Tree from Which the Wood Samples Were Taken.  70  F i g u r e 9. Four 0.9  71  Meter B o l t s Cut  from the S e l e c t e d Tree.  F i g u r e 10. Cross S e c t i o n s of the S e l e c t e d Tree Showing C o l o r Variations. .  73  F i g u r e 11. R a d i a l Pieces S p l i t from a Quadrant and in the I s o l a t i o n of Microorganisms (A) and E x t r a c t i v e s (B and C).  75  xi  Used  F i g u r e 12. C a l i b r a t i o n Curve: Absorbance vs F e r r i c Chelate C o n c e n t r a t i o n s .  Gamma-thujaplicin  82  F i g u r e 13. Growing WR1, WR2 and WR3 C u l t u r e s I s o l a t e d from D i s c o l o r e d WRC Heartwood (Piece A ) .  86  F i g u r e 14. WR1, WR2 and WR3 C u l t u r e s Used i n the Wood Block Bioassay.  88  F i g u r e 15. P e t r i D i s h Prepared f o r T o x i c i t y T e s t s .  101  F i g u r e 16. I n h i b i t i o n of Porta al bipel I uci da Baxter by B e t a - t h u j a p l i c i n at S i x t e e n D i f f e r e n t Concent r a c t i o n s .  103  F i g u r e 17. Inhibition  of Porta  albi  pel Iuci  da  Baxter  by the Unknown Compound, Methylated and T h u j a p l i c i n s .  Thujaplicins  105  F i g u r e 18. Histogram (No.1) of Percentage Frequency D i s t r i b u t i o n for S t e r i l e Wood and WR1, WR2 and WR3 Fungi I n f e c t i o n s vs. Sample Age. 114 F i g u r e 19. Histogram (No.2) of Percentage Frequency D i s t r i b u t i o n s for S t e r i l e Wood and WR1, WR2 and WR3 Fungi I n f e c t i o n s v s . Sample Age. 115 F i g u r e 20. TLC P l a t e s of BE E x t r a c t i v e s from WRC D i s c o l o r e d and L i g h t - s t r a w C o l o r e d Heartwoods.  126  F i g u r e 21. Low R e s o l u t i o n Mass Spectram of the Unknown Compound.  1 47  F i g u r e 22. Proton  152  NMR  Spectrum of the Unknown Compound.  xii  F i g u r e 23. Broad Band and Attached Proton Test Carbon-13 Spectra of the Unknown Compound.  156  F i g u r e 24. High R e s o l u t i o n Mass Spectrm of the Unknown Compound.  1 63  F i g u r e 25. Chemical S t r u c t u r e of " T h u j i n " .  167  F i g u r e 26. UV Spectrum of the Unknown Compound.  168  F i g u r e 27. V i s i b l e Spectrum of the Unknown Compound.  169  F i g u r e 28. F o u r i e r Transform IR Spectrum of the Unknown Compound.  170  xiii  ACKNOWLEDGEMENTS  I  wish  to  appreciation Adjunct  express  my  to my r e s e a r c h  Professor,  Canada Corp.  sincere  gratitude  supervisor,  U.B.C.,  Research  Dr.  E.  Scientist,  and my academic s u p e r v i s o r Dr.  and P.  deep Swan,  Forintek  J . W. Wilson,  P r o f e s s o r , D i r e c t o r of F o r e s t r y Graduate S t u d i e s , U.B.C. t h e i r f i r m guidance, kind a d v i c e ,  for  understanding and constant  encouragement throughout the course of t h i s  study.  I a l s o express my s p e c i a l acknowledgement to Dr. B. van der  Kamp,  Associate Professor,  for h i s guidance, the  (Forest Pathology) U. B. C.  rewarding d i s c u s s i o n s ,  b i o l o g i c a l phase of the r e s e a r c h .  to Dr.  S.  Z.  for t h e i r  U.B.C., members of the s u p e r v i s o r y  i n t e r e s t and support  technologists,  technical study.  Special  elemental  thanks  given  to J .  Forintek  assistance during  Chemistry,  Canadian F o r e s t  and Dr. F. G. H e r r i n g , P r o f e s s o r , Department  I am a l s o i n indebted Clark,  Many thanks are due  Chow, D i r e c t o r of Research,  Products L t d . of Chemistry,  and a s s i s t a n c e i n  the  to me.  Nault, Canada  B.  D a n i e l and J .  Corp.  experimental  for  their  phase of the  are extended to the Department  U.B.C. f o r p r o v i d i n g s p e c t r o s c o p i c analyses;  committee,  t o F o r i n t e k Canada Corp.  xiv  services  of and  for providing  working  space and  Corlett,  research f a c i l i t i e s ;  Identification  Service,  and  to  Dr.  Biosystematic  M.  Research  I n s t i t u t e , A g r i c u l t u r e Canada, Ottawa, f o r i d e n t i f y i n g fungi  i s o l a t e d during the I  am  People's  very  grateful  Republic  to the  Ministry  of  of China and The U n i v e r s i t y  of a N a t i o n a l Overseas Graduate S c h o l a r s h i p  first  three  study. Education, of  British  Columbia, Canada f o r t h e i r generous f i n a n c i a l support form  P.  i n the  (for  the  year) and a U n i v e r s i t y Graduate F e l l o w s h i p ( f o r three  years),  respectively.  Finally,  I  would l i k e to thank my  l o v e , understanding  and  parents  for  their  encouragement which have been a main  source of s t r e n g t h .  xv  INTRODUCTION  Western the  most  important  (B.C.) and woods. fact  (WRC)(Thuja  pii  cat a Donn.) i s one  c o n i f e r o u s woods of  i s recognized  as one  British  that  fallen  floor  WRC  of the world's most  t r e e s may  f o r c e n t u r i e s with  the t h i n sapwood l a y e r .  areas  of B.  C,  there  sometimes  little  On  durable  remain  on  the the  apparent decay except  the other  is s t i l l  of  Columbia  I t s n a t u r a l - r e s i s t a n c e to decay i s i n d i c a t e d by  forest in  redcedar  hand,  in c e r t a i n  a s e r i o u s problem  heartwood decay, e s p e c i a l l y i n mature and  of  overmature  WRC  trees.  "On the B. C. coast e x t e n s i v e columns of butt and trunk r o t , o f t e n a s s o c i a t e d with basal scars, are common. In some areas in the i n t e r i o r of the p r o v i n c e , standing WRC t r e e s consist of little more than a shell of sapwood, the heartwood being completely destroyed by decay. In B. C. as a whole, the amount of decay expressed as a percentage of the t o t a l gross volume of wood i s greatest for WRC (32%). T h i s i s more than with any other major c o n i f e r (average 12%)" (van der Kamp, 1975). This  discrepancy It  needs to be e x p l a i n e d  i s long understood from WRC  the  water-soluble  the  wood  extractives properties,  much  contain the  durability.  two  main  research.  studies  that  i n the heartwood give These  groups  s t e a m - v o l a t i l e tropolone 1  further  chemical  m a t e r i a l s contained  of i t s  by  water  with  soluble  fungicidal  d e r i v a t i v e s and  a  mixture  of  n o n - v o l a t i l e phenolic  Gardner, 1954). group  of  worthwhile choosing  the  f o r WRC  heartwood  to  known  Wood  events.  thujaplicins, decay  are  and  decay fungi as t e s t  decay  Studying  i s the f i n a l chemical  chiefly  resistance.  p o i n t out that these r e s u l t s  well  1961).  (Barton  B i o l o g i c a l t e s t s have shown so f a r that one  tropolones,  responsible  derivatives  are  It i s  based  material  r e s u l t of a  (Roff,  sequence  responses of the t r e e  living  It  i s p o s s i b l e that before  t r e e s the t o x i c i t y  extractives Therefore,  i s already the  Investigations this  involves  1975).  a  The  fungi  decay attack  f a c t o r of t h u j a p l i c i n s and  a l t e r e d by e a r l y  toxicity  under c e r t a i n c o n d i t i o n s  these  of  to the  f i n a l organisms i n t h i s sequence may misrepresent the process.  on  attacking  other fungi.  of t h u j a p l i c i n s to v a r i o u s  fungi  remains an important problem.  of WRC decay i n B. succession  major  C.  of organisms  heart-rotting  have shown that (van  der  Kamp,  organisms  are  * Basidiomycet  es  .  The  heart-rotting  of  on  the c o a s t ,  i n decreasing  *:  The  names used through out the t e x t  fungal  order  fungi  of d e t e c t i o n  i n Appendix  1  according  frequency, those  Their current  names  t o Compendium of  Plant  Disease and Decay Fungi i n Canada 1960-1980 (Ginns, 2  WRC  are  appeared i n the o r i g i n a l p u b l i c a t i o n s . are l i s t e d  living  1986).  Poria  are  al bipel  asiatica  ( P i l a t ) Overh. Forties  and  I uci da  interior  pi ni of  Lloyd.  (white  asiatica  ring  r o t ) and Fomes  (white p i t t e d trunk  ( P i l a t ) Overh.,  an  r ing  pi ni  interesting  In  the  important organisms  are  Poria  rot).  weirii  (Thore) L l o y d .  Murr.  done  by at  der Kamp (1975) i n d i c a t e d  early  fungi  in  several  identified  Ki rschstei WR3  ni el I a  thuji  na  They were not  the  The work  fungi  He found three  samples.  is  which  and  may  different finally  He gave the code names  as:  um  (possibly  sp.)  WR2  WR1  (Peck) Pomerleau & E t h e r i d g e . ) and  (unknown).  extractives, the  arise  especially  on  the r o l e  of  the  t h u j a p l i c i n s , and how  t h u j a p l i c i n s and d e s t r o y the n a t u r a l  WRC  heartwood  fungi  toxins,  detoxify rendering  heartwood s u b j e c t t o decay. Mechanisms  fungi are  time.  Cyl i ndrocephal  Questions  the  properties.  coastal  at that  (possibly  that  stages caused wood d i s c o l o r a t i o n  a l t e r the e x t r a c t i v e  1946).  q u e s t i o n i s which fungus  to i n t e r a c t with s t e r i l e heartwood t i s s u e s .  attack  (yellow  (Buckland,  first  van  Poria.  rot),  (brown c u b i c a l pocket and butt r o t )  the p r o v i n c e the most  Poria  However,  Baxter.  of t h u j a p l i c i n t o x i c i t y to c e r t a i n groups of  and some f u n g i ' s d e t o x i f y i n g unknown.  e f f e c t s on  thujaplicins  Nothing has been r e p o r t e d on these  3  matters.  According  to  reasonable the  key  the  to  chemical  assume t h a t  functional  actions.  This  derivatives  Testing  whose  To various  of  require  investigate fungi  toxicity  and t o  and i n s e c t i c i d e s  of  further  After  carried  Laboratory  (Forintek  available  to  provides  materials  not  for  is is  these  been  of  proven  thujaplicin  activity  structures  of  the  of  has  been  detoxified  or  information.  thujaplicin toxicity  mechanisms research  of  its  on  selective  on new  fungicides  Also,  it  WRC h e a r t w o o d  commercial use.  could  extractives  Our knowledge  in  this  on  WRC  developed.  out  separate  of  importance.  t h i r t y years  extractives  group  stimulate  fully  almost  has  toxicity  effects  utilization  not  still  group  responsible  could provide valuable  technical  field  keto-enolic  it  clarification.  to  little  is  the  examine  w h i c h now have  thujaplicins,  however,  chemical  the  could serve  stimulate  reactive  keto-enolic  thujaplicins  aspects  of  g r o u p w h i c h may be  b l o c k e d and a n a l y s i s  These  the  assumption,  experimentally.  biodegraded  structures  i n the  Canada  of  chemical  Western  Corp.),  Forestry the  these e x t r a c t i v e s . for  studies  examining  Research  techniques In  turn,  decay  are this  resistance  mechani sms. Current  understanding  of  the  4  role  of  WRC  heartwood  extractives this  decay  understanding, With  the  in  such  research 1)  trees  at  attac  the  fungi  early  thujaplicins  questions  in  mind,  must  the  interaction  which  be  three  in  living  between  attack  stages,  Separate,  which  form  fungi;  and  from  WRC  and  trees  selectivities  analyse,  to  in  the  To  improve  answered. objectives  thujaplicins  heartwood reveal  operation  of  to  achieve  these  1)  The  toxicity  between  fungi.  hypothesis  as:  A)  hypotheses  two  in  of  the  and living  role  against  of fungal  organisms  treatment  to  various  be  tested  major  products  with  isolated  of  the  toxic  fungi. in  this  study  in  objectives. of  WRC  with  the  mechanism  w i l l  thujaplicins  consequent  Sterile  microorganisms  after  thujaplicins  major  are  identify  chemical  Two  There  and  thujaplicins  Examine  3)  such  inadequate.  k; 2)  order  is  to:  Examine known  several  questions  are  lesser  resistance  a  test  heartwood high  detoxify  hypotheses  is  tolerance  fungi  for  invaded to  thujaplicins  5  to  this  varies  major  f i r s t  by  thujaplicins  and  and  destroy  these  natural  t o x i n s o f WRC  t o f u r t h e r decay B)  heartwood,  rendering  t h e wood  subject  by t h e same o r new o r g a n i s m s ; a n d  Thujaplicins are highly  t o x i c to the  al bi pel I uci da B a x t e r .  well  known  fungi  s u c h a s Poria  a n d Poria  weirii  Murr.  a n d t h e d e c a y c a u s e d by t h e s e f u n g i  those  a r e a s i n w h i c h t h u j a p l i c i n s have d i s a p p e a r e d or  i s restricted  to  been  deactivated. 2) fungi  The s e l e c t i v i t y is  different  fungal  There major  based  of t h u j a p l i c i n t o x i c i t y  on a c t i v i t y  of t h e  to various  keto-enolic  group  in  environments.  a r e a l s o two c o n s e q u e n t  t e s t hypotheses  for this  hypothesis as: A)  fungi  Deactivation might  hydrolysis B) might ions  arise  of  t h i s g r o u p by  from  oxidation,  Activated  polymerization  t o x i c i t y of t h u j a p l i c i n s t o decay  i n s i d e t h e wood,  of a metal c h e l a t e  or with metal ions  enzymes, o r f o r m a t i o n  amino-acids of the fungi, between  attacking or  of t h u j a p l i c i n s ; and  be due t o t h e f o r m a t i o n  fungal  early  with  metal  associated  with  o f amine t h u j a p l i c i n s a l t s w i t h  and f o r m a t i o n  t h u j a p l i c i n and f u n g a l  t h e s e mechanisms d e a c t i v a t i n g  fungi  protein,  fungal  6  of hydrogen  bonding  w i t h any or a l l of  growth.  LITERATURE REVIEW  2.1.  Importance of western redcedar to the B r i t i s h Columbia forest  industry  Western tree  redcedar (WRC)  {Thuja  piicat  a Donn.),  a major  s p e c i e s i n the f o r e s t s of northwestern North  America,  particularly  in the c o a s t a l areas c f B r i t i s h Columbia  is  the l a r g e s t  one  of  trees in  the  Pacific  Region.  f r e q u e n t l y reaches h e i g h t s of 45 to 60 metres and of  2.5 metres .or more (Barton,1962).  B.C.  86.7% of WRC  i n a l l of Canada, as 876 m i l l i o n m  million  (Canadian  m  constitutes  11.3%  Forestry  resource ( M i n i s t r y of F o r e s t r y ,  1980).  It  diameters  accounts  for  out of  1010  3  Statistics,  (913.5 m i l l i o n m ) of the  (B.C.)  1980). B.  C.  It forest  The t o t a l annual  WRC  3 harvested i n B.C. interior  and  i n 1980 amounted to 2.03 m i l l i o n m 3  6.42  million m  in  (Table 1) ( M i n i s t r y of F o r e s t r y , WRC  the  coast  i n the  respectively  1980).  a l s o p r o v i d e d v a l u a b l e f o r e i g n exports as Grade I,  II, and I I I lumber (Table 2). I t c o n s t i t u t e d 10.4% (3.891 3 3 million m out of 37.418 m i l l i o n m ) of the t o t a l lumber exported from B.C. i n 1982 ( S t a t i s t i c s Canada, 1983). The WRC  t r e e s r a r e l y occur i n e x t e n s i v e pure stands but  7  Table 1. The S i g n i f i c a n c e of WRC i n B. and Timber Cut i n 1980. Forest district  Forest  C.  resource  as a F o r e s t  Resource  Timber cut coast  interior  (million m ) Prince  George  23.5  P r i n c e Rupert  329.8  1 .08  0.33  Vancouver  432.0  5.34  0.06  0.08  Cariboo  20.5  0.18  Kamloops  56.0  0.52  Nelson  51 .7  0.36  Tatal  913.5  6.42  2.03  Source: M i n i s t r y of F o r e s t r y , (1980). Table 2. The Volume of D i f f e r e n t Grades of WRC B. C. i n 1980.  3  Grade 1 2 3 Total Source: M i n i s t r y of F o r e s t r y ,  Log Exports from  Volume (m ) 27,288.9 42,144.8 95,123.2 164,556.9 (1980).  are commonly a s s o c i a t e d with s e v e r a l other the  Pacific  western  coast  hemlock,  firs.  In  forests,  it  S i t k a spruce,  grows  tree species. with  Pacific  the I n t e r i o r of B.C.,  In  Douglas-fir,  silver  and  noble  i t i s u s u a l l y found  with  western white pine, western l a r c h , western hemlock, Douglasfir,  and  Engelman spruce  WRC its  (Bolsinger,  wood i s used i n a v a r i e t y of products,  w e l l recognized  service  reputation  conditions.  shingles,  siding,  The  poles and  outdoor f u r n i t u r e ,  It  a l s o used as a p a r t i a l  pulping  with other  of higher  main products  stock, is  1979).  species  posts,  paneling  durability  under  i n c l u d e shakes  fence m a t e r i a l , and  casket  wood f u r n i s h  Jiang,  1970).  WRC  i s known to have e x c e l l e n t paper-making p r o p e r i t e s . making pine  t e s t s with WRC, bleached  indicated  Kraft  that WRC  hemlock, pulps  ranked f i r s t  properties.  Due  ranked f i r s t  i n o p a c i t y and  higher  tear  other  smoothness.  southern  Thomas,  in burst,  f i n e n e s s of i t s  pulp  f o l d and fibers,  It also  1961) tensile  WRC  also  contributed  strength.  Although the  to the  and  for  Paper-  D o u g l a s - f i r and  (Murray  and  many s p e c i a l items.  (15 to 30%)  (Swan and  because of  WRC  demand f o r WRC western  can  produce a very  as p u l p i n g m a t e r i a l  Canadian s p e c i e s , 9  good in  such as  quality comparison Douglas-fir  pulp, with and  western hemlock i s r e l a t i v e l y discourages higher As  use  of  and yield  a  longer  evaporators  pulping  metal c h e l a t e s these  Douglas-fir  of  due  (Gardner,  better u t i l i z a t i o n The  WRC  in WRC  The  (Barton  and  of  problem  and  encountered of  found,  it will  l e a d to  heartwood  c o s t of p r e - e x t r a c t i o n could be  in  extractive  If the commercial use  by p r e - e x t r a c t i o n of WRC  of a  before  compensated  with the p r o f i t generated by e x t r a c t i v e byproducts.  10  that  digesters  to the formation  be  pulp  MacDonald,1971).  mild-steel  can  its  density.  overall  i s only 60%  1963).  is  requirement  material  another p r o c e s s i n g  extractives  pulping.  in  i t s low  chemical  time are needed.  corrosion is  main reason which  with  a r e l a t i v e l y higher  pulping  from  Accelerated  Kraft  together  per^ u n i t volume of raw  obtained  The  t h i s wood as p u l p i n g m a t e r i a l  e x t r a c t i v e s content,  a consequence,  lower.  2.2. The c h e m i c a l c o m p o s i t i o n of WRC  The  structural  cellulose, same  hemicelluloses  proportion  structural extracted  as  of  An  wood,  t o t a l hot  woods.  which  can  i n western hemlock and  the  taste,  heartwood.  c o l o r of WRC  they a f f e c t the d u r a b i l i t y , and  other p r o p e r t i e s  extractives properties The extraction  permeability,  (Gardner et  distinguish  the  al,  as wood  heartwood i n  work  in WRC  was  Douglas-fir. f a r out  responsible In  of for  addition,  ease of  1966).  its  (1950)  water e x t r a c t i v e s  They are  non-  wood i s  Lewis'  to the amount p r e s e n t .  the  normally  are d e f i n e d  proportion  and  The  be  heartwood e x t r a c t i v e s a f f e c t u t i l i z a t i o n  odor,  namely  occur in roughly  e x t r a c t i v e s content.  3) showed that  WRC  lignin,  wood,  unusual c h a r a c t e r i s t i c of WRC  almost twice as much as The  WRC  by water, or organic s o l v e n t s ,  heartwood  (Table  and  of  i n other c o n i f e r o u s  components  extractives. high  constituents  wood  pulping  In essence,  WRC  appearance  and  from a l l other woods. WRC  heartwood e x t r a c t i v e s may  be  volatile  fractions  fraction  consists  obtained by  hot  separated r e a d i l y i n t o v o l a t i l e and by  steam  distillation.  mainly of a c l a s s of  c a l l e d tropolone d e r i v a t i v e s .  In WRC  1 1  The  organic  heartwood,  water non-  volatile compounds from  old  Table.3. Chemical Composition of WRC, Hemlock and D o u g l a s - f i r Wood.  Species  Alpha cellulose  Hemicellulose  Western  Lignin  T o t a l water extractives  Ash  WRC  47.5  13.2  29.3  10.2  0.24  Western hemlock  48.8  14.7  28.8  5.3  0.47  Douglas-fir  53.8  1.3.3  26.7  5.92  0.28  Source: Lewis,  (1950).  12  growth, 0%  the content of t h i s group of compounds ranged  at the p i t h to 1.2%  (MacLean and Gardner, Appendix 2.  in the outer straw c o l o r e d 1956b).  Their  (1986) i n d i c a t e d  by  using more s e n s i t i v e a n a l y t i c a l methods,  of  tropolone  showed  that  ranged  from  growth  trees,  be  His  t h i s group of  0.014% to 1.774% i n heartwood, the  amounts v a r i e d  from  in that  higher amounts  detected.  i n o l d growth t r e e s ,  heartwood  r e s u l t s are shown  Recent research by Nault  d e r i v a t i v e s can  from  results  components  while i n  0.011%  to  fast  0.525%  (Appendixes 3, 4 ) . There volatile  are  at  least  fraction  gamma-thujaplicins  of  nine  WRC  extractives,  (Gardner,  1958);  and Barton,  1958);  and  and  thujate  (MacLean and  methyl  substances carvacrol  present methyl  components  Gardner,  fungi,  thujaplicins  as  The  thujaplicinol  is  hydroxyl  a  and  MacDonald,  t h u j a p l i c i n containing  group  which  considerably  13  Other and  chemical  1.  are h i g h l y t o x i c to wood  (Barton  acid,  nezukone  destroying  t h e i r t o x i c i t y being of the order as that of  pentachlorophenate  vicinal  thujic  1956b).  1970).  s t r u c t u r e s of these are shown in F i g u r e The  steam  a l p h a - , b e t a - and  beta-thujaplicinol,  (MacLean,  the  b e t a - d o l a b r i n (Gardner  have been i d e n t i f i e d ether  in  sodium  1971). an  Beta-  additional  modifies  its  Figure  1.  The  Source:  Volatile  Barton  and  Components  MacDonald,  of  WRC H e a r t w o o d  (1971).  14  Extractives.  reactivity  and  toxicity  (Roff,  1961).  The  non-tropolone  components of the steam v o l a t i l e o i l , t h u j i c a c i d and  methyl  thujate  Thujic  are a l s o seven-membered  acid  is  with  low  a colorless,  the  odourless  fungal t o x i c i t y .  for the c h a r a c t e r i s t i c carpet  ring  c r y s t a l l i n e organic  Methyl t h u j a t e i s  odour of WRC  b e e t l e and  compounds.  and has  case-making  clothes  acid  responsible  some t o x i c i t y to moth  (Barton,  the  relative  1962). Gardner  and  concentrations  Barton  (1958)  of these compounds.  s l i g h t l y higher c o n c e n t r a t i o n thujaplicin  is  thujaplicinol  present is  only i n minor amounts.  fractions.  They  heartwood.  U n t i l now,  eleven  and MacDonald,  1959;  1960;  thujaplicatin  1959; 1966);  account  the  total  1960;  mainly  for 5 to 15% of  the  l i g n a n s have been i d e n t i f i e d  1971).  They  are:  plicatic  acid  1966); p l i c a t i n (Gardner et  thujaplicatin  methyl ether  dihydroxythujaplicatin  beta-  heartwood e x t r a c t i v e s are  polyphenolic  al,  of  The  i n only t r a c e amounts (Table 4 ) .  n o n - v o l a t i l e WRC  (Gardner et  one-tenth  Alpha-  B e t a - d o l a b r i n , nezukone and c a r v a c r o l  methyl ether are present  (Barton  B e t a - t h u j a p l i c i n has a  than gamma-thu'japlicin.  approximately  t h u j a p l i c i n content.  The  measured  (Gardner et  (MacLean and  (MacLean  15  and  al ,  Murakami, MacDonald,  al,  1966); 1966a); 1967);  Table  4. Composition of Steam V o l a t i l e O i l from WRC Heartwood. components  Butt  oil(%)  wood(%)  21.1  0.17  thujic acid  10.4  0.08  tropolones beta-thujaplicinol gamma-thujaplicin beta-thujaplicin alpha-thujaplicin beta-dolabrin  68.5 8 24 35 1 0.04  0.56 0.07 0.20 0.30 0.01 0.0003  methyl t h u j a t e , other  neutrals  Source: Gardner and Barton,  (1958).  16  hydroxythujaplicatin 1966b);  methyl  dihydroxythujaplicatin  Murakami,  plicatinaphthol  gamma-thujaplicatene apoplicatitoxin  methyl  (MacLean  and  and  ether  (MacLean  are shown i n F i g u r e 2.  Of  these,  the  mixture or 1 to 5% O.D. which  main component  1969a);  The  beta-  chemical  The chemistry  of  (Gardner el  al,  (30 to 40%  of  wood b a s i s ) i s a very strong  was heat and l i g h t  and  MacDonald,  1970); and  1973).  i n t e r e s t i n g l i g n a n s has been d i s c u s s e d  1971).  Murakami,  MacDonald,  (MacDonald and Barton,  (MacDonald and Barton,  s t r u c t u r e s of these  acid  (MacLean  1967); p l i c a t i n a p h t h a l e n e (MacLean and  1969b);  these  ether  sensitive.  It  the  organic  is  called  plicatic acid. The of  water s o l u b l e (organic s o l v e n t i n s o l u b l e ) p o r t i o n s  WRC  extractives  c o n s i s t i n g of simple and  are  sugars,  hemicelluloses.  present  Small  non-phenolic L-arabinose, amounts of  (Barton and MacDonald,  The  formation  extractives  and  p e c t i c compounds protein  varying  WRC  concentration  outer  (more h y d r o x y l a t i o n and l e s s O-methylation) with  ring  (nezukone  to  by  from  metabolism  1969).  explained  heartwood  to  at,  been  also  heartwood  age (Swan et  has  are  1971).  and r e l a t i v e amounts of  their pith  carbohydrates,  changes  The h y d r o x y l a t i o n of the  thujaplicins  17  to  in  tropone  beta-thujaplicinol)  F i g u r e 2. The  Non-volatile  Components  of  WRC  Heartwood  Extractives.  methyl  ether  >  -CH,  *o  HO-  CH O' 3  OH XIV  Dih^droiylhujOpNcotin  XV  Y OH  H O '  0CH3  OH XVI  Hyd'oxytnujoplicatin methyl  methyl  ether  XVII  Plicotlnophthslene  OH  XVIII Plicotinophthol  XIX Y - T h u j c p l i e o f e n e  Source: Barton and MacDonald,  (1971). 18  x  x  "OCH3  OH  Dihydroxylhujopltcotin  ether  ^  B-apoplicatitoxin  appeared  to occur much f a s t e r ,  heartwood boundary. thujaplicins' system.  i n h i b i t i n g a f f e c t on the O-methylation  hydroxylation  to  thujaplicatin  to be formed via  hydroxy-thujaplicatin  to  plicatinaphthol  plicatin  to  (Swan and J i a n g ,  formed  thujaplicatin  During  were thought  ether  1970).  and  the  past  of  three  WRC  column,  combinations  of  complex mixtures  mass  and  hydroxy-  dihydroxy-thujaplicatin  decades,  large  scale  e x t r a c t i v e s has provided much  useful  the  i t s chemistry and methodology.  vapour-phase these  and  techniques,  such as paper,  thin  liquid-phase,  and  separations  for s t r u c t u r a l determinations, UV,  With the  of  l i k e l i g n a n s have become p o s s i b l e .  and  IR  spectroscopy,  d i s p e r s i o n , nuclear magnetic resonance NMR),  acid  ether,  these methods of s e p a r a t i o n together with  visible,  dihydroxy-  On the other hand,  methyl  a i d of modern types of chromatography,  instruments  a main  (Swan and J i a n g , 1970).  i n f o r m a t i o n on both  of  to  plicatic  thujaplicatin  methyl  investigation  layer,  enzyme  sequence from the f i r s t member of the f a m i l y ,  thujaplicatin  methyl ether  sapwood-  They a l s o d i s c u s s e d the p o s s i b i l i t y of  Lignans of WRC  methylation  and wholly at the  spectra  The  powerful as f o r  use new  example  optical  rotary  (proton and  carbon-13  and X-ray c r y s t a l l o g r a p h y has  19  very  lead  to  better al,  understanding of these e x t r a c t i v e s t r u c t u r e s  1969).  20  (Swan  et  2.3.  Fungi  and  decay  in  WRC  Because i t i s an organic m a t e r i a l , several  f o r c e s of degradation,  insects  and marine b o r e r s .  by  fungi.  Fungi  heterotrophic  wood i s s u b j e c t  including  fungi,  to  bacteria  Decay in wood i s caused mainly  contain  organisms,  no  so  chlorophyll they  nourishment from energy s u p p l y i n g ,  must  and  obtain  are their  t i s s u e b u i l d i n g , organic  substances p r e v i o u s l y produced by green, c h l o r o p h y l l bearing plants.  They must consume preformed organic matter.  They  may  l i v e as saprophytes which d i g e s t and consume dead p l a n t s  or  animals,  fungi may of  plants  within  filaments,  and  plants  called  i n the  hyphae ,  liberated  from  environment  the  where  and  Fungi u s u a l l y  form  causing  of  minute  parts  enter  and  threads  or  which c o n s i s t of c h i t i n (septa).  fungal  tubes  D i g e s t i v e enzymes are  cell  into  the  immediate and  then  cell.  such as t r e e s , logs and  to  Alternately,  food molecules are s o l u b l i z e d  absorbed i n t o the fungal Wood,  wastes.  a s s i m i l a t e t i s s u e s or  animals.  with o c c a s i o n a l c r o s s w a l l s  subject  or t h e i r  l i v e as p a r a s i t e s and  living  ramify  their parts,  manufactured products i s  fungal decay which d i s c o l o r s and significant  economic  21  loss  destroys and  it,  various  u t i l i z a t i o n problems. approximately roughly  6.3%  Ontario  22  Basham and Morawski  m i l l i o n cubic  British  Columbia,  percentage average  f e e t of timber,  of the annual harvest,  because of heart  of  for  the  major  amount  conifers,  decay  name and  producing  types  of  wasted a n n u a l l y  decay  expressed was  while  it  scale,  f o r WRC  of as  12% was  on 32%  1957).  the Basi di omycetes.  the Hymenomycetes  are  of This  the  decay  i n c r e a s i n g l y seen  i n the subject of wood decay.  wood decay fungi are  in  In the province  that of a s u b d i v i s i o n c o n t a i n i n g  species,  in l i t e r a t u r e  was  fungi belong to the most advanced c l a s s  fungi i n the e v o l u t i o n a r y class  of  which  volume of wood  ( B r i t i s h Columbia F o r e s t S e r v i c e , The  was  rot or s t a i n .  the t o t a l gross  (1964) found that  Two  recognized:  principal  Brown-rot  and  Whi t e - r o t . Brown-rot secrete  preferably  attack  l e a v i n g the l i g n i n matrix  i n decayed wood,  brown in c o l o r .  s t a r t to metabolize l i g n i n and  The  White-rot  soft.  22  of  the and fungi  P r e f e r a b l y they  hemicelluloses,  i s degraded only at a l a t e r stage  degraded wood i s white and  They  n e a r l y undigested  degrade l i g n i n , as w e l l as p o l y s a c c h a r i d e s .  fraction  softwoods.  enzymes which decompose the p o l y s a c c h a r i d e s  c e l l wall, result  fungi  the c e l l u l o s e  (Liese,  1970).  Brown extremely living  and white r o t s are commercially and  important as they are the types found i n  t r e e s and cause  Soft-rot Fungi  fungi  imperfecti  and l i g n i n . cell  wall  serious structural mostly  which are able to degrade  Ascomycetes  polysaccharides  i n c o n t r a c t with the brown and white  been  considered  as  a  wood  lumen,  destroyer  Although s o f t - r o t d e s t r o y s wood,  rot  soft  progresses  slowly  into  wood  from  the  i n wood i n s e r v i c e and i s found  in  11  However,  because and  t h i s type of rot in  both  and hardwoods.  a l a r g e s c a l e i n v e s t i g a t i o n of decay and decay initiated  i n 1943,  localities  localities  in  Basi di omycet es B. C.  1970).  surface,  is  most important  has  i t i s o r d i n a r i l y much l e s s  go as deeply.  in WRC  rot  (Liese,  commonly does not  In  fungi  than brown and white r o t s in standing t r e e s  softwoods  or  Because of t h e i r p r e f e r e n t i a l growth w i t h i n the  hyphae develop i n s i d e the c e l l  it  standing  failure.  belong to the  whose  serious  ecologically  from  on  the were  1943  a t o t a l of 615 t r e e s were examined  the coast and  interior.  trees  in  list  1946).  WRC  of these fungi  of in is  From the study, s i x  fungi have been r e p o r t e d a s s o c i a t e d with brown c u b i c a l  23  eight  species  on l i v i n g and dead  A check  shown as Appendix 5 (Buckland,  110  Seventy-seven  collected  to 1945.  fungi  rots  of  l i v i n g WRC  trees.  Polyporus  and  Two  Poria  of these,  balsameus  Peck.  were  asiatica of  Overh.  considerable  importance.  Another  four which o c c u r r e d i n f r e q u e n t l y were  Fomes  a  Cke.,  pi ni col  Coni ophor a  course  cer ebelI  r o t s of importance localized  al bi pel  I uci  (Thore)  Lloyd.  and  as  importance  this  principal somehow  fungi  occured  on  organisms,  us  spp.  been  Poria  found  of  responsible  al bipel  the the  The  annosus  Omphali  (Romell)  Buckland  I uci  da  a  former was  the  coast and the l a t t e r  this  i s unknown.  interior  and  campanella  Egel.  WRC  Poria  but  causing  as  decay i n WRC  24  the  wood  were  of  weirii  the Murr.  heart-rotting  only s i g n i f i c a n t l y  i n the i n t e r i o r .  of  (Fr.)  concluded that  f o r decay i n  Baxter.  were  four  with the g e o g r a p h i c a l l o c a t i o n  coast and  Basi di omycet es  us  pini  Sacc.  were i d e n t i f i e d as Fomes  ni grol imi tat  Poria  Fomes  (Peck)  (Fr.) Q u e l . ,  producing  WRC.  Murr.,  subacida  the  significance  in  weirii  Fr.,  During  four of p o s s i b l e  investigation,  associated Poria  tree.  and  Poria  mellea  Quel, and Fomes From  Meruit  major wood d e s t r o y i n g f u n g i .  Armillaria  Cke.,  and  schweinitzii  four fungi capable  have  Baxter.,  secondary  (Fr.)  areas  da  identified  Polyporus  a Perso.  of i n v e s t i g a t i o n ,  white in  (sw)  important  The  wood  reason  in  on for  service  appeared  to  be  trees.  Southam  (1965),  Eslyn  al  totally different and  Ehrlich  (1970),  (1983) and S e t l i f f  from those  (1950),  Duncan  C l a r k and Smith and C s e r j e s i  rather  research  i n standing t r e e s ,  natural  1979).  decay  important  full  attacking  the  the other hand, of  was  Wood  (Merrill in  the  possibly  d i r e c t i o n and (Wilkes,  an  pattern  1982  a,  b). to  at the end of t h i s sequence w i l l not lead to  pioneer  organisms  The  d i s c o l o r e d margins  roles  began  they were i s o l a t e d  fungi were c o n s i s t e n t l y  decayed  heartwood  processes  s c i e n t i f i c c u r i o s i t y because  decay  More and  variation  understanding of the t o t a l p r o c e s s .  the  the  t o x i c i t y of n a t u r a l p r o t e c t i v e components  non-hymenomycetous  from  in  fungi never occured alone  f a c t o r governing the rate.,  those organisms a  established  i n s l a s h or i n wood i n use.  resistance  microbial  Studying  On  30  shakes.  the f i n a l r e s u l t of a sequence of events  Shigo,  of  Lombard  area of f o r e s t pathology i n the 1970's.  decay was and  and  (1985) r e p o r t e d about  i n t e r e s t i n g concept was  more evidence suggested that decay either  standing  (1979), S c h e f t e r , et  s p e c i e s of fungi found i n s e r v i c e p o l e s and A  in  of decay columns,  to  of draw  consistently while  recovered only from  wood  advanced  wood.  Manion  and  Zabel  (1979)  25  postulated  that  these  "pioneers" trees,  had  and  an  then  after  i n f e c t i o n s occurred study  done  i n i t i a l detoxifying a  as other  by S h o r t l e  by both "pioneers"  affected  by  the  preconditioning  and  period,  out  that  f a c t o r s a s s o c i a t e d with the defense  new The  successful  decay fungi appeared to  research  done by van  der Kamp  decay  fungi were i s o l a t e d from the  brown  stained  finally  sound  i d e n t i f i e d , but  Poria  levels  hypothesis  (1975),  three  straw-colored,  wood areas of  WRC.  They  were given code numbers WR1,  al bi pel Iuci da  zone at the edge of the the  standing  be of  tree. In  WR3.  in  decay fungi became a c t i v e .  (1979) p o i n t e d  invasion  role  Baxter, was  sound wood.  that decay of WRC  organisms.  26  non-  red  and  were  not  WR2  and  i s o l a t e d from a narrow These r e s u l t s supported  involved a succession  of  2.4.  Tropolones,  Tropolone  tropolone  derivatives  seven-membered carbon  2-hydroxy-2,4,6-cycloheptatrien-1-one 1945,  enolone  Dewar  (1945a,  b,  s t r u c t u r e which was  and  of  T h i s was  as  They  thujaplicins.  In-  and  system and  were 1933,  l a t e r an isomer  s t r u c t u r e of such a  the f i r s t  discovery  to be c o n c l u s i v e l y  alpha-,  beta-,  Anderson  and  (m.p.  the  isomeric  and  gamma-  Sherrard c  (1933)  i o 1 2 ° 2 ' *P* H  m  52-52.5°) that c r y s t a l l i z e d  from the mother l i q u o r on standing f o r a long p e r i o d of (Anderson Gripenberg cultivated third and  and Gripenberg,  1948).  In 1948,  Erdtman  (1948) obtained from heartwood of the same i n Sweden,  isomer of m.p.  besides the substance 34°.  gamma-thujaplicins  melting points ( H i l l i s ,  of m.p.  the i n c r e a s i n g order  1962).  27  time and tree  82°, a  They were c a l l e d as alpha-, in  he  derivatives.  d i s c o v e r e d a t o x i c " p h e n o l i c " substance, in WRC  seven-membered  tropolone d e r i v a t i v e s were  thujaplicins.  terms  derivatives.  the i n c e n t i v e that l e d to l a t e r  simplest of these and  characterized  its  a novel aromatic  some dozens of n a t u r a l t r o p o l o n e s The  properties  r i n g compounds,  c) proposed a  gave the name of tropolone to the parent system.  their  and tropolone d e r i v a t i v e s are generic  a p p l i e d to unsaturated  In  and  of  betatheir  its  Beta-thujaplicin,  a l s o known as h i n o k i t i o l because  presence  essential  in  (Chamaecyparis subject  the  taiwanensis  complex,  hinokitin.  hinokitiol  ring  study  hinoki"  Suzuki.)  was  Nozoe  found  independently  an unsaturated  i t s i d e n t i t y with  by d i r e c t comparison  et  1969),  and pigments (Nozoe,  1950), et  alkaloids 1956).  of the f a m i l y Cupr es s aceae In Table 5,  beta-thujaplicin  al,  fungal  They can metabolites  1966; Ng et  (Santavy,  al, 1968;  1950), g l u c o s i d e s  WRC and s e v e r a l other s p e c i e s  are r i c h i n t e r p e n o i d t r o p o l o n e s .  s e v e r a l kinds of important  t e r p e n o i d tropolones  from the heartwoods and e s s e n t i a l o i l s of some t r e e are these was  listed  (Nozoe,  1956).  chromatography heartwoods  by  Zavarin  (Table 6 ) .  varied  species  The q u a n t i t a t i v e a n a l y s i s  t r o p o l o n e s corresponding provided  carbon  (Nozoe, 1950).  i n t e r p e n o i d s (Nozoe,  al ,  on  Nozoe, with a number of  proposed  (Just and W i l l i a m s , 1962; Blank Blank  (1956)  As a r e s u l t of e x t e n s i v e research  Tropolones are d i s t r i b u t e d widely i n nature. be  the  from a l k a l i n e h y d r o l y s i s of i t s red i r o n  s t r u c t u r e and l a t e r  was proven  "Taiwan  i n Japan.  i n the p e r i o d 1940-1947,  associates,  et  Masamune  of much independent  obtained h i n o k i t i o l  o i l of  of  to s p e c i e s  et The  al  (1959)  of  Cupressaceae  of  using  tropolone contents  paper i n these  g r e a t l y between t r e e s , with age of t r e e ,  28  Table 5. List of Some Trees Containing Terpenoid Tropolones.  Botanical name  Geographical location  Common name  Chamaccyparis taiwaTaiwan-hinoki• nensis M A S A M U N E et  Formosa (Taiwan)  Type oi tropolone"  a.  P  References  (133. 134)  S U Z U K I  C. formosensis M A T S U M . C. nootkatensis S P A C H . Thuja plicata D . D O N Thuja plicata D . D O N T. occidentalis L . T. occidentalis L . T.Slandishii C A R R . Thujopsis dolabrata Z I K B . et Zucc. Libocedrus decurrens  Benihi* Alaskan Yellow cedar Western Red cedar  Formosa U .  S . A .  U .  S . A .  (trace)  Nk  (133) (37)  «• P-{14, Y 15, 80, 246) (80. 92) (93)  Northern White cedar Nioi-hib?.* Nezuko* Hiba*  Sweden Sweden  a, y a. y  Japan Japan Japan  (126) p ("7) P <*. (796) P  Incense cedar  U .  P-  S . A .  T O R R E Y  Cupressus macrocarpa  Monterey cypress Juniperus chinensis L . Byakushin*  New Zealand  H A R T W .  * Japanese common name.  •  Japan  a.  p. P.  Y (245- 247)  Nk (57. 246) Nk (128)  * • a, p\ y: Thujaplicins; Nk: Nootkatin.  Source: Nozoe, (1956).  29  Table 6. Q u a n t i t a t i v e Analyses of Tropolones  0Thuja- Thujaplicin plicin  Species  Cuprcssus pypmaea (Lcinm.) Sarg. C. targenlii, Jeps. C. abramsiana, C. B. Wolf C. govenxana, Gord. C. ariionica, Greene C. gempervirent, L.' C. macrocarpa, Uartw. '* ' C. toruhsa -' C. macnabiana * Chamaeeyparis lawtoniana* (A. Murr) Pari Ch. noolkalcnsis,' (D. Don) Spach ' Ch.tyoidei*(BS.P.) Ch.formosensia* Mateum.' ' Ch. taiwanensis,' Masam. ct Su*. ~ Ch. obluta,' Sieb. et Zucc.» » 1  -  ,,M  ; o  0.008  —  —  0.0003 0.2  —  +—  +  +•  1  , (  ! !  — —  1  .  —  —  0.1 0.03 0.04  — —  1  l,  T*  Thujapliein — —  0.002 — —  —  —  —  —  +—  +  +—  +  +  —  es s aceae. 6Tliujaplicinol  .Nootkntin  Dolnbrin  0.1 0.1 0.02 0.0008 0.008 2.Q 0.2 *  0.001 0.01 0.001 0.0004  —  —  0.005  —  —  —  0.0003  —  —  —  —  —  + +  —  — —'-  — —  —  —  0.1 — — —  —  —  0.0002  i n Cupr  mucin 0.4 —  —  0.03  Totai  0.1  —  — —  —  —  — •  — —  —  1.4 0.17 0.07 0.02 0.01 2.2 0.2 Unkn. Unkn. 0.0002  Clirom. Chrom. Chrom. Chrom. Chrom. Chrom. & Prep. Chrom. & Prep. Unkn. Unkn. Chrom.  0.1 Unkn, Unkn. Unkn.  Chrom. & Prep. Chrom. Chrom. & Prep. Chrom. & Prep. Prep.  T-10  T-4.5  0.4  0.3  —  —  0.01» 0.05» —  —  — —  — —  — —  — —  —  — —  —  —  —  —  — •  —  — —  —  —  —  —  —  —  —  —  —  —  —  —  —  —  —  —  —  — —  —  —  —  —  — —  —  —  —  —  —  — •  —  Method of  T-O.l  T-U  Analysis  • Numbers denote the Approximate amount offttropolone in %, based on dry weight of wood. ' In this case it has not been determined which of the two compounds is present, M  -  —  —  —  —  —  —  T-ll  T-10  —  —  —  or whether both o r e . ' These analyses hove been taken from the literature.  TABLE II" or-  Thujaplicin  Species Papuaecdrui torriccllentii, Li Junipcrut occidcnlalit, Hook / . communis, L. /. monospcrma, (Englm) Sarg oslcospermn, (Torr) Littlo /. deppcana, Stcud. J. mexieana, Sprong. • /. virginiana, L.* /. chinenais," L.* 1  1  0Thujaplicin  V Thujaplicin  Nootkntin  Dolabrin  0ThujaPygmacin plicinol  T-4.5  T-O.l  —  0.07  —  _  —  —  —  —  —  _  0.05  0.12  — —  —  —  —  —  —  — •  —  —  —  —  —  —  0.0003 0.008  —  —  —  0.0000  —  —  —  —  — —  — —  — —  — —  —  —  —  —  —  —  0.005 0.1G 0.0001 0.043  —  — — —  0.001  0.0002 0.0001  0.003  —  0.003 0.008  — —  0.04  —  Traco  0.0001 —  0.0002  —  —  —  —  —  —  —  —  —  —  —  —  —  — —  —  —  —. —  —  —  —  —  —  0.0004  —  0.008  —  —  —  —  —  —  —  0.0084  • Numbers denote the approximate amount of tropolone in %, based on dry weight of wood. * These analyses have been taken from the literature.  Source:  Total  Zavarin el al , ( 1959).  Method of Analysis Chrom. Chrom. Chrom. Chrom. Chrom. Chrom. Chrom. Chrom. Prep.  tree  growth  s i t e and p o i n t of sampling  within  the  They  may be i s o l a t e d from wood i n the l a b o r a t o r y  steam d i s t i l l a t i o n or by e x t r a c t i o n with organic The and  phenols.  the  properties  The  acidity,  i s midway  phenols. varies  The  by  solvents.  of  enols  enhanced by the adjacent  between  considerably  The  characteristic  that of  carboxylic  a c t u a l a c i d i t y of each  substituents  latter.  either  hydroxyl group of t h u j a p l i c i n s i s e n o l i c i n nature  possesses  group,  stem.  and  carbonyl acids  individual  and  compound  depending on the presence or absence of  and the c h a r a c t e r  acidity  increases  and the p o s i t i o n of slightly  from  the  alpha- to  gamma- t o b e t a - t h u j a p l i c i n s with pKa values of 7.8, 7.3, and 7.1 r e s p e c t i v e l y The  (Raa and Goksoyr, 1965).  keto-enolic  possibilities thujaplicins  for  s t r u c t u r e of t h u j a p l i c i n s gives chelation.  Color  reactions  with s o l u t i o n s of f e r r i c and c u p r i c s a l t s  highly c h a r a c t e r i s t i c .  Treatment with f e r r i c s a l t  g i v e s an immediate b r i l l i a n t red c o o r d i n a t i o n is  insoluble  chloroform. complex  i n water but r e a d i l y s o l u b l e An  excess  of  ferric  ion  in  benzene  converts  i s formed,  1956a).  With c u p r i c s a l t s ,  of are  solutions  complex,  t o a green d e r i v a t i v e which i s very s o l u b l e  (MacLean and Gardner, complex  great  which and  the red i n water a green  which i s i n s o l u b l e i n water but s o l u b l e  31  in benzene, are  chloroform  readily  useful  in  crystallized isolation  tropolones. large  and  A  commercial  of  copper complexes  solvents  characterizations  copper  collect  preparing screens  in  al,  1957).  Decomposition of the copper c h e l a t e s with hydrogen  sulphide  deposit  the  natural  copper  chelate  to  of  are  green  thujaplicin  kilns  used  and  of  but expensive method  thujaplicins  drying  The  from organic  and  simple  samples  alcohols.  et  (Gardner  y i e l d s an o i l y mixture of the t h u j a p l i c i n s . has  proven to be a convenient method of o b t a i n i n g  samples of In  tropolone  (Nozoe et  al , 1953,  (Barton,  1976).  beta-dolabrin  15 18°2' H  iron  and  absorption the be  from m  *P*  et  Gardner and Among  them,  yellow 95°.  cedar.  1957),  beta-dolabrin nootkatin  both b e t a - t h u j a p l i c i n o l and while  Nootkatin  nootkatin  has  the  i t s u l t r a v i o l e t and  i n d i c a t e d that  to decay fungi  32  i t was  was  together  with  with  infrared  a tropolone.  nootkatin  was  formula  I t s c h a r a c t e r i s t i c c o l o r complex  copper s o l u t i o n s and  toxic  al,  were i s o l a t e d from WRC,  spectra  important  Barton, 1958), and  t h u j a p l i c i n s found in WRC, very  s e v e r a l other  d e r i v a t i v e s have been c h a r a c t e r i z e d such  as b e t a - t h u j a p l i c i n o l (Gardner,  isolated  research  thujaplicins.  a d d i t i o n to t h u j a p l i c i n s ,  terpenoid  C  T h i s procedure  Like  considered  to  chamic  and  chaminic  a c i d s , which were assumed to be r e s p o n s i b l e f o r the  d u r a b i l i t y of yellow cedar Tropolone also isolated  the  1976).  d e r i v a t i v e s were found not only i n t r e e s , but from the m e t a b o l i t e of microorganisms, such  from Sepedonium 1965).  (Barton,  chrysospermum  There were two  organism.  One  (Bull.) Fr.  et  (Divekar,  tropolone d e r i v a t i v e s i d e n t i f i e d  of them,  named  sepedonin,  as al,  from  ^11 12^5' H  3,6,9-trihydroxy-methyl-1,3,4,7-tetrahydrocyclo-heptapyran7-one), predominated i n growing c u l t u r e s and was be the  d i r e c t product  derivative in  the  chemical  of metabolism.  of sepedonin,  was  probably  c u l t u r e f i l t r a t e during the (Divekar et  dehydration  Tropolone monomers.  derivatives  Only  one  al,  trivial  name  utahin.  heartwood of Juniper i s a yellow,  m.p.  313°.  symmetrically molecules  us  utahensis  oxidative  Chemically,  and  nature  Norin,  Lemm.  inactive,  process  by  mostly  as  and N o r i n ,  tropolone  1968).  isolated by Runeberg  from  a Its the  ( 1 960).  high m e l t i n g compound,  c o u p l i n g of two  i n v o l v e d the  simple  tropolone  1968).  and  33  j  in  formation of u t a h i n probably  (Baggaley  spontaneously  isolation  Utahin was  optically  The  formed  d i t r o p o l o n o i d has been r e p o r t e d as  compound so f a r (Baggaley is  other, an anhydro  1965).  exist  natural  It  The  b e l i e v e d to  its  derivatives  undergo  facile electrophilic  s u b s t i t u t i o n and t r o p e n o i d compounds i n  general submit more e a s i l y to n u c l e o p h i l i c s u b s t i t u t i o n s well  as  to  rearrangement  to benzenoid  compounds  as  (Nozoe,  1956). The e n o l i c hydroxyl i n the t r o p o l o n e r i n g of  i t s acidity,  tropolone (Figure  are  and hence the a c i d i t y d i s a p p e a r s when  i s e s t e r i f i e d or e t h e r i f i e d .  or  (Doering,  resistant  stabilized  its  the  sodium  1951;  in  Raa and Goksoyr,  the form of t h e i r  Poh  Poh,  (1974)  specifically  and  benzene,  reported rapidly  ether  1965).  methyl  Tropolones  since  conjugated  with  they  acid  are  cations  primary  labile amine  that  with  tropolones  reacted  a l l non-aromatic  such as carbon  primary  tetrachloride  to y i e l d the corresponding yellow s o l i d amine  salts  tropolones  and non-aromatic  as  with  the  1974).  tropolone  reaction.  salt  or a non-aromatic  amines i n non-polar s o l v e n t s , and  methyl  However, t r o p o l o n e s are h i g h l y  presence of a l k a l i  (Nozoe, 1956;  or s i l v e r  to hot c o n c e n t r a t e d a c i d s ,  ( F i g u r e s 3B and 3C). in  The  3A) c o u l d be obtained by t r e a t i n g a t r o p o l o n e  diazomethane, iodide  i s the source  (Figure  3D).  The  reaction  primary amines i s an  between acid-base  Since secondary and t e r t i a r y a l i p h a t i c amines are  b a s i c as primary a l i p h a t i c amines (pKa about  34  10),  they  Figure  3.  S t r u c t u r e s of M e t h y l E t h e r , C o n j u g a t e d and Amine S a l t s of T r o p o l o n e .  35  Acid  Cations,  are  expected  to  demonstrated tropolone  that  salts  benzene,  also  react  non-aromatic  with  tropolones.  secondary  were s o l u b l e i n carbon  whereas non-aromatic primary  and  Poh  tertiary  tetrachloride  amine tropolone  and salts  were i n s o l u b l e i n these s o l v e n t s .  Because aromatic  are  they do not a b s t r a c t the  weak bases (pKa l e s s than  5),  amines  a c i d i c proton of a tropolone to a s u f f i c i e n t e x t e n t . amine  tropolone s a l t s r e a c t r a p i d l y with aqueous  give back the o r i g i n a l amines and  the  tropolones  (Poh,  tropolone  ring.  r e p o r t e d (Nozoe,  gave  first  rapidly  isopropyl-3-hydroxytropolone  cleaved  formation  of  levulinic  acid  oxidation. tropolone 3- and  to  a  It  cis,  dihydroxy was was  a l s o produced a l s o found  5- p o s i t i o n s (Nozoe et  then  Because  al,  beta-thujaplicin  in hydroxylation  1953).  acid  In the  acid  was  (probably only  the  formed under the  36  of  beta-isopropyl-  that p e r s u l f a t e o x i d a t i o n  alpha-thujaplicin,  isopropyl-muconic  was  case  i t yielded beta-isopropyl-levulinic  beta-isopropyl-muconic For  that  (hinokitiol)  acid.  during  degrade  1956)  which  intermediate,  or b e t a - t h u j a p l i c i n r e s u l t e d  gamma-thujaplicin  form).  cis-muconic  to  1974).  with a l k a l i n e hydrogen peroxide can  hydrogen peroxide o x i d a t i o n of b e t a - t h u j a p l i c i n  and  bases  Oxidation  I t was  These  in  its  compound  of of of acid  trans beta-  influence  of  hydrogen  p e r o x i d e . The r e a c t i o n process of hydrogen  peroxide  o x i d a t i o n i s shown as F i g u r e 4. Tropolones substitution. expected The  3,  were found to e a s i l y undergo The  5,  positions  to  be  s i m i l a r to  e s p e c i a l l y phenols. easily  in  tropolones (by  halogenation  (Nozoe,  observed  and  of  azo  1955).  tropolones  benzenoid  coupling  compounds,  nitrosation;  hydroxymethylation;  The e l e c t r o p h i l i c  and  substitution  i n f l u e n c e d by the s t e r i c e f f e c t i n the tropolone  1956)  that  series.  troponoids  such as halogens and  methoxyl  It  of was  containing  groups,  could  e l i m i n a t e these s u b s t i t u e n t s i n the form of an anion  then  either  s u b s t i t u t e with  rearrange t o benzenoid s t r u c t u r e s By  means  ultraviolet  of  of  (UV),  (NMR) s p e c t r a , deal  (Pauson,  as  s u b s t i t u t i o n s taken p l a c e  acid);  1956).  groups  (Nozoe,  substituents easily  sulfamic  were markedly  neighboring  those  were:  were  s u b s t i t u t i o n of  Electrophilic  sulfonation  processes  substituted  and 7 i n the t r o p o l o n e r i n g  mechanisms of e l e c t r o p h i l i c  seemed  the  easily  electrophilic  the  another  measurements,  (IR), nuclear magnetic  and x-ray and e l e c t r o n d i f f r a c t i o n , structural  i n f o r m a t i o n about  n a t u r a l compounds was a v a i l a b l e .  37  or  (Figure 5).  modern p h y s i c a l infrared  substituent  this  such  as  resonance a great group  of  F i g u r e 4. O x i d a t i o n of H i n o k i t i o l with Hydrogen P e r o x i d e .  CC, Hinokitiol  ICXXXI1.)  (CXXXMl.) • • 1 voprof>y!-ru.cumuconu: »cid.  I OH O  1  HO  r \ f '  -<  O H  fCXXXV.)  Source: Nozoe,  I / O  COOH  /""V'' -<  /—COOH  ° -<  ICXXXVI.J  ICXXXVU.)  (1956).  38  C  .  -<  H  a  COOH  (CXXXVII1.)  Figure  5.  Anionic  S u b s t i t u t i o n and Rearrangement of T r o p o l o n e s .  Source: Nozoe,  (1956).  39  The  UV  extent,  to  wavelength Hillis  s p e c t r a of t r o p o n o i d s were those  range  (1962),  intense region  benzenoid  compounds  but d i f f e r e n t  with  intensity.  to  some  the  As  same  noted  by  the UV a b s o r p t i o n e x h i b i t e d a maximum of very  a b s o r p t i o n ( l o g e, (Region  similar,  4.25-4.7) i n the 228 to 270 urn  1) and two bands of somewhat l e s s e r  intensity  (loge. , 3.5-4.0) i n the 300 to 340 and 350 to 380 urn regions (Region  2 ) . The band i n Region 1 was common t o tropones and  tropolones.  I t was h a r d l y a f f e c t e d by the presence  groups  shifted  but  halogen,  slightly  to  longer  of a l k y l  wavelength  hydroxyl or e s t e r groups were p r e s e n t .  IR s p e c t r a of t r o p o l o n e s have been r e p o r t e d i n studies  (Nozoe,  1956;  the spectrum a t 1265, have  C-H to  Gardner et  al,  1957).  (Gardner  et  al,  1440, 1468, 1558, 1618, and 3165 c m  1957).  weak  of benzenoid  intensity  stretching carbon  compounds and appeared as a  i n the region 3060 t o 3010 cm . 1  v i b r a t i o n of tropones  appeared at 1645  the  similar band The cm  of C=0  1  in  t e t r a c h l o r i d e and with higher i n t e n s i t y at 1651 cm  i n the gaseous s t a t e . l o c a t e d at 1613,  -1  tropolone  Besides those bands,  s t r e t c h i n g v i b r a t i o n of the t r o p o l o n e r i n g was that  several  The bands i n  been c o n s i d e r e d t o be c h a r a c t e r i s t i c of the  nucleus  when  1  For t r o p o l o n e s , the c a r b o n y l band was  1620 and 1628 cm  40  1  (solid,  s o l u t i o n , gas,  respectively)  indicating  toward the lower This  frequency  observation,  tropolone (3600  was  cm  )  1  indicated  a displacement  of 20 to  25  region as compared with  tropones.  as w e l l as the f a c t that the OH band  d i s p l a c e d from the normal hydroxyl to  cm  a  lower  the presence  frequency  region  of i n t r a m o l e c u l a r  of  frequency  (3165  hydrogen  cm bonding  (Nozoe, 1956). NMR  spectroscopy  assignment report  and  i s a very u s e f u l method f o r s t r u c t u r a l  isomer  to  proton  NMR  tropolone d e r i v a t i v e s .  ( H)  ring  completely  protons.  multiplets, ( B a g l i and  of  simplicity, tropone and  larger some  These which  st-Jacques,  protons were  chemical s h i f t s proton  decoupled  and  often  1972;  B a g l i and  be in  non-  exhibited to  analyse  On the other hand, greater  spectral  carbon-13 NMR  tropolone d e r i v a t i v e s have been found  s t u d i e s (Weiler,  range  coupled  tedious  1978).  NMR  1  T h i s might  s p e c t r a f o r the v a r i o u s s t r o n g l y  second-order  al,  systematic  d i f f i c u l t i e s of the small chemical s h i f t  equivalent  because  No  has been found g i v i n g i n f o r m a t i o n on proton  s p e c t r a of tropone and due  characterization.  st-Jacques,  data  of  in several  1978;  Bagli  et  1979). The  solvent  chemical for  the  s h i f t s i n ppm  from TMS  tropone nucleus seemed  41  using very  CDClg simple  as and  distinguishable. C-3  and  C-6:  Jacques, carbons 7  135.8,  1978).  C-4 and C-5:  134.4 ( B a g l i  The carbon-13 chemical  of v a r i o u s s u b s t i t u t e d tropones et  (Bagli  between  They were C-1: 187.7, C-2 and C-7: 141.7,  the  al,  1979).  two  tautomers (or  There was  and s t -  s h i f t s f o r the r i n g are l i s t e d  a  rapid  resonance  i n Table  equilibrium  structures)  of  t r o p o l o n e , as f o l l o w s :  The  proton decoupled  carbon-13 NMR  s p e c t r a showed that t h i s  compound behaved as a symmetric s t r u c t u r e . four s i n g l e t l i n e s i n the spectrum ( H NMR 1  20  lines).  C-7,  C-4  The chemical  shifts  There were only spectrum of  f o r C-1 and C-2,  C-3  over and  and C-6 had the same v a l u e s r e s p e c t i v e l y (Weiler,  1972). N e i t h e r proton NMR nor carbon-13 NMR s p e c t r o s c o p i c data of  t r o p o l o n e s d e r i v a t i v e s i s o l a t e d from WRC heartwood  have  been r e p o r t e d so f a r . There the  has been much s p e c u l a t i o n on the  natural  Erdtman  tropolones.  (1952)  discussed  biogenesis  With regard t o wood the  42  good  of  tropolones,  possibility  that  Table 7. Carbon-13 Chemical S h i f t s of S e v e r a l D e r i v a t i v e s of 2-methoxytropone i n CDC1-, S o l u t i o n . C-1  Compound  C-2  C-3  C-4  C=5  C-6  C-7  c- 8  H  180. 1 165. 0 112. 2 132. 4 1 27. 6 136. 3 1 36. 3 55. 9  3-Br  9 1 37. 179. 2 162. 9 1 27. 9 1 28.1 1  7-Br  1 73. 6 162. 6 112. 2 1 32. 5 56. 5 8 1 25.1 1 39. 6 1 37.  5-Br  2 1 39. 7 56. 2 179. 2 164. 6 111. 2 1 34. 0 1 22. 6 1 35.  3,7-Br 5,7-Br  3 47 .  0 59. 1 1 38.  173. 1 159. 8 127. 6 137. 7 125. 2 1 38.1 1 39.1 59. 5  2  173. 2 161 .8 111. 5 1 34. 3 1 42. 9 56. 8 5 1 19. 6 1 36.  3  178. 8 164. 0 110. 6 130. 4 133. 2 137. 2 1 35. 6 56. 0  5-Cl 3-CH C0 CH  3  181 .4 164. 4 1 32. 0 1 35. 4 136. 8 1 38. 6 58. 7 5 1 29.  7-CH C0 CH  3  7 164. 1 112. 2 1 32. 2 1 26. 2 56. 1 1 78. 6 137. 5 1 42.  2  2  2  2  3-CH3  2 1 34. 2 58. 0 181. 1 163. 7 1 36. 0 1 37. 5 1 36. 8 1 29.  7-CH3  4 135. 5 1 45. 6 55. 6 179. 1 162. 6 111. 7 1 30. 3 1 26.  3-OCH  7 1 33. 180. 7 154. 5 1 58. 6 1 27. 8 1 29. 2 140. 5 58. 6  7-OCH  7 161 .7 114. 1 1 25. 7 1 25. 7 114. 1 161. 7 56. 2 1 73.  3  3  6-OCH  3  177. 8 165. 4 108. 2 1 29. 9 1 24. 0 163. 2 113. 2 55. 0  5-OCH  3  2 54. 9 5 132. 1 1 36. 178. 6 159. 0 112. 9 107. 1 1 58.  Source: B a g l i  et al,  (1979).  43  tropolones  were  derived  common p r e c u r s o r .  directly  Although  usual i s o p r e n o i d s t r u c t u r e ,  from terpenes  phenols,  had  the  they might be c o n s i d e r e d as  of  such as  carvacrol,  together  with  thymoquinone,  and  thymohydroquinone and with t e r p e n o i d c a r b o x y l i c a c i d s , as r h o d i n i c a c i d and alpha-, beta-chamic a c i d Nootkatin, cedar,  one  has  r e f e r r e d to as a sesquiterpene  (Barton,  1976), which might be d e r i v e d from a  directly  or  by secondary  i n t r o d u c t i o n of  s i d e c h a i n to b e t a - t h u j a p l i c i n In  the  case of microorganism  biosynthesis formed  via  studies  experiments  have  conducted that  Sepedonium  by  sepodonin,  chr ys os permum  methylation and  of  a  rearrangement and  a  his  followed  Fr.,  44  by  tropolones, they  (1969)  metabolite was  a  were  biosynthetic  co-workers  intermediate  cyclization.  isopentenyl  that  tropolone  (Bull.)  tropolone  1962).  Carbon-13  and  polyketide  malonate,  the  suggested  Wright  i n yellow  sesquiterpene  metabolite  the a c e t a t e mechanism.  indicated  acetate  (Hillis,  such  (Nozoe, 1956).  of the tropolone d e r i v a t i v e s found  been  a  t h u j a p l i c i n s d i d not possess  t e r p e n o i d o r i g i n because they were o f t e n found terpenoid  or  formed derived  of by from  stereospecific  2.5.  Biological  The  activities  heartwood  of  fungi,  1944).  i n s e c t s and  It  i s now  parallels  structural  components,  MacDonald,  1971),  Hillis,  other  extractives  trees,  in  contrast  the  attack  sometimes even marine borers  (Wise,  the  presence  such  as  of  lignans  high  (MacRae and  heartwood  extraneous  tropolones  r e s i n s (Richardson,  1972),  to  be remarkably r e s i s t a n t to  g e n e r a l l y accepted that  durability  and  wood  many  accompanying sapwood, may of  of  non-  (Barton  1978), tannins Towers,  and (Hart  1984),  and  phenolics. It  groups  had  been e s t a b l i s h e d f o r a long time that two  of  extractives  (especially  in  thujaplicins)  WRC, and  tropolone water  major  derivatives  soluble  phenols  ( l i g n a n s ) have f u n g i c i d a l p r o p e r t i e s . Rennerfelt He  (1948) t e s t e d the t o x i c i t y of t h u j a p l i c i n s .  found that decay fungi were i n h i b i t e d with  between ppm). fungi that  0.001  and  Phenol, at 0.1  0.002 percent  t e s t e d in the  to 0.2  thujaplicins  percent were about  concentration  of gamma-thujaplicin  same way,  i n h i b i t e d the decay  concentration.  He  1945).  concluded  10 times more e f f e c t i v e  p i n o s y l v i n , the most potent f u n g i c i d e i n hard pine (Rennerfelt,  (10-20  than  heartwood  A f t e r comparison of i n h i b i t i n g e f f e c t s  45  between  thujaplicin  synthetic  product  effective  fungicide  timber, order.  which  sodium has  against  he i n d i c a t e d that  widely  used  blueing  as an  fungi  on  t h e i r a c t i v i t i e s were of the same fungi, Rennerfelt  (1945) a l s o  with beta being the most t o x i c ,  by gamma, then alpha using  durability  become  a  beta-, and gamma-thujaplicins t o be t o x i c i n  concentrations  Rudman  pentachlorophenol,  decay and  In the case of b l u e i n g  showed alpha-, low  and  conducted i n timber  followed  the agar d i f f u s i o n method. research  (Rudman,  on  1962;  causes  of  1963).  natural  A number  of  heartwood e x t r a c t i v e s and r e l a t e d compounds were examined by a  semi-micro  technique which used a wood s u b s t r a t e  form of sawdust. 8  (Rudman,  thujaplicins, all  Results  1963).  of h i s research He  found  i n the  a r e shown i n Table  that  beta  and  gamma  and b e t a - t h u j a p l i c i n o l were the most t o x i c t o  the s p e c i e s  of decay fungi t e s t e d by  comparison  with  other e x t r a c t i v e chemicals a t 1 percent w/w c o n c e n t r a t i o n i n wood sawdust.  He a l s o i n d i c a t e d t h u j a p l i c i n s t o be as t o x i c  as the naphthopyran lapachonon The Endl.) examined  (Rudman, 1963).  a n t i f u n g a l a c t i v i t y of h i n o k i  {Chamaecyparis  extractives  in thujaplicins  and  which  i s rich  proved to be h i g h l y t o x i c  c u l t u r e media of Basi di omycet es  to  was  a cultivation  (Kinjo and Yaga, 1986).  46  obtusa  Table 11 R e s u l t s of T o x i c i t y T e s t s f o r a Number of Heartwood E x t r a c t i v e s (Decay as a percentage of decay i n c o n t r o l ) . C.  Fungi  L. ttpldtut  1779  Pfrcfntapf Concentration w/w ot E x t i a t t l v t In Sawdutt Benttnc  elivacea  0.11  0.33  1.00  0.11  0.33  P. monlicola  L. trabta  7516  7520  1.00  0.11  0.33  7522  1.00  1.00  0.11  0.33  76 65 78 60 100 62 62 104 78 85 113 103  70 103 76 76 65 85 63 100 88 63 64 103  100 68  Derivative* 104 109 B5  1 30 53 101 65 60 11 60 80 78 102 59  100 105 107 06 115 62 104 63  92 110 77 109 60 103 126 103 115 69 104 76  28 69 75 102 66 110 104 120 110 64 65 69  106 64 66 114 104 68 85 86 78 60 67 61  69 62 68 120 115 112 66 65 78 60 64 84  39 76 61 105 107 88 70 79 88 6 71 64  78 76 71 62 68 62 65 68 64 69 102 61  72 45 75 102 63 65 64 68 66 89 88 87  108  48  0  65  44  13  93  0  I  61  61  0  101 102 09  87 103 64  101 107 87  69 105 62  58 78 66  54 104 65  69 67 67  67 66 64  47 63 7  100 78 44  79 69 25  45 78 6  69 106 90 106 109 100  82 107 67  83 102 69  103 62 67  67 46  92 121 12 —  34  104  70 77 60  24 2  0  B7 70 61 71 106 63 75 85  84 67 100  105 100 107  66 117 62 109 no 88 60 78  89 110 81  118 95 62 66  63 63 65 60 72 65 60 30  64 71 88 71  70 68  64 91 65 66  86 69 64 62  83 63 61  74 101 70 68  71 62 68 80  88 69 39 43  87 107 87 64  65 104 66 65  86 65 69 82  60 91 64 107  100 62 42 101  112 113 102  69 113 60  62 30 8  63 53 84  64 7 31  29  75 35 63  60  —  60 68 66 62 92 78 61 66 83 62 9B 104 62 76  63 62 66 69 61 66 88 62 eo 62 69 67 87 87 — — 104 102 I OB 69 83 101  60 69 88 65  80 83 55 84 79 36 81 84 66 61 75 107 83 105 107  86 63 40 61  76 69 67 66 63 84  84 65 77 77 87 74 78 64 86 66 106 82 103 68 86  67 69 103 73  69 too 106 100 79 86  63 89 70 104 66 49 87 85 66 68 69 111 102 100 102 62 80 75 60 84 62 B2  76 62 102 63 63 69  61 80 80 61 79 98  64 63 81 86 64 102  78 BO 81  . 61  eo  87 81 84 85 62 60 79 69 106  66  Naphthopyran  Qulnono  Anlhraq-jinont-2-carboxyllc acid .  .  Coumarlns  es  4  0  —  75 73 BS B9  —  0 0  23  63  66  — —  —  0  StUbenes  BB  89  0  86 66  Ttopolonea —  —  0  0  79 66 83 78 62 65 66 114 89 64 -103 64 87 101 67  79 63 61 61 104 64 85 69 61 67 63 86 80 68  64 61 B6 79 B7 84  88 87 101 68 69 64  66 63 62 66 95  97 90 81 88 106  __ 95 71 15 69 98  71 72 87 71 89 83 61 62 86 65 65 77 106 66 67 63 69 82 89 64 99 87  63 64 96  85 6 82  66 109 97  101 107 86  93 no 88  96 80 13  101 100 63  109 65 90  0  0 0 0  16 74  11 17  II  2  92 73  66 B2  0 0 0 0  0 6  6  81  1 • 10  0  0  0  11  Flavonold* d-CBtfchin Ta*lfolin Rcbiiiftln Arorr.aflf ndrln Aromadrndrin-7-mtthylether  . .  .  -—  64 100 78 107 2-HydTexy-4 6-dimethoxychalkone l  .  es  63 65  Llgnant  —  68  89 76 62 81 59  14  82 62 86 62  '  75 72 81 83 86  60  BB  •  P8 62 —  63 69 —  66 86 —  B3 65 90  52 36 74  0 0 — 0  Commercial Fungicides  0  0  *> Ether-methanol extracted mountain ash ( £ .  rifnans)  88  0  44  0  0  28  2  2  0 6  0 0  0 0  heartwood was uted a i the tubttrate.  Source: Pudman, (1963). 47  Toxicity of WRC  heartwood e x t r a c t i v e s  (1954) also  showed  that  thujaplicins. of  fraction  conducted by Roff and  Atkinson WRC  were  though they were much l e s s  toxic  phenols i n  The t o x i c i t y t o fungi  the same order as that  Atkinson,  of t h i s f r a c t i o n  of z i n c c h l o r i d e  (Roff  phenolic  frequently  However,  fraction  20 to 100 times that  a l s o had a c e r t a i n All  since  WRC  of the t h u j a p l i c i n s  reports  on  known decay f u n g i ,  preservative. the  higher  Basidi  antifungal  These  results  thujaplicins  as  p o t e n t i a l wood p r e s e r v a t i v e  treatment  in  service.  explain  the  simply  because  were  useful  However,  these  r e a l r o l e of t h u j a p l i c i n s i n  thujaplicins  the  and  results  of  non-hymenomycetous  for  trees.  48  considering in  results  interaction "pioneer"  lumber  fail  standing  c o u l d a f f e c t the whole process of microorganism in standing WRC  obtained  omycet es , as t e s t i n g  organisms.  a  was  (Barton,  a c t i v i t y of t h u j a p l i c i n s were based on the r e s u l t s using w e l l  of  t h i s f r a c t i o n of the e x t r a c t i v e s  r o l e as a n a t u r a l  previous  the c o n c e n t r a t i o n  i n the inner heartwood of  1962), i t was apparent that  by  and  1964), which was roughly one two-hundredth of that  of the t h u j a p l i c i n s . the  phenolic  the water s o l u b l e  t o x i c t o decay f u n g i ,  than was  t e s t s of the water s o l u b l e  to  trees, between  organisms progression  The  antibacterial  demonstrated  by T u r s t  activity  and  of  beta-thujaplicins  Coombs (1973).  They showed that  beta-thujaplicin  was  compound.  b a c t e r o s t a t i c f o r g r a m - p o s i t i v e and  I t was  negative s p e c i e s . it  was  a  broad-spectrum  At c o n c e n t r a t i o n s  and  Turst  (1973)  found  a c t i v i t y of b e t a - t h u j a p l i c i n was laboratory  light.  thujaplicin spectral  greater  was  lost  Photochemical  detected  analysis  that  Raa  caused  and  Goksoyr  during  (1965).  the  to  beta-  ultraviolet  e f f e c t s of  had  been  inhibition  the  of  The  beta-  investigated  in  i t s cupric chelate  same manner, but  species.  antibacterial  t h e i r study by  The  ug/ml,  respiration  seemed to  act  l a t t e r compound was  70  times more t o x i c .  Previous that there amount  100  decomposition  of the compound.  by t h u j a p l i c i n and  very much i n the to 100  than  gram-  f o l l o w i n g exposure  t h u j a p l i c i n on the metabolism of yeast by  antibacterial  b a c t e r i c i d a l f o r a number of gram-negative  Coombs  was  work (Barton and MacDonald,  was  a strong  1971)  indicated  r e l a t i o n s h i p among wood  of wood e x t r a c t i v e s and  color,  the  the decay r e s i s t a n c e of  WRC  heartwood.  The  paralleled  v a r i a t i o n i n content of the e x t r a c t i v e s  thujaplicins). light-straw  v a r i a t i o n in the p a t t e r n  The through  color  of  pinkish  WRC and  49  of decay  resistance (mainly  heartwood  ranged  from  tan-brown  to  dark  a  chocolate  brown.  Gardner,  (1956b;  light  to  darker  t r a n s i t i o n was  1958), color  to the  wherever a c o l o r t r a n s i t i o n was  present  with  was  f o r water s o l u b l e phenols.  1985)  distance A and  decay  from the rather  Piirto  natural heartwood  sample,  the  in the wood,  The  These  change  phenomena  Work c a r r i e d out  in  were  recently  resistance  It was  increased  provided  evidence  with  radial  done by  Wilcox  concerning  between c o l o r v a r i a t i o n across decay  resistance  (D.Don.) E n d l . ) . variability  in  of  In t h e i r study,  the highest  a  the  section  and  (Sequoia  redwood  Redwood has  both the shade  used as e x t r a c t i n g s o l v e n t s . c o l o r e d wood had  apparent in  pith.  (1976)  color.  and  a number of decay fungi showed a  i n t e r e s t i n g p i e c e of research  natural  sempervirens  from  much more pronounced than  to the o l d growth wood.  that  relationship the  (1961).  wood a g a i n s t  similar pattern study  and  on t e s t i n g the d u r a b i l i t y of second growth  fast-grown WRC  his  a  the water s o l u b l e phenols.  f u r t h e r observed by Roff (Doyle,  in  the dominant n a t u r a l p r e s e r v a t i v e  t h u j a p l i c i n content with c o l o r was it  r e s u l t s of MacLean  accompanied by a sharp drop i n the content of  thujaplicins, together  According  a wide degree of  and  water and  intensity ethanol  of  were  T h e i r r e s u l t s showed that dark e x t r a c t i v e s content.  50  When the  darkness  of wood c o l o r  increased,  the content of  soluble  extractives  content  of ethanol s o l u b l e e x t r a c t i v e s  the  color  against  They demonstrated that  of the wood darkened,  an  exceptionally  moniicola, in  increased.  increased,  wood  boards tending t o have the highest patterns  WRC  and  contradiction of wood; for  that  were  opposite.  may be due to d i f f e r e n c e  detailed  structural  research  changes  mechanisms of e x t r a c t i v e  be  a  with the darkest  of c o l o r a t i o n of wood v s . decay  redwood  P.  destroyer,  resistance.  or the microorganisms used,  further  chemical  wood  Thus, they g e n e r a l i z e d  r e l a t i v e l y good index of decay r e s i s t a n c e ,  in  the  darkness of heartwood c o l o r appeared to  The  as  that i s ,  weight l o s s of  aggressive  tended to decrease.  redwood,  ethanol-  toxicity  This  kind  i n methods;  of  species  and i n d i c a t e d a  on  and  resistance  the  need  relationship  discoloration, in natural  and  of the  durability.  Heartwood e x t r a c t i v e s of other t r e e s a l s o showed v a r i e d biological activity. extractives  of  the  Schneid were reported some Quel.  wood and  The hexane,  heartwood of Madura (Wang and Hart,  decay f u n g i , Gloeophyllum  c h l o r o f o r m , and methanol  such as Coriolus trabeum  (Pers.  major f r a c t i o n of the e x t r a c t i v e s  51  pomifera  (Raf.)  1983) t o be t o x i c versicolor ex.Fr.) M u l l .  responsible  f o r the  to (L.) The  decay  resistance, mixture  according of  to t h e i r r e s u l t s ,  t e t r a - and  tetrahydroxystilbenes The Fomes  toxicity  annosus  spruce  of e x t r a c t a b l e l i g n a n s Karst.  examined  Hydroxymatairesinol, identified annosus  conidendrin  not  whereas  significantly  Shain  not  than a  sound  and  their  and  of the  hydroxymatairesinol The  effect  for matairesinol,  for hydroxymatairesinol,  concentrations  lower  c o n c e n t r a t i o n s than one  of  but  which was  r e l a t i o n s h i p between s o l u b i l i t y  pinosylvin)  that  concentrations  was more  tested. of  the  inhibitors.  They d i s c o v e r e d that a compound with r e l a t i v e l y low (e.g.  F.  t e s t s using  fungal i n h i b i t o r s and decay r e s i s t a n c e of these  solubility  were  indicated  m y c e l i a l growth.  significant  (1971).  conidendrin  study  and  Norway  Hillis  Bioassay  the former at a l l  suggested  from  and  i n h i b i t o r y at any  inhibited  highly s i g n i f i c a n t inhibitory  in  matairesinol  c o n c e n t r a t i o n was  They  by  matairesinol,  Karst.  was  with  a f f e c t e d heartwood of  from the e x t r a c t i v e s . (Fr.)  tested,  penta-hydroxystilbenes  predominating.  (Fr.)  were  appeared to be a  would have to be  aqueous  active  with r e l a t i v e l y high  at  aqueous  solubility.  of  Doi hi stroma  pi ni  young Pi nus  radiata  Hulbary D.Don.  is a significant  l e a f pathogen  p l a n t a t i o n s in New  52  Zealand.  Bioassays  of  selected  fatty  and  Dot hi stroma  pi ni  1983)  shown that a long c h a i n  have  omega-hydroxy and  Hulbary.  as t e s t organism  f a t t y a c i d s and  7-hydroxydehydroabietic  8,11 ,13-trien-18-oic  acid)  fatty  germination  and  antifungal  mycelial  effects  acid  al ,  (stearic),  resin acids  acids;  13-hydroxypodocarpa-  were h i g h l y  (red  pi ni  oak),  (7-keto  fungistatic.  and  The  Hulbary.  growth in v i t r o .  rubra  using  ( F r a n i c h et  spore  The s i g n i f i c a n t  of s e l e c t e d bark e x t r a c t i v e s  Quercus  Mill.,  acids  oxidized  i n h i b i t e d both Dot hi stroma  compounds  ovata  resin  P.  Carya  of  strobus  (white  pine) were observed on the growth medium of Lenzites (Harun and The  J r , 1985).  et  S. a  k r a f t black  1986).  diethyl  Z.)  Sukatcev  and ver.  l i q u o r s and  by Sameshima and and  were  Hara)  extraction  and  and  extractives  e f f l u e n t s were  steam  al  responsible  f a t t y and for  the  1978;  1980  obtained  toxicity.  by had  (Sameshima et  resin acids  in  reported  distillation  killing activity four  (Betula  birch  found that the e x t r a c t i v e s  Six phenols  extractives  japonica  white  h i s coworkers (Sameshima et  They  ether  Japanese  k r a f t bleach  r e l a t i v e l y higher termite 1980).  (Pinus  b i o l o g i c a l a c t i v i t i e s of Japanese red pine  densiflora pi at yphylI  trabea  al ,  from  the  Their  t o x i c i t y l e v e l s were determined by g a s - l i q u i d chromatography  53  (Sameshima et  al,  1986).  Many works have p o i n t e d out in  s e r v i c e and  trees  were  that the d u r a b i l i t y of wood  the decay r e s i s t a n c e of heartwood in  dependent on the t o x i c i t y of  t h e i r c o n c e n t r a t i o n and  their  specificity  living  i t s extractives, to c e r t a i n s p e c i e s  of f u n g i . As  reported by a number of r e s e a r c h e r s ,  e x t r a c t i v e s had On  no t o x i c e f f e c t  the other hand,  to c e r t a i n s p e c i e s of f u n g i .  e x t r a c t i v e s might have been m o d i f i e d  u t i l i z e d by the fungi as an energy source Nootkatin, heartwood Don)  of  one  (1955)  and  of  (Chamaecyparis  been  found  of  0.005  a g a i n s t a number of  L a t e r i n 1970, stain  about 0.001  to  concentrations percent  of  and C s e r j e s i , nootkatin  from  noolkatensis  be  percent  (D. Nacht  fungistatic and  at  fungicidal  wood-destroying  at  fungi.  s e v e r a l u n i d e n t i f i e d fungi which cause black  in yellow cedar were i s o l a t e d  tolerance  isolated  been t e s t e d e a r l i e r by R e n n e r f e l t and  had  or  in v i t r o .  n a t u r a l tropolones  yellow cedar  Spach.) had  some p h e n o l i c  these 1970).  fungi to n o o t k a t i n was  1970)  and  examined  the (Smith  They found a c o n s i d e r a b l e decrease of s i g n i f i c a n t weight l o s s i n  clear  yellow cedar blocks a f t e r s i x u n i d e n t i f i e d b l a c k s t a i n  fungi  grew  content  (Smith,  on them.  without  They suspected  that the lower  54  concentration  of n o o t k a t i n of  a f t e r the treatment was due to the  nootkatin  by these f u n g i ,  degradation  and t h i s e f f e c t r e s u l t e d i n a  great decrease i n the decay r e s i s t a n c e of the wood to unidentified  wood-destroying  fungi.  No  other  attempts  of  i s o l a t i o n and i d e n t i f i c a t i o n of degraded compounds were made in the study. In whose WRC  1975,  van  der Kamp (1975) i s o l a t e d  code numbers were WR1, on the B.  C.  coast  s t a i n e d sound wood.  WR2  The bioassays  heartwood  concentrations considerable the  quickly to  trace  reduction  of  WRC  early  invaders  reduced  and WR2 even  amounts  brown  invaded  high  and  (van der sterile  thujaplicin  then  caused  a  resistance  of  He supported the h y p o t h e s i s that  involved a succession ( p o s s i b l y WR1)  n a t u r a l e x t r a c t i v e s present  and  fungi were performed  i n the n a t u r a l decay  wood i n t e s t b l o c k s .  decay  red  f o r t e s t i n g the t o x i c i t y  He found that both WR1 and  fungi,  from o l d growth  from l i g h t - s t r a w ,  of t h u j a p l i c i n s to these three Kamp, 1986).  and WR3,  three  of  organisms  somehow destroyed  and the  the toxic  i n the heartwood.  Work done by Edmonds (1976) was s i m i l a r t o the work Shain  and  compounds matairesinol  Hillis catechin  (1971). and  In  addition  leucocyanidin,  and h y d r o x y m a t a i r e s i n o l ,  55  to  the the  by  phenolic lignans:  which were t e s t e d i n  Shain  and H i l l i s '  (1971) work,  were a l s o used by  t e s t organism was a l s o the same: F.  The The  only d i f f e r e n c e was  from the d i f f e r e n t  annosus  tree species,  one was  Edmonds' r e s u l t  that  were  not  capable of i n h i b i t i n g the growth  (Fr.)  Karst.  present  heartwood F.  of  (1976)  annosus  Though l e u c o c y a n i d i n i n h i b i t e d growth of  only small amounts  apparently  annosus  e x t r a c t i v e s from western hemlock  higher c o n c e n t r a t i o n s i n c u l t u r e media,  in  levels.  isolated  from Norway spruce  indicated  at  (Fr.) K a r s t .  that these compounds were  and the other from western hemlock.  fungus  Edmonds.  did  in  wood  extractives,  not show any e f f e c t at n a t u r a l l y  On the other hand, i t was (Fr.) K a r s t .  was  it  the was and  occurring  proved by him that the F.  capable of modifying and  utilizing  these p h e n o l i c e x t r a c t i v e s . Edmonds' (1971). F. to  The c o n t r a d i c t i o n may  annosus  (Fr.) K a r s t . used  Korhonen  colsely differ  r e s u l t c o n t r a d i c t e d that of Shain and  (1978) F.  related  a r i s e from the t e s t  i n both experiments.  annosus  c o n s i s t e d of at  intersterility  groups  which  from each other i n many p r o p e r t i e s .  found mostly on Norway spruce (S group),  was  found only on pine (P group).  56  least  I f the F.  two  average  group  which was  important cause of butt rot of Norway spruce.  organism According  on  One  Hillis  was  the most  The other one annosus  used i n  both  experiments were not the same,  the d i f f e r e n t  results  may by the consequence. Two  of  parasitica the  early  A j e l l o et  stage al.  attacking  and PaeciI  omyces  study of the t o x i c i t y t e s t s of  microcorys  F.  Heather,  Muell.)  1983).  naturally  invaders. occurring  t h e i r growth extractive  enhanced The  extractives  PaeciIomyces et  al.  can spp.  of using  be  used  to  1983).  as the only Their  as  source  utilize by  in nutrient  source of energy.  57  source  of  r e s u l t s suggested  of  was more capable than P. extractives  discolored  solution containing water-soluble  these organisms remove or modify n a t u r a l  which  of  fungi  to a form l e s s i n h i b i t o r y to the Basi di omycetes form  and  extractives  e x t r a c t i v e s was examined  tallowwood heartwood  carbon (Wilkes and Heather, that  (Wilkes  the fungus' a b i l i t y to a c t  a b i l i t y of  heartwood  in a nutrient of  (Eucalyptus  t o l e r a n t to n a t u r a l l y o c c u r r i n g  This tolerance  pioneer  were used i n  tallowwood  c l e a r wood and the m o d i f i e d e x t r a c t i v e s  tissues. as  spp.  ophora  They found that these two m i c r o f u n g i were  apparently quite from  heartwood  Phi al  fungi,  preservatives or even t o a energy.  parasitica  The Ajello  s o l u t i o n as the s o l e  2.6.  P o s s i b l e mechanisms of e x t r a c t i v e  It  has  heartwood insects  been  extractives  either  combinations It  has  heartwood  of  these  of  the  water  fungistatic  80  are  thus  that  white  the  of  the  of  alba  monticola  80  extraordinarily  had been  the of  overcame  PVP and Tween complexes the  and  tannin-  strong.  They  was p r o b a b l y due and f u n g a l  reported  to  proteins,  f u n g a l membrane p e r m e a b i l i t y .  (Zabel,  to  (polyoxyethylene  Ordinarily,  effect  in  The a d d i t i o n  tannin-protein  tannin molecules  of  or  that  Murr.  L . ) was due  ellagitannins.  activity.  been  (1972)  g r o w t h medium t o t a l l y  the  t a n n i n on f u n g i  toxicity  fungi  a hydrogen-bonding t a n n i n - p r o t e i n  oak h e a r t w o o d The  the  ellagitannin  between  which a f f e c t e d  and H i l l i s  ellagitannins.  splitting  have  f o r m a t i o n of  effects  to  enzyme  complex  interaction and  of  of  in  b i o c h e m i c a l l y o r by  f u n g i P.  {Quercus  soluble  effects  capable  suggested  by H a r t  p y r r o l i d o n e ) o r Tween  regenerate  protein  invasion  compounds  processes.  oak  monooleate)  the  the  chemically,  wood-rotting  white  (polyvinyl  sorbitan  prevent  been d e m o n s t r a t e d of  PVP  by some w o r k e r s t h a t  mechanically,  of  inhibition  presence  supposed  toxicity  complex The  toxic  earlier  in  a  of  1948).  podocarpic a c i d  58  (PCA),  kind  lignan, was  deposited  ( B a u c h et  investigated  concentration globosum  ex.  puteana  workers  al,  1977).  F r . ) s t r . and  (Schum.  suggested that  ex.  The p h e n o l i c  important  s t i l b e n e compound  toxic  t h e Si rex  role i n restricting  ( H i l l i s and Inoue, 1968). more  fungi  percent  with  s t r . Those as t h e  and i n s e c t s .  pinosylvin, spp.  formed a f t e r  fungus, could  t h e spread of t h e  Pinosylvin  PCA  Chaetomium  PCA i n t h e t i s s u e m i g h t a c t  sapwood was i n f e c t e d w i t h an  0.25  Fr.) Karst.  mechanical blockage f o r b a c t e r i a ,  spp.  The f u n g i s t a t i c  was 0.05 p e r c e n t o f h e a r t w o o d w i t h  (Kunze.  Coniophora  Dacrydium  i n heartshakes of several  fungus  was f o u n d t o be  i n most c a s e s a n d a more u n i v e r s a l  play  rather  poison  than  i t s m o n o m e t h y l e t h e r , w h i c h was an i n h i b i t o r i n 0.01 t o 0.02 percent  concentration.-  It  was  was p r o p o s e d t h a t  inhibition  effect  due  to  both  a  inactivation  o f t h e e x t r a - c e l l u l a r enzymes.  metabolism  (1965). above  of  y e a s t was r e p o r t e d by  They showed t h a t -4 10  M and c u p r i c  beta-thujaplicin  respiration glucose,  as  Raa  or  sodium  acetate.  59  and  Goksoyr  i n concentrations above the  w e l l as the r e s p i r a t i o n a f t e r  ethanol  and  beta-thujaplicin  t h u j a p l i c i n chelate  i n h i b i t e d t h e r e s p i r a t i o n of baker's yeast,  1  fungitoxin  S t u d y on t h e e f f e c t s o f WRC e x t r a c t i v e on  pinosylvin s  Their  -6 10  M  endogenous  addition  of  experiment  demonstrated  that  chelating  compounds  were  more  far  discussed  the  thujaplicin  and  the  of  its  They  concluded  the  that  free  in  for  same e n z y m e  must  acid  substrates of  but  the The  showed  that  blocked. and  dehydrogenase.  these  two  did  act  not  of  bacteria,  Aeromonas  compounds entirely  and o t h e r  spp.  beta-thujaplicin  inhibition  interfere  with  wall  and c e l l  cell  osmotic  Another  possible  certain  groups  membrane  on  on  system.  case  by  a n d on  thujaplicin  species,  lysis  beta-  acted  was  succinic  that  They  cycle.  respiration  target  been  have  negative  from  which  of  pathway  citric  these  systems,  metal  and o t h e r s ,  inhibition  compounds  the  form  beta-thujaplicin.  respiratory  have  enzyme  (II)  that  speculated  the  iron  one p o s s i b l e  may  could  and a c e t a t e  pathway  they  the  the  system  chelate  In  of  succinate  respiratory  inactivated  the  chelating  part  the  However,  than  possibilities  transfer  inhibition  cupric  with copper,  toxic  glycolytic  electron  beta-thujaplicin  mode of  function  integrity,  pressure of  action  bacteria of  the  (Turst  might  bacteria  and  of  gram-  appeared  death Coombs,  resulted 1973).  beta-thujaplicin  have (Hugo  been and  to  to  to  disrupt  Bloomfield,  1971). Inhibition  of  the  enzyme c a t e c h o l  60  O-methyl  transferase  (COMT)  by tropolones has been r e p o r t e d ( B e l l e a u and  1963).  In the study, the enzyme was p u r i f i e d and a c a t e c h o l  methylation presence  reaction  was  c a r r i e d out with  of tropolone d e r i v a t i v e and without  derivative.  The  inhibitory  activity  d e r i v a t i v e s toward the COMT was observed. that  t r o p o l o n e s acted as s p e c i f i c  enzyme. for  complex  methionine The  the  tropolone  of  tropolone  I t was confirmed  i n h i b i t o r s of the  tropolones  and  proposed s-adenosyl  mechanism by which WRC t h u j a p l i c i n s i n h i b i t  was  presence  between  (COMT)  (Figure 6 ) .  unknown.  t h i s matter,  toxic  i n the  A s t r u c t u r e s i m i l a r t o that for COMT was  the  fungi  COMT  Burba,  decay  No experiments have been r e p o r t e d  so f a r . The most common hypothesis was that the  of the r e a c t i v e k e t o - e n o l i c group was necessary  of  minerals  have  been  shown  t o be  g r e a t e r i n d i s c o l o r e d and decayed wood i n l i v i n g t r e e s healthy  clear  (Shigo and Sharon, heartwood ion  for  action. Concentrations  in  in  wood of the same t r e e s 1970).  on  than  wt/wt  basis  R e n n e r f e l t (1962) compared sound  and decayed heartwood of spruce stems as t o metal  contents.  He  found  a . remarkable  potassium  and c a l c i u m content  suggested  that a c t i v e accumulation  increase  i n decayed  61  of metal  i n the  heartwood. ions i s  He  caused  Figure  6.  The Complex S t r u c t u r e between Tropolone, S-Adenosyl Methionine.  *:  Dot l i n e s represent the complex.  hydrogen  Source: B e l l e a u and Burba,  (1963).  62  Magnesium and  bonds between p r o t e i n s and  by l i v i n g hyphae from the t r a n s p i r a t i o n stream. Shigo  (1974)  manganese,  found that the  concentration  S a f f o r d and  of  potassium,  c a l c i u m and magnesium were a l l higher i n decayed  heartwood. It  was  decayed  not c l e a r  so f a r i f metal ion  accumulation  heartwood was due to the metabolic a c t i v i t y  in  of  the  fungus or a defence mechanism of the t r e e , or both. Metal for  elements,  especially  microorganisms to grow.  either  to  variety  iron,  Iron,  because of i t s  accept or donate e l e c t r o n s , of  oxidation  reduction  were very important ability  participated  reactions,  in  making  a it  i n d i s p e n s a b l e f o r a v a r i e t y of enzymatic r e a c t i o n s i n almost all  organisms.  Microorganisms  secondary m e t a b o l i t e s (Moody,  1986).  such as c a t e c h o l and hydroxamate available iron  to  oxygen atoms. form  siderophores  a  uptake  stable  capture i r o n ,  removal  of  of the  the i n t a c t iron  type  siderophores  These  at  complex  compounds,  (Neiland,  to be of any use,  contained  (Moody,  ferric  1981). it  Once  must  be  T h i s was accomplished e i t h e r by i r o n - s i d e r o p h o r e complex the  cell  surface  by  or  1986).  63  C u r r e n t l y , two  by  membrane  r e c e p t o r s , which had the s p e c i a l f u n c t i o n to recognize siderophore complex  as  siderophores,  These oxygen atoms can bind  t r a n s p o r t e d i n t o the c e l l . the  produced  iron-  possible  mechanisms  f o r the removal of i r o n from a siderophore  considered.  Hydrolysis  removal  in  or,  (Raymond et  situ  of the siderophore followed reduction  al ,1984).  was d i s r u p t e d  and r e l e a s e  of  were  by i r o n  the  iron  I f the process of t r a n s l o c a t i n g i r o n  by other f o r c e s ,  the microorganisms would  not  survive. Cowling and Brown (1969) s t a t e d that the wood c e l l capillary  s t r u c t u r e was  too small  decay enzymes i n t o wood. rot the  Therefore,  fungi to i n i t i a t e decay rapid,  to allow  the m i g r a t i o n  the a b i l i t y  i n the secondary c e l l  widespread depolymerization  wall of  of brownwall  and  of the c e l l u l o s e d i d  not appear to be a t t r i b u t a b l e s o l e l y to enzymatic mechanism. From the r e s u l t s of a s e r i e s of s t u d i e s 1973;  1974a;  1974b),  that  fungi may  via  an  Koenigs  have attacked  H20 iron _  2  (1972a;  proposed f o r the f i r s t  rate  of  hydroperoxy  non-enzymatic  agents.  system.  of  which  H  2^2  P °duced by r  H  2^2  were  I t has been e s t a b l i s h e d  extracellular much  decomposition  radicals,  The  t  the  (Buchanan  brown-rot  rate  hydroxyl  o  active et  Schmidt  et  al  (1981) demonstrated  64  on  and  oxidizing  al , 1976)  fungi  of  dependent  that  decomposed  more r a p i d l y i n the presence of F e ( I I ) as compared  Fe(lII).  time  c e l l u l o s e and p a r t l y decay wood  c e l l u l o s e o x i d a t i o n by the iron-H2C>2 system was the  1972b;  that  to  oxalic  acid,  which  naturally  was  a  fungal  occurring  secretion  ferric  ferrous  (Fe I I ) s t a t e .  (1981)  found  that  product,  i r o n (Fe I I I ) i n  reduced  wood  Experiments conducted by  oxalic  was  also  one  of  the  products  cellulose.  T h i s endogenous o x a l i c a c i d p a r t i c i p a t e d i n the  system, Thus,  of  more  which the  ferric  of  Such  a  H  2^2  (II) reaction  -  2  2  i r o n s to f e r r o u s  accelerated  system  established.  the H 0 F e  Nicholas  degradation  reduction  in  acid  t o the  the whole degradation n  o  n  _  e  system  n  z  y  i  n  a  t  i-  could  decay  c  attack.  I f tropolone d e r i v a t i v e s  Fe(III)  in  chelates, Among tropolone with stable  the wood's s u s c e p t i b i l i t y  the  wood  such as copper,  were c a l c u l a t e d to be 14 X 1 0 constants  1954a;  1 4  in a  structure,  process  to  a characteristic t o combine etc.  ion,  thus  form  and 7 X 1 0 . 1 1  of  rapidly to form a  c o n s t a n t s of CuT  of the tropolone metal  d e r i v a t i v e s were a l s o reported workers (1953;  ferric  The s t a b i l i t y  well  system w i l l be blocked.  d e r i v a t i v e s was t h e i r a b i l i t y  chelate.  formation  the  WRC heartwood e x t r a c t i v e s ,  metals,  was  i n WRC can capture e i t h e r  the non-enzymatic H 0 i r o n 2  process.  to subsequent enzymatic  or F e ( I I ) i n 2  i n the  have functioned  p r e p a r a t o r y way by a c t i n g on the wood pore increasing  irons  with  2  and  FeT  2  The data f o r complexes  and  e a r l i e r by Bayant and h i s co-  1954b; 1954c).  65  S i m i l a r c h e l a t e s can  be  formed w i t h c a t e c h o l .  Some s t r u c t u r e s o f t h e s e  c o m p l e x e s a r e shown i n F i g u r e thujaplicins' metal  toxicity  7.  One v e r y  t o decay f u n g i  c h e l a t e s w i t h those  irons,  the other  hand,  concentrations  basic  mechanisms  Their  preventive  direct  t o x i c a c t i o n , or t h e i r  their  chemical  probably  such p r e s e r v a t i v e i n use i s  thujaplicins In to e a s i l y the  may  the  cation's  be  system.  i t has  The  most  copper-chromium-arsenic  1978).  Therefore,  more t o x i c  to  thujaplicin  decay  fungi  than  themselves.  addition to the c a p a b i l i t y  of tropolone d e r i v a t i v e s  form metal c h e l a t e complexes,  k e t o - e n o l i c group should  mechanism o f t o x i c i t y  be t a k e n  i s considered.  l a r g e m o l e c u l e s i n t h e wood c e l l  other  a c t i v i t i e s of  i n t o a c c o u n t when The a b i l i t y  to utilize  which i n t u r n depends  t h e enzymes w i t h w h i c h t h e f u n g u s i s e q u i p p e d .  66  the  w a l l d e p e n d s on t h e a b i l i t y  of t h e fungus t o d i g e s t t h e m o l e c u l e s , on  preservatives.  i s due t o  t o t h e f u n g a l enzyme  (Richardson,  at  i s one o f t h e  m o d i f i c a t i o n t o t h e s u b s t r a t e so t h a t  common  chelates  effects  apparent, r e p e l l e n t a c t i o n , or  resistant  metal  This  o f t h e m e t a l c o m p l e x wood  become  (CCA)  of  succession.  t o fungal growth.  behavior  formation  m e t a l i o n s show t o x i c  higher  salts  i s the  a c t i o n of  which a r e e s s e n t i a l f o r  fungal s u r v i v a l or for microorganism On  likely  chelate  Enzymes  Figure  7.  The Chemical Chelates.  S t r u c t u r e s of the WRC  Extractive  Fe  +3  I beta-thujaplicin ferric  chelate  Fe  J 3  catechol  ferric  Source: Barton and MacDonald,  chelate  (1971).  67  +3  Ferric  are  built  up  combination tropolone 1974)  and  of p r o t e i n s of  various  derivatives  which i n  turn  amino-acids.  consist The  of  the  ability  of  to form amine-tropolone s a l t s  a l s o to form hydrogen bonds with p r o t e i n s  p o s s i b l e mechanisms f o r t o x i c  action.  68  (Poh, provide  MATERIALS and METHODS  3.1.  Sample s e l e c t i o n  The  WRC  wood sample f o r t h i s study was  c o l l e c t e d at  U.B.C. Research F o r e s t , Maple Ridge, B. C. Lake area, trees  which has WRC  were  examined  samples  which  pattern)  and  found, but The  only one  84 cm  the t r e e was  years.  m b o l t was  order  to  Sections at  cut and  growth zones  (Figure 9) and  transport  split  i n t o four lengthwise  then  In order  stored  U.  trees  B.  C.  m  in  campus  height tree  throughout The  a  age  of  four  0.9  one  0.9  sections  in  and stump.  laboratory.  i n a c o l d room immediately  to maintain the wood f r e s h n e s s ,  69  were  The  height,  removed,  them to the U.B.C.  were l a b e l l e d and  (general  height.  S t a r t i n g at breast  the F a c u l t y of F o r e s t r y ,  arrival.  approximately 30  removed from 20 m above the  b o l t was  obtain  felled.  with a s s i s t a n c e of a hand l e n s . 420  Several  to  variation  i n diameter at breast  m length b o l t s were cut  Each  was  determined by counting  cross-section  years o l d .  Several desirable  of these was  sampled  i n the Gwendoline  power borer  were observed for c o l o r  tree  was  using a 30 cm  decay l e v e l .  -(Figure 8) and age  t r e e s over 350  the  (2°C) after  two  of  F i g u r e 8. Tree from Which the Wood Samples Were Taken  70  Figure  9.  Four  0.9  M e t e r B o l t s Cut  from the S e l e c t e d  Tree.  the most r e p r e s e n t a t i v e  s e c t i o n s taken at breast height  were  kept i n a p l a s t i c bag i n a f r e e z e r at about -5°C. The Figure  c o l o r p a t t e r n of the t r e e c r o s s - s e c t i o n i s shown i n 10.  The  wood  patterns  for  this  sapwood,  light-straw  color  species. i n outer  variation  followed  I t ranged  from  heartwood,  typical white  in  through p i n k i s h ,  brown and tan-brown i n inner heartwood, t o dark-brown i n the c e n t r a l area. pocket This  of can  In a d d i t i o n to t h i s normal c o l o r p a t t e r n ,  pink-brown wood be  seen  was observed i n  clearly  (Figure  heartwood to inner heartwood r e g i o n .  10B)  heartwood  to  height  injury  quadrant  with  samples  for  scar at breast the  d i s c o l o r e d pocket  comparison  d i s c o l o r e d and sound WRC  of  72  the  outer  and can be t r a c e d  (Figure  10A).  provided  very  chemical  heartwoods.  in  quadrant.  T h i s d i s c o l o r e d pocket  was surrounded by straw-colored an  one  a  composition  The good of  Figure  10.  Cross Sections Variations.  10A: C r o s s s e c t i o n a t breast height.  of  the S e l e c t e d  Tree  Showing  10B: C r o s s s e c t i o n a t 0.9 ra above b r e a s t h e i g h t .  73  Color  3.2. I s o l a t i o n of microorganisms  Microorganisms  were i s o l a t e d  b o l t taken at breast amount  of  large the  height.  from one quadrant of  The quadrant i n v o l v e d a l a r g e  l i g h t - s t r a w c o l o r e d heartwood on one s i d e and  amount of d i s c o l o r e d heartwood on the other same  growth  radially  zones.  lengthwise  into  The quadrant was two  sections  side  further  and  one  Pieces A,  surface  of  B and C (Figure  11).  colored  (16 inner  dark-brown Piece and  growth zones);  of  inner  White  colored  Reddish c o l o r e d  (134  A was used i s o l a t i n g microorganisms,  split  zones:  Light-straw  heartwood  the  radial  heartwood (53 growth zones);  colored  outer  light-brown  and  Brown  growth  to  zones).  while Pieces  B  C were used i n e x t r a c t i v e s t u d i e s . Fungi  grow  naturally  normally as saprophytes  occurring  animal  or  plant  or  parasites  products.  number of n u t r i e n t s , the  fungus.  cultivation  of fungi  on  These  products can be extremely complex m a t e r i a l s which provide  of  of  exposed  Piece A c o n s i s t e d of f i v e c o l o r  c o l o r e d sapwood (7 growth zones); heartwood  The  a  split  s e c t i o n s , i n c l u d i n g the d i s c o l o r e d pocket, was f u r t h e r into  the  a  i n c l u d i n g some unknown, f o r the growth A  primary  requisite  for  laboratory  i s s e l e c t i o n of a s u i t a b l e substratum.  74  Figure  1 1.  Radial Pieces S p l i t from a Q u a d r a n t and Used in the Isolation of Microorganisms (A) and E x t r a c t i v e s (B and C ) .  A  B  C  75  There  are  two  types of media  n a t u r a l media and malt e x t r a c t and  used  s y n t h e t i c media. yeast  extract,  in  N a t u r a l media,  autoclaved  laboratory  and  used  experiments.  directly  for  fungal  they  are  consists  of  magnesium  sulphate,  composition  composition.  chemicals  and  medium which may  (such or  as  biotin)  concentration.  A  which This  Unfortunately,  i s o l a t i n g and  developing  "normally"  importance, t h e r e f o r e ,  fungi as  are  bench. were  of  known of  i s s u i t a b l e for  In  the  grow  present  from heartwood of  possible  the n a t u r a l medium was  was  of  the most  preferred.  A f r e s h uncontaminated r a d i a l wood surface A was  as  asparagine,  some fungi may  study,  as  in  medium  i s the only type  or not at a l l on s y n t h e t i c media.  tree  synthetic  D-glucose,  poorly  sampled  growth  for studies  be p r e c i s e l y d u p l i c a t e d and  physiological studies.  which  A disadvantage of n a t u r a l media i s  they can never be p r e c i s e l y d u p l i c a t e d unknown  such as  These n a t u r a l media  that  of  work,  are the m a t e r i a l s on  fungi would o r d i n a r i l y grow in nature. are  laboratory  from Sample  exposed under s t e r i l e c o n d i t i o n s on a laminar a i r flow Small c h i p s of wood removed from the placed  in P e t r i dishes  on a c u l t u r e medium  of 30 ml of 2% agar, 3% malt e x t r a c t and There were four c h i p s  radial  i n each d i s h .  76  0.5%  surface  consisting  yeast  extract.  A minimum of four  chips  were  removed  dishes  from  were  every f i v e annual  maintained  incubator f o r about  at  room  rings.  The  temperature  in  Petri a  dark  two months with r e g u l a r examination.  3.3. Determination of e x t r a c t i v e s Wood  specimens used  f o r e x t r a c t i v e s d e t e r m i n a t i o n were  obtained from P i e c e s B and C ( F i g u r e 11). into  small  according  wood s l i c e s and then d i v i d e d to  growth zone and c o l o r  Pieces into  were  four  variation.  cut  groups  The  five  years  old  groups were: 1.  White  colored  sapwood from 413 to 420  (S-P); 2. D i s c o l o r e d outer heartwood from 303 to 412 years o l d (D-O); 3.  Light-straw  colored  outer heartwood from  303  to  412 years o l d (L-O); 4. D i s c o l o r e d inner heartwood from  198 to 302 years o l d  (D-I); and 5.  L i g h t - s t r a w c o l o r e d inner heartwood from  198 to 302  years o l d ( L - I ) . The to  pass  samples  samples were a i r - d r i e d , and ground i n a W i l l e y a fifty-mesh screen. were determined  Moisture content  by oven-drying.  77  of  They were  mill these  57.5%,  31.36%, 27.45%, 28.22% and 23.56% f o r S-P, D-0, L-0, D-I and L-I,  respectively. Nearly  ten grams oven-dry  (O.-D.) b a s i s of  wood  meal  f o r each sample was e x t r a c t e d (using four thimbles with of 33 X 88 mm four  hours  i n Soxhlet e x t r a c t i o n apparatuses) with reagent  300  ml  were  concentrated to 40°C.  (BE)  The r a t i o of s o l v e n t t o wood  s o l v e n t to ten grams of wood (O.-D.).  solubles  f o r twenty-  grade benzene and ethanol  r a t i o of 2:1 as s o l v e n t .  size  transferred  to  a  The  pre-weighed  at was  solvent  flask  and  by means of a r o t a r y vacuum evaporator at  35°  The c o n c e n t r a t e s were then d r i e d under vacuum i n a  desiccator  u n t i l constant weight was o b t a i n e d .  The y i e l d s  of d r i e d e x t r a c t s were c a l c u l a t e d , as: % dried extract  =  wt. of ( f l a s k + s o l u b l e ) - wt. of f l a s k X 100 wt. of wood sample (O.-D.)  For  determining  extractives, diluted  each  tropolone d e r i v a t i v e content  concentrated  t o 50 ml with the same BE mixture.  Gardner and Barton  (1958),  were  the  used,  (1984).  extractive  with  in  mixture  the was  The methods of  and MacLean and Gardner  m o d i f i c a t i o n as suggested  (1956a) by  Nault  F i v e ml of each e x t r a c t i v e mixture was t r a n s f e r r e d  to a c l e a n beaker and f u r t h e r d i l u t e d to 10 ml with BE (2:1) solvent.  The d i l u t e d s o l u t i o n s were e x t r a c t e d with 78  10 ml 1  N LiOH to remove a c i d i c compounds. A small separatory was  used  with  shaking  extraction.  The  H S0  then  2  and  4  extracted (3:2)  acetate  solution  fresh f e r r i c chloride solutions.  The  solution  was  chelates  ml  twice  with  of  solvent.  acetate was  well was  were then  (1% w/w Fe) s o l u t i o n .  of f e r r i c a c e t a t e and  removed  and  the  in  tropolone layer  The this  of  (0.537 M)  organic  saved.  The  volumes  and  4N of  which was  made from equal  shaken  ml  Thujaplicins  t h u j a p l i c i n s were d i s s o l v e d  The  f o r each  10  (0.179 M) and sodium a c e t a t e  mixture  (chloroform:hexane)  layer.  minute  i n the chloroform:hexane s o l u t i o n  mixed with 5 ml of f e r r i c ferric  one  aqueous l a y e r was a c i d i f i e d with 3 ml  chloroform:hexane dissolved  f o r about  funnel  ferric organic  aqueous l a y e r was washed with an a d d i t i o n a l 10  of chloroform:hexane and the washings were added to the  organic  layer.  The f e r r i c c h e l a t e s o l u t i o n was made up to a  f i n a l volume of 100 ml. Nault that the  (1984) found  from c a l i b r a t i o n curves  he generated  the absorbance of both beta- and gamma-thujaplicins at same c o n c e n t r a t i o n s were  gamma-thujaplicin this  study  being  available.  was  the  selected  same.  as a standard  due to only a small amount of  79  Therefore, solution  only in  beta-thujaplicin  A  pure  Forintek  gamma-thujaplicin  Canada  thujaplicin  ferric  measurements method  as  Corp.  at  was  supplied  Standard s o l u t i o n s of  c h e l a t e were  the  f o r the  sample  prepared  the  for  The  standard  c o n s i s t e d of a s e r i e s of c o n c e n t r a t i o n s of  gamma-  absorbance  same time by using e x a c t l y extractives.  by  the  same  solutions  gamma-thujaplicin  (Table 9 ) . According t h u j a p l i c i n was of  Nault's  results  hours  ferric  10 both  the  acetate. 2  Due  to  i n t h i s study were measured  higher  ml of s o l u t i o n  the 100 ml f e r r i c  nm  spectrophotometer.  sample  with  solution taken  c h e l a t e s o l u t i o n and then d i l u t e d to  standard s o l u t i o n s and 425  reacted  f o r each sample was  ml f o r s p e c t r o s c o p i c measurement.  the  chelated  On the b a s i s  a f t e r the t r o p o l o n e s o l u t i o n s were  concentration, from  (1984),  at the maximum a f t e r one day.  h i s r e s u l t s , a l l absorbances  24  in  to  The  absorbances  sample s o l u t i o n s were  region  on  a  Beckman  The chloroform:hexane  of  measured Model  (3:2) solvent  24 was  used as a blank. A c a l i b r a t i o n curve was  prepared a f t e r r e c o r d i n g the UV  absorbance  f o r the standard gamma-thujaplicin  solutions  (Figure  12).  The  BE  (2:1)  f e r r i c chelate  extractives  t h u j a p l i c i n contents i n the f i v e samples were measured.  80  and  Table 9 . C o n c e n t r a t i o n S e r i e s f o r Standard Solutions.  C o n c e n t r a t i o n s of standard gamma-thujaplicin chelate solution (g/IOOml)  Gamma-thujaplicin  Absorbance  5.10X10"  3  1.190  3.40X10"  3  0.789  2.55X10  -  3  0.640  1.25X10  -  3  0.301  5.10X10'  4  0.121  0.00  0.000  81  Figure  12.  C a l i b r a t i o n Curve: Absorbance vs. Gamma-thujaplicin F e r r i c Chelate C o n c e n t r a t i o n s .  Concentration (g/l00mlX10-3)  82  3.4.  I d e n t i f i c a t i o n of WRC heartwood Thin l a y e r chromatography  extractive  (TLC) has been  fractions found to be a  convenient method f o r s e p a r a t i n g and i d e n t i f y i n g the v a r i o u s components  of  WRC heartwood  reasonable  choice  cellulose.  Whatman K6F s i l i c a  of  extractives.  absorbents,  It  allowed  such as s i l i c a  a  g e l or  g e l (40A) p l a t e s , 20 X 20 cm,  250 urn t h i c k n e s s and 20 X 5 cm,  250 urn t h i c k n e s s , were used  in t h i s study. The L-O,  e x t r a c t i v e s obtained from D-O,  D-I,  and L-I were spotted on a 20 X 20 cm K6F p l a t e a short  distance of  WRC heartwood  (1 cm) above the edge and were e l u t e d by the  mobile phase benzene:ethanol  (9:1) which was p r e - s e l e c t e d  a c c o r d i n g to e a r l i e r experiments. covered  the  rest  flow  The mobile phase  of the p l a t e by  capillary  slowly  flow,  which  c a r r i e d sample components upward through the absorbent  bed.  Development of the chromatogram was stopped when the l e a d i n g edge of the mobile phase neared the top of the p l a t e . TLC  experiments were repeated many times i n  o b t a i n the best r e s u l t s . was  performed  D e t e c t i o n of chromatographic  by v a r i o u s methods.  compounds  were marked d i r e c t l y on  compounds  that  were  order  naturally  Some s t r o n g l y the p l a t e .  fluorescent  b r i g h t zones when the p l a t e s were examined under 83  zones  colored  Some were  to  other  seen  as  ultraviolet  light.  For  compounds  which  were  non-colored  and  non-  f l u o r e s c e n t , two s p e c i a l d e t e c t i o n techniques were a p p l i e d . One  detection  i o d i n e vapor  method was to expose the TLC  f o r about two minutes.  The i o d i n e vapor  not r e a c t with the o r g a n i c compounds, condensed i n the s o l u t e zones, organic molecules zones  appear  plate  to does  but i s p r e f e r e n t i a l l y  a p p a r e n t l y being bound to the  p u r e l y by van der Waals f o r c e s .  as yellow spots a g a i n s t a  white  Thus, the background.  The advantages of t h i s d e t e c t i o n method are that the process is  entirely  r e v e r s i b l e and u n i v e r s a l .  removed from the i o d i n e tank, minutes.  T h i s technique  Once the p l a t e  the spot disappears i n a  the  few  i s p a r t i c u l a r l y u s e f u l when s o l u t e  zones must be recovered f o r f u r t h e r a n a l y s i s . corresponding • to  is  components i n t h i s  C o l o r e d spots study  could  be  d e t e c t e d q u i t e w e l l a f t e r the treatment. A second of  d e t e c t i o n method was to spray an equal  concentrated  followed  by  s u l f u r i c and n i t r i c a c i d s  on  the  mixture plate,  h e a t i n g the p l a t e f o r two to three minutes  to  r e t a i n the c o l o r bands. Individual extractive means of R  chromatographic  components c  spots  were l o c a t e d  values.  84  and  corresponding characterized  to by  3.5.  Wood block  This  bioassay  experiment  was  experiments  (see  Section  microorganism  experiments showed that there were three e a r l y  stage a t t a c k i n g f u n g i . assigned block  (Figure  test  colored  was  section  .  The  Code Nos.  One  WR1,  found  with d i s c o l o r e d and  that  there  heartwood  heartwood.  Therefore,  bioassy  experiment (light-straw  the above three The  was  a  which  WR2  was  cut  major  surface.  range  with  the  wood  sound WRC  was  heartwood,  compound not  (see it  present  present  in  in sound  o b j e c t i v e of the wood block  c o l o r e d ) heartwood a f t e r  changes  of  infection  by  fungi.  lengthwise  was  were  light-straw  to study the chemical  wood sample was  stick  and WR3  fungi.  obtained from the same p i e c e  f o r d e t e r m i n a t i o n of heartwood e x t r a c t i v e s . was  of  a n a l y s i s experiments  new  another  earlier  isolation  of the purposes of  chemical composition  3.4)  of  to examine decay r e s i s t a n c e of  discolored  sound  The  3.2).  (L-0) heartwood to these three From  was  13)  based on the r e s u l t s  i n t o a 6.5  cut i n t o 0.5 All  X 6.5  cm  long  The  wood p i e c e  stick.  cm b l o c k s with c r o s s - s e c t i o n on blocks were i n the same  a very small v a r i a t i o n  85  in  height  used  The the  growth  zone  within  stem  F i g u r e 13.  (maximum  30 cm).  All  blocks  the  constant until  The blocks were were  temperature  equilibrium  nearest  0.001 g.  to O.-D.  numbered  dried in a (25°C)  humidity  and then  at  humidity  Then,  the  O.-D.  content  a  (70%)  weighed  to  the  A f t e r weighing the b l o c k s , ten were  to determine the moisture  equilibrium.  chamber  and r e l a t i v e  was reached,  sequentially.  taken  of the blocks at  weights of the t e s t  blocks  were c a l c u l a t e d . The  fungi used i n t h i s experiment were WR1,  which were i s o l a t e d  i n the e a r l i e r experiment  and WR3  ( S e c t i o n 3.2.)  as  described.  3%  malt e x t r a c t and 0.5% yeast e x t r a c t i n 25 mm deep  Petri dishes.  Cultures  WR2  In order  were grown on media with 2% agar,  to keep the fungi growing evenly on  the s u r f a c e of the c u l t u r e media, colony  a w e l l growing WR1  was t r a n s f e r r e d i n t o a s t e r i l e c o n t a i n e r  sterilized  distilled  dispensed  in  the  water.  The  s o l u t i o n and  then  mycelium  WR1  WR1  mycelium evenly.  P e t r i d i s h e s were ready  and WR3, to  of  WR1  was  to  the  The metal  wire  was moved a c r o s s the e n t i r e s u r f a c e s e v e r a l  spread  fungal  containing  transferred  c u l t u r e media s u r f a c e by a metal wire loop. loop  glass  times  A f t e r about t h r e e weeks, f o r use (Figure 14A).  For  to the WR2  small c h i p s of the fungal c u l t u r e were t r a n s f e r r e d  the g l a s s P e t r i d i s h e s and spread as evenly as p o s s i b l e .  87  F i g u r e 14.  A f t e r about ready  three weeks, WR2  groups each  sound heartwood blocks  (L-0) were d i v i d e d i n t o  with four b l o c k s i n each group. group  distilled  were s u r f a c e s t e r i l i z e d water  groups assigned  were  A l l the  by  placed  content  of 70%.  surface  The  of  fungi  in  i n the dark  other two groups of wood b l o c k s served as  sets of c o n t r o l samples.  the  the  c o n t a c t i n g the c u l t u r e media.  P e t r i d i s h e s were p l a c e d i n an incubator  25° C.  Sterile  Seven of the nine  on top of the growing  P e t r i d i s h e s without  nine  b l o c k s of  flaming.  was p i p e t t e d on the main  b l o c k s to g i v e a moisture  on  P e t r i dishes were a l s o  f o r use ( F i g u r e s 14B and 14C). The  The  and WR3  at two  Four b l o c k s of one set were p l a c e d  the top of the c u l t u r e media i n the P e t r i dishes l a c k i n g  growing f u n g i .  Another four blocks were p l a c e d i n the empty  Petri dishes.  These two set of c o n t r o l samples were s t o r e d  in the same incubator with other groups d u r i n g the treatment period.  The d e t a i l s of each treatment  Group 1 (1-1,  1-2,  were:  1-3, 1-4), Group 2 (2-1, 2-2, 2-3,  2-4) and Group 3 (3-1, 3-2, 3-3, 3-4) were t r e a t e d with WR2  and WR3,  WR1,  r e s p e c t i v e l y , f o r an e i g h t week p e r i o d ;  Group 4 (4-1, 4-2, 4-3, 4-4) was t r e a t e d f i r s t with for four weeks and then Group 5 (5-1,  5-2,  t r e a t e d with WR2  WR1  f o r four weeks;  5-3, 5-4) was t r e a t e d with WR1 f o r  89  four weeks and then t r e a t e d with WR3 Group 6 (6-1,  6-2,  four weeks;  6-3, 6-4) was t r e a t e d with WR2 f o r  four weeks and then t r e a t e d with WR3 Group 7 (7-1, 7-2, 7-3, 7-4) and WR3  f o r another  f o r another  four weeks;  was t r e a t e d with WR1,  WR2  one a f t e r another at four week i n t e r v a l s ;  Group 8 (8-1,  8-2,  8-3, 8-4) was p l a c e d on the top of  c u l t u r e media f o r eight weeks (as c o n t r o l l e v e l 2); and Group 9 (9-1,  9-2, 9-3, 9-4) was p l a c e d i n empty P e t r i  d i s h e s f o r eight weeks (as c o n t r o l After and  treatments,  dried  (25°C)  the b l o c k s were cleaned by a  i n the humidity chamber at  and  reached.  l e v e l 1).  r e l a t i v e humidity  A f t e r weighing  (70%)  constant  brush  temperature  u n t i l equilibrium  the b l o c k s to the nearest 0.001 g,  nine b l o c k s which were taken from the nine r e s p e c t i v e were oven-dried and the O.-D. were  then  obtained.  was  groups  weights of a l l t r e a t e d blocks  The weight  l o s s of each group  after  treatment was c a l c u l a t e d . Statistical out.  carried  Due t o inhomogenous v a r i a n c e f o r the nine groups  percentage weight was  a n a l y s i s of a l l the data was then  used.  were then  loss,  O.-D.  the square root data t r a n s f o r m a t i o n  A n a l y s i s of v a r i a n c e and m u l t i p l e  range  tests  wood  block  performed.  Extractive  analyses  for  each  90  group  of  samples earlier.  were  performed  using  the  methods  as  The r e s u l t s were compared with those from  samples.  91  described control  3.6.  Isolation  and  discolored  identification  heartwood  According to 3.4.  and  unknown D-l)  and  but  not  in in  Isolation,  the chemical s t r u c t u r e  Solvent  the  WR2  WR1  or  treated  WR3  (D-0,  heartwood  treated  heartwood  p u r i f i c a t i o n and d e t e r m i n a t i o n of  f o r t h i s compound was attempted.  fractionation  discolored  solvent.  from  (Section  i n d i s c o l o r e d heartwood  A 400 gram sample of a mixture of outer (D-l)  compound  i t was demonstrated p o s i t i v e l y that a new  extractives  a)  unknown  extractives  compound was present  extractives.  an  r e s u l t s of p r e v i o u s experiments  3.5.),  extractives,  of  heartwood  was e x t r a c t e d  A f t e r evaporation of the s o l v e n t ,  (D-0) and inner with  BE  (2:1)  the e x t r a c t i v e s  were d r i e d i n a d e s i c c a t o r .  To f r a c t i o n a t e the e x t r a c t i v e s ,  the  were  dried  BE  extractives  dissolved  f i l t e r e d t o remove i n s o l u b l e m a t e r i a l s . materials acetate ethanol. were  were  dissolved  insolubles The ether,  were  in  ethyl  f i l t e r e d and  in  The ether  92  and  insoluble  acetate.  The  then  dissolved  e t h y l a c e t a t e and ethanol  examined by TLC with BE (9:1) as  ether  ethyl in  extractives  developing  solvent.  It was found that the new compound was present mainly i n the ethyl  acetate  soluble f r a c t i o n .  Further  separation  was  accomplished by using column chromatography.  b)  Column chromatography  Elution based  on  types  absorbents, silicate, acid  column chromatography was used with of  compounds  such as c e l l u l o s e , s i l i c i c acid,  (silica  and a c i d s .  to  gel) i s About  be  separated.  starch,  fluorisil  absorbent  sugars,  Among magnesium  and alumina, only s i l i c i c  u s e f u l f o r the s e p a r a t i o n of  150 grams of s i l i c a  esters  g e l ( B i o - s i l A,  200-  400 mesh) was oven-dried at 130°C f o r four hours and kept i n a desiccator u n t i l  i t was c o o l .  D i s t i l l e d water was  added  in the r a t i o of 50 ml water to 100 gram s i l i c a g e l . A column 50 mm plug and  i n diameter was used i n t h i s  study.  of g l a s s wool was p l a c e d i n the bottom of the  A  column,  a l a y e r of sand was added on top of the plug to p r o v i d e  an even base f o r the absorbent column. were chosen by t r i a l experiments.  The e l u t i n g  Because the l e a s t  s o l v e n t used i n t h i s experiment was benzene, packed desired  by adding the absorbent s l u r r y amount  of s i l i c a  polar  the column was  i n benzene u n t i l  g e l had been p l a c e d .  93  solvents  After  the the  column had the  been prepared,  the  solvent  top of the column by d r a i n i n g  l e v e l was  i t from the  lowered  to  bottom of  the  column. The  dried ethyl acetate soluble  heartwood  extractives  which  the  was  extractive added  to  the  extractives the for  every  100  gradient  mixtures of and  elution  was  used  thickness, was  (9:1)  solvent,  dissolve  absolute  amount  gram of d r i e d The  increasing  to  of  gel  remove  eluted  percentage (50:50). each  and mainly  e l u t i o n with a BE  the  g e l TLC  r e s u l t s showed  present i n the  (85:15) solvent  94  that  fraction  fraction  mixture.  p l a t e s of the  of This  Four f r a c t i o n s were f i n a l l y c o l l e c t e d . silica  in  s t a r t i n g with  the BE  of  extractives  column was  polarity,  the  carefully  the amount of s i l i c a  an equal mixture  f r a c t i o n s were run on K6F  compound  The  increasing  gradually  give  individually.  urn  top of the column.  discolored  s o l u t i o n was  grams of s i l i c i c a c i d .  benzene to  i n BE  that would  extractive  column with a maximum of one  ethanol  four  The  solvent  added depended on  with solvent pure  were d i s s o l v e d  least polar  mixture.  f r a c t i o n of  The 250  unknown  obtained  by  c) Thin l a y e r  chromatography  P r e p a r a t i v e t h i n l a y e r chromatography was used i n experiment others  to  f u r t h e r separate the unknown  compound  i n the f r a c t i o n s c o l l e c t e d by column  described  i n the S e c t i o n above. 20  plates, fraction  X  20 cm,  from  chromatography  Whatman PLK5F s i l i c a  1 0 0 0 urn t h i c k n e s s  this  were  used.  obtained by column chromatography which  the unknown compound i n the d i s c o l o r e d heartwood  gel The  contained (D-0 and D-  I) e x t r a c t i v e s was a p p l i e d on the p l a t e as a band across the plate  i n s t e a d of s p o t s .  for  developing  ultraviolet  light  the  A BE ( 9 : 1 ) s o l v e n t system was used plate.  Following  development,  (both short and long wavelength) was then  employed to l o c a t e the band of unknown compound. was scraped from the p l a t e with c a r e . from  The  band  The m a t e r i a l scraped  the p l a t e was c a r e f u l l y ground t o very f i n e  particles  and then the unknown compound was leached from the absorbent ( s i l i c a powder) using e t h a n o l . This  procedure  was  unknown compound c o l l e c t e d  repeated  many  times.  i n t h i s way was a p p l i e d t o  TLC p l a t e s again u s i n g the same s o l v e n t system, gain unknown  higher sample p u r i t y . compound  obtained  A l l the thick  i n order to  The eventual t o t a l amount of in  95  this  way  was  about  ten  milligrams. Quantitative performed with  measurement of t h i s unknown compound  a l s o by p r e p a r a t i v e  TLC.  An ethanol  0.06745 g vacuum d r i e d BE e x t r a c t i v e s  was  plate.  The  recovered new compound was vacuum d r i e d and  was then  solution  applied  p l a t e was developed by BE ( 9 : 1 )  the  The  was  on  solvent.  the  yield  caculated.  d)  Crystallization  and  elemental  analysis  of  the  unknown compoud  Crystallization preparative dissolved evaporated added  to  TLC  method  sample  was  i n 5 ml of e t h y l a c e t a t e and then the solvent  was  with the  was  nitrogen solution.  heptane was about 5 ml.  of the unknown compound p u r i f i e d by the  1:3.  attempted.  The  gas t o about 2 ml. The r a t i o of  The f i n a l  ethyl  Heptane acetate  was to  s o l u t i o n was evaporated t o  The s o l u t i o n was kept i n a f r e e z e r  f o r about 5 weeks  u n t i l c r y s t a l l i z a t i o n was a c h i e v e d . A crystallized analysis Dept.  sample was submitted f o r micro-elemental  which was c a r r i e d out by the M i c r o - a n a l y s i s  of Chemistry,  w i l l be d i s c u s s e d  U.  B.C..  Lab.,  The r e s u l t of t h i s a n a l y s i s  later.  96  e) Mass spectrometry  Mass  spectra  of  d i s c o l o r e d heartwood  this  unknown  B.  C.  samples urn  from  the  (D-0 and D-I) e x t r a c t i v e s were o b t a i n e d  from the Mass Spectrometry Center, U.  compound  Department of Chemistry,  A t o t a l of four s p e c t r a were o b t a i n e d .  The four  used f o r these mass s p e c t r a were p u r i f i e d  TLC p l a t e s at d i f f e r e n t  times.  Two  from 1000  preparations  were  used to o b t a i n low r e s o l u t i o n s p e c t r a , and these two s p e c t r a were  identical.  resolution  mass  The  ionization  spectra  was  temperature  100°C.  The  for other  p r e p a r a t i o n s were used to o b t a i n higher r e s o l u t i o n mass range scanned f o r these higher r e s o l u t i o n  was  from  temperature was  Both (NMR)  Chemistry, Because  of  U.  ionization  resonance spectrometry (proton and  NMR)  proton and carbon-13  spectra  spectra  150°C.  f) Nuclear magnetic carbon-13  The  two  spectra.  The  30.9984 to 692.9569 mass u n i t s .  low  nuclear  were taken by NMR B. C.  magnetic  Services,  resonance  Department  of  The machine used was a V a r i a n XL-300.  the small amount of sample  97  available,  Fourier  transform NMR  was  sensitivity.  The samples were prepared i n d e u t e r o c h l o r o f o r m  solution  with  standard.  employed i n a l l cases i n order to  tetramethylsilane  Because only about  (TMS)  as  enhance  an  internal  5 mg of sample was  available,  the carbon-13 s p e c t r a were run o v e r n i g h t .  g) V i s i b l e and u l t r a v i o l e t  A  Beckman  Canada  Corp.  unknown  at  a  Model 24 spectrometer l o c a t e d was  used  compound.  solvent.  The  The  for q u a l i t a t i v e sample  was  at  Forintek  analysis  dissolved  in  of  sample  was  of 250 ug/ml, at  25  ug/ml.  while  the  The  the  ethanol  sample prepared f o r the v i s i b l e spectrum  concentration  spectrum  spectrometry  was  ultraviolet  reference  beam  c o n t a i n e d the solvent as a p p r o p r i a t e blank.  h) I n f r a r e d  spectrometry  P r e l i m i n a r y IR spectrometry of the sample was u s i n g a Perkin-Elmer 680 the  s e r i e s IR spectrometer.  low machine s e n s i t i v i t y ,  obtained  even  unsatisfactory  98  Because of  spectra  though an 80 minute scan time was  more s a t i s f a c t o r y IR spectrum was  performed  used.  were A  run on a F o u r i e r transform  IR  spectrometer  small  amounts  plate the  Department  (about  20 u g )  were  pipetted  onto  plate.  When  solution crystal  i n the  was c a r e f u l l y  the  of  Chemistry, U.B.C.  unknown compound i n  the  surface  ethanol  secured  of  was  between  cell.  99  of  a  silver  evaporated, the  metal  the  Very ethanol  chloride crystal  retainers  of  3.7. T o x i c i t y t e s t bioassays  Poria  al bipe11 uci da  microorganism it  Baxter.  was  t o be used i n t o x i c i t y  selected  as  the  t e s t b i o a s s a y s because  i s the most common decay organism i s o l a t e d from the  heartwood. large  Because  amount  of the great  of the  new  difficulty  compound,  technique  was used i n s t e a d of using  methods.  The  containing extract  following  obtaining particular  standard t o x i c i t y  were performed  in Petri  test dishes  30 ml of 2% agar, 1% malt e x t r a c t and 0.5% yeast  c u l t u r e media.  solidified holes  bioassays  for  WRC  A f t e r the c u l t u r e media  i n the P e t r i d i s h e s ,  were made and  solutions  four uniform 5 mm diameter  the c u l t u r e media i n s i d e  these  holes  were c a r e f u l l y removed as shown i n F i g u r e 15.  a) T o x i c i t y of t h u j a p l i c i n s  A  series  different were a t : 1.25;  of  beta-thujaplicin  concentrations 5.00;  1.00;  4.00;  0.940; 0.1  solutions  at  sixteen  was prepared i n 10% e t h a n o l .  They  3.75; 3.33; 2.50; 2.00; 1.89; 1.67;  0.830;  0.625; 0.500; 0.469; and 0.417  mg/ml.  Then,  ml of each s o l u t i o n was t r a n s f e r r e d  syringe  i n t o i n d i v i d u a l h o l e s as shown i n F i g u r e  100  15.  by Four  Figure  15.  P e t r i Dish Prepared for T o x i c i t y  101  Tests.  Petri  dishes  were  were used f o r each t r i a l .  dishes  l y i n g on a laminar flow a i r bench without a cover  two  hours to allow e v a p o r a t i o n of ethanol and d i f f u s i o n  solution  to the media.  evenly growing Poria in  The P e t r i  the  center  regularly. growth  A plug from a P e t r i d i s h  al bipel  of the  After  96 hours,  inhibition  for  the  The  plates  were  c o n c e n t r a t i o n s were observed (Figure  different  16).  placed  examined  d i f f e r e n t p a t t e r n s of sixteen  of  containing  I uci da Baxter, fungus was  dish.  for  fungal solution  There were three  r e p l i c a t i o n s of t h i s experiment. The  same experiments were c a r r i e d out by using  thujaplicin  and  a mixture of  beta- and  gamma-  gamma-thujaplicin  solutions.  b) T o x i c i t y of the unknown compound  The t o x i c i t y t e s t comparison  between  f o r the unknown compound was based on  e f f e c t s with b e t a - t h u j a p l i c i n  unknown compound on fungal growth 0.125 was  mg b e t a - t h u j a p l i c i n / 0.1  inhibition.  assumes  an  average  of WRC  1 02  used. T h i s  heartwood.  t h u j a p l i c i n content of 0.6%  wood.  the  A s o l u t i o n of  ml 10% ethanol was  e x t r a c t e d from about 20 mg O.-D.  and  in  This O.-D.  F i g u r e 16. Inhibition  of  Poria  albipel  I ucida  Baxter.  by  B e t a - t h u j a p l i c i n a t Sixteen D i f f e r e n t C o n c e n t r a t i o n s . C o n c e n t r a t i o n s are (mg b e t a - t h u j a p l i c i n / m l e t h a n o l ) : 1:5.00; 2:4.00; 3:3.75; 4:3.33; 5:2.50; 6:2.00; 7:1.89; 8:1.67; 9:1.25; 10:1.00; 11:0.940; 12:0.830; 13:0.625; 14:0.500; 15:0.469; 16:0.417.  103  The  amount  heartwood  was  extractives  of unknown compound i n 20  i s o l a t e d as f o l l o w s .  comprised  heartwood.  On  urn TLC p l a t e and the  unknown  The s i l i c a  was  dissolved  i n 0.1 ml 10% ethanol solvent  syringe holes  mg/ml  weight of WRC  A 3.15 mg sample of  leached by e t h a n o l .  compound  The d r i e d compound and then  i n t o one hole i n the P e t r i d i s h . (left  band  and r i g h t ) were f i l l e d  In each p l a t e ,  with 0.1 ml of  with 0.1 ml of unknown compound s o l u t i o n .  48  the  were  1.25  bottom)  were f i l l e d  relative inhibiting effects  was  transferred  b e t a - t h u j a p l i c i n and other two holes (up and  hours,  was  powder was ground and the  compound  two  the BE  (D-0 and D-I) heartwood BE e x t r a c t i v e s was run on  recovered by s c r a p i n g .  by  WRC  T h e r e f o r e , f o r 20 mg O.-D. heartwood, about 3.15  discolored 1000  O.-D.  average,  15% of the t o t a l O.-D.  mg of BE e x t r a c t i v e s would be p r e s e n t .  a  mg  After  compared  (Figure 17A). At  the  same  time,  four  solutions  of  the  compound were prepared a t higher c o n c e n t r a t i o n s of 1.67,  and  1.25 mg/ml and t e s t e d by the same method  unknown 5,  2.5,  (Figure  17B) . c) T o x i c i t y of methylated t h u j a p l i c i n Ethanol  solutions  of  5  104  mg/ml  beta- and  gamma-  F i g u r e 17. Inhibition of Poria al bi peI I ucida Baxter, by the Unknown Compound, Methylated T h u j a p l i c i n s and T h u j a p l i c i n s .  1 7A  17B 1 7A: Left and r i g h t holes filled with 0.1 ml of 1.25 mg/ml betathujaplicin; top and bottom f i l l e d with unknown compound. 1 7B: Unknown compound at four higher c o n c e n t r a c t i o n s : 5.00,2.50,1.67 and 1.25 mg/ml. 1 7C: Left and r i g h t holes filled with 0.1 ml of 1.25 mg/ml betathujaplicin; t o p and bottom filled with the same amount of methylated b e t a - t h u j a p l i c i n .  1 7 C  105  thujaplicin  mixtures  methylated  by  diazomethane.  were  addition After  of  prepared. a  double  The  solution  volume  of  shaking the s o l u t i o n f o r two  the  solvent  was evaporated by h e a t i n g at  The  methylated t h u j a p l i c i n s were d i s s o l v e d  about in  was  etheral minutes,  60°-70°C.  20%  ethanol  solvent  at a c o n c e n t r a t i o n of 5 mg/ml.  sample  of the s o l u t i o n was t r a n s f e r r e d to each hole i n the  culture holes  media  After  A total  of  four  were made on each d i s h i n which two of the h o l e s were  filled 0.1  i n a prepared P e t r i d i s h .  A 0.1 ml methylated  with methylated t h u j a p l i c i n s and the other two  ml of 1.25 mg/ml beta- and 48  compared  hours,  the  gamma-thujaplicin  relative  (Figure 17C).  106  inhibiting  with  mixture.  effects  were  3.8.  Examining o r i g i n of the new  Earlier unknown  experiments ( S e c t i o n 3.4.)  compound  following 3.5.),  WR1  appeared in  treatment.  however,  compound.  The  components  did  of  The  not t e l l  present  individually  WRC under  Sapwood  was  (S-P)  extractives  wood block  bioassay  the o r i g i n of  extractives  treatment  with  WR1  only  unknown  that major  were to  new  (Section  this  so designed  heartwood  examined  find  their  compound.  used i n t h i s  was  showed that the  heartwood  experiment was  r e l a t i o n s h i p with t h i s new  sapwood  unknown compound  experiment.  White  colored  cut i n t o small p i e c e s with the dimensions  40 X 5 X 2 mm.  The  on each p i e c e .  A l l the sapwood p i e c e s were e x t r a c t e d by  (2:1)  t r a n s v e r s e s u r f a c e was  s o l v e n t i n a shaker f o r four  sapwood  hours.  the l a r g e s t  The  p i e c e s were d i v i d e d i n t o s i x sets with  face BE  extracted  four sapwood  p i e c e s w i t h i n the s e t . The sample  first for  set of sapwood p i e c e s was  extracted  sapwood  and  used as a  these  were  control left  in  storage. The distilled moisture  second  set of sapwood p i e c e s  water was content  air-dried  and  p i p e t t e d onto the s u r f a c e s to keep  the  high  enough f o r  1 07  was  fungal  growth.  These  sapwood  p i e c e s were p l a c e d on top of evenly growing WR1  P e t r i d i s h e s without  d i r e c t contact with the c u l t u r e  The  d i s h e s were examined r e g u l a r l y and a f t e r  WR1  could  The  sapwood  with a  on the top s u r f a c e s of  p i e c e s were taken  thujaplicin  pieces  were  thujaplicin  ethanol  evaporated.  The  1%  samples.  pieces.  from the dishes and  cleaned  t r e a t e d the  that there was  of  soaked  solution  in  concentrated  until  the  amount of t h u j a p l i c i n  O.-D.  The  weight o.f the  sapwood  pieces  were  set  then  content high enough f o r fungal growth. was  of  had was  sapwood  air-dried  moisture  WR1  beta-  i n the s o l u t i o n  third  of WR1.  ethanol  water was  the  same  an a d d i t i o n  distilled  days,  the  s o l u t i o n before being p l a c e d on top of the  sapwood  about  days, the  t h i r d set of sapwood samples was  as the second set except  The  media.  brush.  The way  be observed  15  in  and  p i p e t t e d onto t h e i r s u r f a c e s to keep the  observed  on the top  After  surfaces  of  15 the  pieces. The of  same experiments were performed f o r the  gamma-thujaplicin  mixture  beta- and  (Set 5), and p l i c a t i c a c i d  All enough  (Set 4),  solutions  gamma-thujaplicin  (Set 6 ) .  s i x s e t s of samples were ground to wood meal to  pass  a 40-mesh  screen.  108  They  were  fine  extracted  separately extractive A  with  BE  solutions  multiple  spectral  study  (2:1)  internal of  a  Perkin-Elmer  technical  for  reflection  hours.  the r e a c t i o n  infrared  The  and t h u j a p l i c i n s was a l s o  680  series  meaningful  (MIRIR)  of m i c r o - s e c t i o n s  IR  results.  109  of  WRC  performed  spectrometer).  d i f f i c u l t i e s were encountered.  f a i l e d t o produce  24  were examined by TLC.  sapwood with fungus WR1 (by  solvent  Some  T h i s measurement  3.9  Detection  of  metal  chelates  from  discolored  WRC  heartwood toluene e x t r a c t i v e s  About 350 g of d i s c o l o r e d WRC heartwood (mixture of D-0 and  D-I)  After  was  e x t r a c t e d with toluene s o l v e n t  evaporating  most of the s o l v e n t ,  f o r 24 h r .  one-third  of the  toluene e x t r a c t i v e s was d i l u t e d t o 50 ml with t o l u e n e .  This  toluene  solution  NaOH  (twice)  and then was washed with  (twice).  was  e x t r a c t e d with  Theoretically,  15 ml of  the m a t e r i a l s  toluene f r a c t i o n should be thujone, and other non-phenolic are  was observed,  of  0.1%  distilled remaining  water i n the  the new unknown compound  components s o l u b l e i n t o l u e n e ,  a l l weakly c o l o r e d .  color  15 ml  A very unusual  which  dark r e d  solution  which i s a c h a r a c t e r i s t i c of  Fe(III)-  thujaplicin chelate. Fifty of  6  ml of the toluene f r a c t i o n was r e a c t e d with 2  N NaOH and 2g/25 ml (H 0) of Na S f o r 3 2  stirring. precipitates Analytical A  Dark  precipitates  were vacuum f i l t e r e d  through  hr.  while  resulted.  The  a l a y e r of C e l i t e  F i l t e r - A i d , and then oven d r i e d .  solution  extractives  black-green  2  ml  of 0.06 N H SO^ was added to  obtained  2  after  filtration.  d e t e c t e d with a change of s o l u t i o n c o l o r ,  1 10  the  toluene  The r e a c t i o n  was  from dark red to  brown  to a f i n a l  stopped,  as  solution  was  yellow-white.  soon as a  The  addition  white c o l o r appeared.  extracted  ethyl  The  by e t h y l ether and then  ether e x t r a c t i v e s were washed with d i s t i l l e d from  water.  ethyl After  the  thujaplicins  were detected as the r e a c t i o n products i n on a TLC p l a t e .  1 1 1  the  the  was  entire  evaporating  e t h y l ether s o l u t i o n s  ether  of ^ S C ^  extractives, the  RESULTS AND  4.1.  Microorganism  DISCUSSION  isolation  and  distribution  in  WRC  heartwood Microorganisms i s o l a t e d  i n t h i s study supported e a r l i e r  f i n d i n g s r e p o r t e d by van der Kamp (1975). fungi  consistently  There were three  i s o l a t e d from wood samples examined  in  They  were  labelled  WR3  respectively  (Figure  13).  A t o t a l of 328 attempts were made  to  these fungi from WRC  this  study.  isolate  as  wood  WR1,  WR2  chips.  and  Among  c h i p s , only those which y i e l d e d pure c u l t u r e s of WR1, WR3  were  fungi  counted.  from  The i s o l a t i o n  v a r i o u s c o l o r e d zones of the WRC  given i n Table 10. the  occurence  infections Figures  18  frequency f o r the  and  The percentage frequency histograms  of  sample 19.  WR1,  WR2  age i n the  tree  There were 26  p a i d to these organisms. isolated  three is  V a r i o u s organisms appeared from them,  never  WR2 or  heartwood  f o r s t e r i l e wood,  versus  328  WR1,  WR2  from the sapwood.  and WR3  sapwood  are  fungal  shown  chips  used.  but no a t t e n t i o n and WR3,  in  was  however, were  Among 26 c h i p s ,  12 were  was the slowest  growing  sterile. Among fungus  WR1,  isolated.  WR2  and WR3,  After  WR1  about two 1 12  months  incubation,  a  Table 10. Frequency of Microorganism I s o l a t i o n s on Malt Agar from WRC Heartwood Samples v s . Sample Age. age  412-396  395-382  381-362  361-342  341-324  color  L-0  R-0  R-B-0  R-B-0  R-B-0  attempts  36(%)  24(%)  32(%)  38(%)  36(%)  sterile  28(77.8)  8(33)  8(25)  6(15.8)  5(13.9)  WR1  4(11.1)  16(67)  14(43.8)  13(34.2)  12(33.3)  WR2  0(0)  0(0)  10(31.2)  13(34.2)  8(22.2)  WR3  0(0)  0(0)  0(0)  0(0)  7(19.4)  age  323-294  293-264  263-234  233-198  color  B-I  B-I  B-I  B-I  attempts  32(%)  38(%)  34(%)  32(%)  sterile  3(9)  2(5.3)  3(8.8)  1 (3)  WR1  7(22)  10(27.8)  2(5.6)  0(0)  WR2  8(25)  6(16.7)  4(11.1)  4(12.5)  WR3  14(44)  14(38.9)  23(63.9)  27(84.5)  "attempts" r e p r e s e n t s the number of c h i p s . The numbers i n b r a c k e t s represent the percentage of s t e r i l e , WR1, WR2, or WR3 c h i p s p r e s e n t . L-O: R-O: R-B-O: B-I:  L i g h t - s t r a w c o l o r e d outer heartwood. Red-colored outer heartwood. Red-brown c o l o r e d outer heartwood. Brown-darkbrown inner heartwood.  11 3  F i g u r e 18. Histogram (No.l) of Percentage Frequency Distribution for S t e r i l e Wood and WR1,WR2 and WR3 Fungi I n f e c t i o n s vs. Sample Age.  100  Legend  90 >> O  EZ3 STERILE  80  m  c  Q) D  70  E l  (D  60  |  0)  50  D  40  c  30-  /  20-  /  cr  cn -t—  o d) QL.  10 0  I  Sporothrix  sp.  thujina | />/w a/ ophora  sp,  llHI  Age of the Growth Z o n e in Year from the Pith  Figure  19.  Histogram (NO.2) o f P e r c e n t a g e F r e q u e n c y D i s t r i b u t i o n s f o r S t e r i l e Wood and WR1, WR2 and WR3 F u n g i I n f e c t i o n s vs. Sample A g e . 100  80  STERILE  60 40 20 H 100-  Sporot hri x  0<D 1-  U_ (D  40H 20 H  100 H  Ui  21  XZZZ2K. t huj i na  CO  8  C  60-  2  40H  Q_  sp.  0  20  100-1 sp.  Phialophora  80 60 40 20  H  , 6 V ,6V,6V;6> o>  l<v  fc  l<v  fc  A g e of the G r o w t h Z o n e in Year from the Pith 115  living very  WR1  culture  from some of the  characteristic  appearance.  c o l o r , with a maximum r a d i u s on  the  the  cultures  c o n i d i a with few WR2  ( 1975)  13B)  identified  Pomerleau  of  WR2  (Figure  &  had  a mixture of black and Cultures  of  Identification Agriculture comfirmed van WR2  as  Service,  der  Etheridge.  On  the  WR2  because  isolated  der Kamp  and  ni el I a of  the  tentatively  above.  and  for f i n a l  ei ni el I a basis  hyphae.  WR1 was  Van  i t s color  was  WR3  were  sent  the  B i o s y s t e m a t i c Research  including pointed  which  of  color.  Ki rschstei  as  i r r e g u l a r shapes and  features  was  WR2  identification.  Kamp's (1975) t e n t a t i v e  Ki rschst  fresh  grey.  WR1,  Canada  in  13A).  Etheridge  to the one  13C)  a  c o n s i s t e n t l y dark black i n  i d e n t i f i e d by Smith (1970) as the WR3  to  spores produced in the c u l t u r e .  (Peck)  resemblence  After transfer  developed i n t o a convoluted mass  was  tentatively  t huji na  white-greyish  a  f o r a developed colony  hyphal segments (Figure  (Figure  There were no  I t was  of 30 mm  malt e x t r a c t medium.  medium,  wood chips developed  thuji of  na  Institute, The  report  identification  (Peck)  described  to  Pomerleau  colony  and  i n c r u s t a t i o n s on s u r f a c e  116  der  Kamp's  &  hypha of  t e n t a t i v e l y i d e n t i f i e d as Sporothrix  q u i t e d i f f e r e n t from van  on  report  the sp. as  Cyl i ndr oce phal urn.  hyphomycete the  WR3  i s suggested  like culture.  following  Phialophora  text,  to be Phialophora  In order to a v o i d c o n f u s i o n ,  Sporothrix  K.  sp. ,  sp. w i l l be used i n s t e a d of WR1,  respectively.  The  original  in  t huj i na and WR2 and  WR3  c u l t u r e s of WR2 and WR3 are  a v a i l a b l e at I d e n t i f i c a t i o n Service, Institute,  sp.,a  A g r i c u l t u r e Canada,  B i o s y s t e m a t i c Research  Ottawa, with system number:  DAOM 196473 and DAOM 196486 r e s p e c t i v e l y . An obvious p a t t e r n of microorganism d i s t r i b u t i o n heartwood Figures  was observed i n t h i s study. 18  and  19 that i n WRC  I t can be seen from  heartwood,  sterile  occurred mainly i n growth zones from 396 t o 412, a  straw-colored  Sporothrix which  i n WRC  heartwood area adjacent  to  wood  which was  the sapwood.  sp. appeared mostly i n growth zones 382 t o 395, corresponded t o the r e d d i s h c o l o r e d  c a l l e d the edge area of the d i s c o l o r a t i o n  heartwood,  also  K. t huji na  zone.  occurred mostly i n growth zones 342 t o 361 and was found i n both reddish-brown and brown c o l o r e d zones of the heartwood. Phialophora  sp. was found from growth zones 198 t o 324 and  with g r e a t e s t frequency a t growth zones of 198 t o is  233.  It  c l e a r that the occurence of these microorganisms i n WRC  discolored first  heartwood f o l l o w e d the order that Sporothrix  moved outwards from the p i t h ,  11 7  then K.  t huji na  sp. and  then Phialophora An  sp.  i n t e r p r e t a t i o n of t h i s  pattern  was  microorganism  given by van der Kamp (1975) and i s a Sporothrix  s u b j e c t of the present study. which  has  compounds, a c t i o n would Phialophora  al bipel  sp.  the c a p a c i t y to d e s t r o y or a l t e r such  sp.  further  is a  fungus  natural  toxic  as t h u j a p l i c i n s and perhaps o t h e r s .  This  facilitate  appears to a f f e c t more  distribution  K.  the i n v a s i o n by K.  thujina  thujina  Phialophora  and  and then sp.  the wood i n such a manner that  susceptible I uci da Baxter.,  to  decay  fungi,  such  which o r d i n a r i l y would be  by n a t u r a l l y o c c u r r i n g t o x i c compounds.  1 18  i n turn  it  becomes  as  Poria  inhibited  4.2. WRC  Extractive differences  several  amount of wood e x t r a c t i v e s f a c t o r s i n c l u d i n g age,  tree,  genetic  geographic  differences  comparison s o u n d and tree  radius  at  the  same  samples:  Discolored  heartwood  (D-l);  Light-straw  was  1.  i n the  there  inner  Table  within  the  and  study,  a  between  same  tree,  Difference  has  2.  the  higher  extractions  WRC  thereby along  the  f o r f i v e groups  of  (D-0); Discolored  colored  The the  were t h r e e  sapwood;  a  this  made w i t h i n t h e  heartwood  11.  b a s e d on  Heartwood  Within  (2:1)  Light-straw  content than  has  level  within  variations  in extractive composition  outer heartwood  colored  determined  summary,  In  sample  by  l e v e l were e x a m i n e d .  y i e l d s of BE  given  1962).  some s o u r c e s o f v a r i a t i o n .  a t one  The  (Hillis,  is affected  the  seasonal  d i s c o l o r e d h e a r t w o o d was  and  in trees  p o s i t i o n of  location,  of d i f f e r e n c e s  eliminating  are  discolored  heartwood  The  the  b e t w e e n s o u n d and  outer heratwood ( L - I ) and  concentration O.-D.  weight  same g r o w t h z o n e s ,  concentration  of BE  119  BE  of  (S-P)  extractives  of  (2:1)  wood.  In  study: extractives  discolored  extractives  (L-O);  Sapwood  r e s u l t s from t h i s  a much h i g h e r  inner  heartwood  than  light-  Table 11. Benzene ethanol (BE) (2:1) E x t r a c t i v e s and T h u j a p l i c i n (TH) Content of the WRC Heartwood. Age  Code  BE extract ives content (O.D. %)  TH content (O.D. %)  Unknown compound (O.D. %)  413-303  D-0  15.45  0.230  0.291  303-198  D-I  12.04  0.120  0. 187  413-303  L-0  12.55  0.730  None  303-198  L-I  9. 1 1  0.552  None  420-413  S-P  3.20  0.012  None  D-O: D-I: L-O: L-I: S-P:  discolored discolored light-straw light-straw sapwood.  outer heartwood. inner heartwood. c o l o r e d outer heartwood. c o l o r e d inner heartwood.  120  straw c o l o r e d heartwood; and 3.  Across a r a d i u s , BE e x t r a c t i v e s increase  heartwood to outer There other  e x t r a c t i v e s i n the c e l l w a l l  reported  a  content in  to  heartwood.  higher  the sapwood of WRC  abies  in  other  and  (66.5%)  (L.) Karst.)  Theander,  r e l a t i v e to the  species,  1974).  have  Gardner and  (1954)  cellulose  heartwood.  (2.6%) Similar (Picea  such as Norway  spruce  been  (Johansson  found  reported  and in  sapwood rays but not i n the heartwood where i t seemed to  be  replaced  in  phenols.  The  that s t a r c h was  is  common  by  They  and  increase when sapwood  Barton  holocellulose  polyphenolics  (54.2%) and a much lower e x t r a c t i v e content  results  inner  heartwood.  i s evidence that amounts of  transformed  from  formation  of  phenolics  heartwoods i s part of the t r e e defence mechanism. sapwoods,  however,  physiologically 1967). due on  The  the  when d i s t u r b e d  parenchyma  by fungal  may  infection  react (Shain,  lower e x t r a c t i v e content i n sapwoods might  t o n a t u r a l l y higher living  living  In l i v i n g  sapwood.  r e s i s t a n c e t o microorganism  In heartwoods, the accumulated  e x t r a c t i v e s can prevent such a t t a c k s .  Results  be  attack phenolic  obtained  in  t h i s study agree with these concepts. The  color  of  WRC  heartwood  121  is  subject  to  marked  variation.  Because  extractives  contained t h e r e i n ,  darker also  c o l o r of the wood i s g e n e r a l l y  study.  in  WRC  There  a c c o r d i n g to r e s u l t s  the  (D-0)  greater  inner  discolored greater There  formed  outer sound  (L-O)  measured  This  is  in  this  that  WRC  outer  extractive  content  heartwood,  an e x t r a c t i v e  while content  ( L - l ) heartwood (Table  p a r t l y due  The  11).  amount appears to be  to separate these v a r i a b l e s . showed  that  the amount of  with d i s t a n c e  Nault,  of  partly  to environmental f a c t o r s , and  i t is  In t h i s study, BE  (2:1)  known p a t t e r n s  the  extractives  from the p i t h to the most  heartwood which f o l l o w s  The  (2:1)  (2:1)  heartwood had  a  evidence of f a c t o r s a f f e c t i n g the amount  MacDonald, 1971;  3.3.).  a BE  in heartwoods.  i n h e r i t e d and  increased  (D-I)  that  sound heartwood taken  study found  than sound inner  is  extractives  results  than the  to  comparing  This  heartwood had  2.90%  difficult  in d i s c o l o r e d and  same growth zones.  discolored  obtained  have been no previous r e p o r t s  e x t r a c t i v e contents  2.93%  i t i s u s u a l l y true  c o l o r denotes a higher e x t r a c t i v e c o n t e n t . true  from  due  recently  (Barton  and  1984).  t h u j a p l i c i n contents of f i v e groups of samples were by The  a  c o l o r i m e t r i c method as  r e s u l t s are  extractives within  listed  i n Table  the heartwood,  122  described 11.  (Section  S i m i l a r to  BE  t h u j a p l i c i n content  increased  from  possessed the  trace  was  thujaplicin different  heartwood.  Sapwood  only  amounts of t h u j a p l i c i n s i n comparison  (2:1)  observed,  however,  concentration from  discolored  in  outer  to  heartwood. It  a  p i t h to  that of  heartwood  vs. BE  that  the  heartwood  pattern  coloration  (2:1) e x t r a c t i v e s .  light-straw  colored  total  heartwood outer  contained  heartwood  discolored It  those highly  while the inner  and  generally  accepted  that  the  straw-  the  d u r a b i l i t y of WRC heartwoods  high  parallel  inner  the e x t r a c t i v e s mentioned i n t h i s sense only  toxic  to  certain  groups of  dominant n a t u r a l p r e s e r v a t i v e the e x t r a c t i v e s .  Hence,  heartwood  decay the  However, i t i s worthwhile to point  components or f r a c t i o n s i n the e x t r a c t i v e s  discolored  colored  light  had 4.0 times the content of  presence of e x t r a c t i v e s . that  light-straw  heartwood.  i s now  resistance  heartwood  3.2 times as much t h u j a p l i c i n s as the  d i s c o l o r e d heartwood,  colored  out  The outer  BE  heartwood had  much higher t h u j a p l i c i n content than d i s c o l o r e d the same growth zones.  was  Although  (D-0 and D-I) had a higher  e x t r a c t i v e content,  of  fungi.  which  In  WRC  mean are the  i s the t h u j a p l i c i n f r a c t i o n i n  a higher BE e x t r a c t i v e content i n  (D-0 and D-I)  123  does  not  necessarily  represent  a  higher  light-straw lower  toxicity  colored  heartwood  Discoloration colored  heartwood  advanced  decay.  changed  concentration indicates  content this  of  tropolone  a  of  same  As  decreased, have  microorganisms  causing  this  the  of  the  resistance The discolored  the  to  the  heartwood  decrease heartwood  of  the  to  has  when  than  content.  prior wood  to color  thujaplicin  observation process  process),  a  light-straw  the  the  strongly (in  this  extractive  changed,  particularly  for  result  of  this  the  which  suggests  change,  structurally new  It  thujaplicin heartwood further  1 24  was  is  in  by  not  that  certainly of  natural  attack.  concentration the  or those  expected  terms  fungal  the  altered  content  thujaplicin  extractives  that  compounds  discoloration.  of  the  zones,  This  been  heartwood  properties  of  a  it  discolored  heartwood  discoloration  compounds.  though  The  resistance  brown,  attack  fungi.  than  changes  growth  of  thujaplicin  colored  somehow  biochemically  change  higher  to  the  L-I),  decay  remarkably.  during  might  and  wood  brown  the  groups  extractives  its  the  heartwood  thujaplicins  affected  to  into  content  degraded  BE  microorganism  the  group  (L-0  light-straw  that was  of  dropped  certain  higher  due  In  from  it  of  possesses  discolored  case,  heartwood  concentration  heartwood,  to  only  in major  phenomenon finding  observed is  that  in in  t h i s study.  addition  composition  present  was  p r e v i o u s l y unknown  a new,  discolored detected confirmed these  TLC  to  TLC  samples  normal  extractives.  T h i s new  The  same  were  Nault  obtained  (1986)  content  these  Four  was  samples used f o r Nault  (2:1) e x t r a c t i v e s  indicated  that  three  out  had  of  of BE  of  Thin l a y e r chromatography was i n t h i s study to separate and  components (both  on  components.  long wavelengths)  exposed to i o d i n e vapor.  a  of  lower  these four  four  samples  spot.  the most important  method  i d e n t i f y the v a r i o u s positions  the TLC p l a t e s were marked  short and  (1986).  discoloration.  the  The  and  in  observation  samples  and c e r t a i n degree  extractive  there  was  from  analysis  heartwood  chemical  compound  c o n t a i n e d the unknown compound chromatographic  used  interesting  compound c l e a r l y present  ( F i g u r e 20).  experiments  thujaplicin The  the  by repeated experiments.  According  more  in l i g h t - s t r a w c o l o r e d heartwood,  heartwood by  to  A  under  of UV  WRC the  light  then the p l a t e s were  The R^ values f o r a l l spots on  the  experimental p l a t e s were c a l c u l a t e d and are l i s t e d  in  12.  heartwood  The  R^  values  e x t r a c t i v e s using BE MacDonald  and  of  known l i g n a n s  in  WRC  Table  (9:1) e l u t i n g s o l v e n t were p u b l i s h e d by  Swan (1970).  The  125  known components  in  the  Figure  20.  TLC P l a t e s of BE E x t r a c t i v e s from WRC L i g h t - s t r a w C o l o r e d Heartwoods.  «o  m>  MS?  3s**.  3fn~  m  D-0  L-0  D-I 126  L-I  Discolored  and  Table  12. TLC R Values f o r Four WRC Heartwood Using BE (9:1) Solvent. f  Spot #  Extracts  D-0  D-I  L-0  L-I  1  0.00  0.00  0.00  0.00  2  0.05  0.05  0.05  0.05  3  0.09  0.09  0.09  0.09  4  0.14  0.14  0.14  0.14  5  None  None  0.17  0.17  6  0.19  0.19  0.19  0.19  7  0.25  0.25  0.25  0.25  8  0.30  0.30  0.30  0.30  9  0.33  0.33  0.33  0.33  10  0.35  0.35  0.35  0.35  1 1  0.40  0.40  0.40  0.40  12  0.45  0.45  0.45  0.45  13  0.48  0.48  0.48  0.48  14  0.79  0.79  None  None  15  0.86  0.86  0.86  0.86  D-O: D-I: L-O: L-I:  D i s c o l o r e d outer heartwood. D i s c o l o r e d inner heartwood. L i g h t - s t r a w c o l o r e d outer heartwood. L i g h t - s t r a w c o l o r e d inner heartwood.  127  volatile  fraction  identified solvent  by  by  TLC.  values of the  heartwood  spots on  extractives  were  pure compounds with BE  (9:1)  values  in Table the  of  known  13.  in e x t r a c t i v e  sound  were  identified  in  samples.  Since  extractive  of  between The  sound  values.  In  plates,  only  the  major  i d e n t i f i e d by p r e v i o u s work,  expect the  R^ L-I  obvious.  both  and  addition  there  were  components i t is  samples (Table  12,  represent a s i g n i f i c a n t  Spots # 4,6,  and were  reasonable  presence of some unknown spots.  the unknown spots occurred i n both sound and  not  and  unknown spots which appeared on both d i s c o l o r e d  i s o l a t e d and to  L-0,  composition  these known component spots on the  several  D-I,  d i s c o l o r e d heartwood e x t r a c t i v e s are  components  heartwood  made.  d i s c o l o r e d heartwood e x t r a c t i v e s by R^ to  WRC  A comparison of the  p l a t e s of D-O,  values was  differences  sound and known  R^  listed  known R^ The  WRC  running against  e x t r a c t i v e s are  to the  of  As  long  as  d i s c o l o r e d wood and  9), they d i d  d i f f e r e n c e between the  two  kinds  extractives. Special  spots  a t t e n t i o n was  appearing  on  the  e x t r a c t i v e samples (D-0, heartwood e x t r a c t i v e s  p a i d to one plates  D-I)  (L-0,  of  of the  discolored  which was  L-I).  128  new,  not  T h i s new  unknown heartwood  found i n spot has  sound an  R  f  Table 13. TLC R Values of Known WRC Using BE (9:1) S o l v e n t . f  Extractives  Name of the compound  R^ value  Lignans: Plicatic  acid  0.00  Plicatin  0.05  Plicatinaphthol  0.10  Dihydroxythujaplicatin  0.15  Plicatinaphthalene  0.16  Dihydroxythujaplicatin methyl ether  0.17  Thujaplicatin  0.21  Gamma-thujaplicatene  0.23  Hydroxythujaplicatin methyl ether  0.25  Thujaplicatin methyl ether  0.30  *  V o l a t i l e Fraction : Beta-thujaplicinol  0.35  Beta-thujaplicin  0.40  Gamma-thujaplicin  0.45  Thujic  0.47  acid  Methyl t h u j a t e 0.85 Source: MacDonald and Swan, (1970). *The data obtained by u s i n g pure samples i n t h i s 129  study.  value  of 0.79 which i s c l e a r l y d i f f e r e n t  compounds reported this  new  3.6c)  (Table  13).  discolored discolored  that  heartwood heartwood  the  concentration  (D-0) (D-I),  other  known  Q u a n t i t a t i v e measurement of  compound by the p r e p a r a t i v e TLC  showed  from  was  0.291%  i t was  method of  it  and  (Section in  outer  for  inner  0.187%.  i n v e s t i g a t i o n of t h i s compound was c a r r i e d out.  Further A detailed  e l u c i d a t i o n of i t s s t r u c t u r e , an e v a l u a t i o n of i t s t o x i c i t y to  certain  fungi and a p o s s i b l e mechanism of formation  t h i s compound i n d i s c o l o r e d heartwood are d i s c u s s e d  130  of  later.  4.3.  Biological and  thujina  and c h e m i c a l r o l e s  Thujaplicins responsible may  for  interact  tree. fungi  In  this  study,  effects sound  WRC  the fungi  wood of  heartwood  content  and  sequentially  natural  which  contained  total  extractives,  BE  the  stage  heartwood.  sp.) The 18  sp.  may b e  the  first  attack  were  frequency  Figures  with  living  attacking  10,  fungi  on p e r c e n t a g e  thujaplicin,  decay  different  tree,  early  and  19)  fungus  b y K.  b i o a s s a y was d e s i g n e d t o  blocks,  the experiment  the l i v i n g  within  thujina  following.  three  with  of  heartwoods,  a n d Phialophora  heartwood,  block  these  i n WRC  which are  (Table  Sporothrix  sp.  extractives  existing  kinds  discolored  the s t e r i l e  The  three  thujina  of  a n d Phialophora  fungi  K.  WRC t r e e s  resistance  K.  that  attacking  various  sp.,  Sporothrix  living  heartwood  decay  sp.,  from  indicated  in  natural  with  distribution  keep  and other  (Sporothrix  isolated  sp. in  Phialophora  of  as close fresh  a high  cut  (2:1) extractives  orders.  light-straw of  The r e s u l t s  and t h u j a p l i c i n  131  the  losses  of  extractives  individually,  as possible to  concentration  was u s e d .  weight  BE ( 2 : 1 )  resistance attacking  examine  In  and  order  to  real  conditions  colored  heartwood,  thujaplicins of  contents  weight of  and  losses,  WRC  sound  heartwood b l o c k s t r e a t e d with Sporothrix Phialophora  sp. are shown i n Table  The  thujina  and  14. a  completely  randomized design experiment with nine d i f f e r e n t  treatments  There  wood  sp. , K.  were  treatment. the  block bioassay experiment was  four Two  of BE  thujaplicin dishes  within  It  is  (2:1)  content  noted  that  e x t r a c t i v e s and  each  were used  there  is  a  in 2.85%  0.026% r e d u c t i o n  for  from the wood blocks i n the empty P e t r i  (Group 9) to the wood, b l o c k s p l a c e d on the top of  culture  media  without  S i m i l a r to chemical loss  measurements  l e v e l s of c o n t r o l treatments  experiment.  reduction  repeated  between  chemical  loss,  growing  on  control  levels  (Group  well  8).  weight  (difference:0.635%-  T h i s o b s e r v a t i o n t e l l s one as  it  l o s s , there i s a l s o a s i g n i f i c a n t  the two  0.021%=0.614%).  fungi  as a weight l o s s  that there i s a due  to  direct  contact between the wood blocks and water c u l t u r e media. t h i s experiment, the  culture  growing however,  treatment  media,  fungal  blocks d i d not d i r e c t l y  Mass  or  chemical  p r o x i m i t y between the b l o c k s and c u l t u r e media. taking  a c o n s e r v a t i v e approach,  132  the  exchange,  between t e s t i n g blocks and c u l t u r e media are  p o s s i b l e through t r a n s l o c a t i o n by fungi or due  In  contact  but were p l a c e d on the s u r f a c e of  cultures.  the  still  to very c l o s e Therefore,  a l l c o n c l u s i o n s drawn from  Table 14. Sound thujina  Results of Weight Loss and E x t r a c t i v e Analyses of WRC Heartwood Blocks Treated with Sporothrix sp., K. and Phialophora  Group No. (n=4)  sp.  Weight Loss Mean (*) SD.  BE EXT (O.-D.)%  TH Thujin (O.-D.)%  (8 weeks with 1.401  WR1) (d)  0.172  10.84  0.081  (8 weeks with 1.552  WR2) (d)  0.110  8.75  0.412  (8 weeks with 0.896  WR3) (c)  0.107  10.51  +  0.509  (4 weeks with WR1, then 4 weeks with WR2) 5.476 (f) 0.256 9.36  0.090  +  (4 weeks with WR1, 3.629 (e)  then 4 weeks with WR3) 0.659 10.97  0.10.1  +  (4 weeks with WR2, 1.250 (d)  then 4 weeks with WR3) 0.143 9.21  0.426  (4 weeks with WR1, with WR3) 5.561 ( f )  then 4 weeks with 0.616  WR2  9.01  then 4 weeks 0.079  +  8: ( c o n t r o l l e v e l 2: 8 weeks with c u l t u r e medium) 0.635 (b) 0.260 10.93 0.587 9: ( c o n t r o l l e v e l 1: 8 weeks i n empty P e t r i 0.021 (a) 0.016 13.78  dishes) 0.613  *: a,b,c,d,e and f represent s i x homogeneous subsets among nine O.-D. weight l o s s means a c c o r d i n g to Duncan's M u l t i p l e Range Test with 95% confidence l e v e l . BE EXT: Benzene ethanol e x t r a c t i v e s . TH:  Thujaplicins 133  t h i s experiment w i l l be based on comparing treatment with that of the second c o n t r o l l e v e l The  chemical  thujaplicin  analysis  provide  of BE  (Group 8 ) . (2:1)  strong evidence  results  extractives  for b i o l o g i c a l  and  r o l e s of  the three e a r l y a t t a c k i n g f u n g i . Sporothrix  Since isolated is  sp.  heartwood  chemicals  earlier  It  experiment  Sporothrix  reducing  that natural  thujaplicin  attack  of  to  was  very  than  heartwood, interacts  other  was  plays  a  of the  WRC  but,  in  sp.  this  role  in  heartwood. and  1/7  of  the  f a c t that a high p r o p o r t i o n  of  the  c o u l d not be  had  detected  suggests  (Table  form of the  l o s s of  thujaplicins  This  f u n c t i o n s uniquely  1 34  exposed  thujaplicin  the weight l o s s of the  14).  that  Sporothrix  showed that the blocks  a considerable  at t h i s stage  small  with  i n v o l v e d (Groups 1, 4, 5,  changed the chemical  sp.  it  isolated  major  reduced to approximately  The  organism  the l i g h t - s t r a w c o l o r e d heartwood by  Sporothrix  Sporothrix  sp.  wood block bioassay  content, was  sp.  content  somehow The  major  suggested by o b s e r v a t i o n s  decay r e s i s t a n c e  amount.  thujaplicin  sp.  is  time Sporothrix  initial  only  to b e l i e v e that t h i s fungus  microorganisms.  7)  the  near the edge of the d i s c o l o r e d WRC  reasonable  Every  was  blocks  demonstrates  in d e t o x i f i c a t i o n of  that the  compounds t o x i c to other f u n g i , It  Sporothrix  sp.  w a l l components. Sporothrix  time,  growth may not o r i g i n a t e  have the c a p a c i t y  the  to degrade  to a form with much lower t o x i c i t y .  this  reaction  Sporothrix  was  a  the  At the same  i t s e l f might p r o v i d e the energy source  extractives,  new  previously  unknown compound  from d i s c o l o r e d heartwood  f r e s h l y cut n a t u r a l chemical  discolored  i t was p o s i t i v e l y shown that present  (Figure  d i s c o l o r e d heartwood samples were taken  The  4,  there  only  in  20). The sound directly  from  wood.  composition  of  BE  (2:1)  extractives  obtained from each t r e a t e d wood group were examined by This  alter  sp. growth.  extractives and  enzymes  or  During the chemical comparison of sound and heartwood  needed  from wood c e l l  There are p o s s i b i l i t i e s that sp.  thujaplicin  for  thujaplicins.  i s a l s o suggested by t h i s r e s u l t that the energy  for  in  i n t h i s case  TLC.  same new unknown compound was found again i n Groups 1, 5  These contain groups  and 7 of t r e a t e d wood  blocks  wood  block e x t r a c t i v e s  were l i g h t - s t r a w  colored  (Table and  t h i s new compound before the treatments. of  treatment,  samples but  the  underwent common  different  points  treatments s t a r t e d with Sporothrix  are  sp.,  135  14).  d i d not  These  four  sequences  of  that  all  four  and a l l four showed  major  losses  unknown  of t h u j a p l i c i n .  compound  only  in  The  those  appearance of t h i s heartwood  extractives  treatments i n v o l v i n g Sporothrix  obtained from the  spp,  not  in e x t r a c t i v e s obtained from treatments with K.  or  Phialophora  sp. ,  K. thujina  or  new  but thujina,  then Phialophora  and  sp. ,  i n d i c a t e d that t h i s compound i s a s p e c i a l product y i e l d e d interaction  between  sound  Sporothrix  Although responsible  for  occurrence  of the  heartwood  sp.  reduction new  that  the  of t h u j a p l i c i n content  unknown compound,  new  compound may  e x t r a c t i v e components, Sporothrix  sp.  plicatic  acid  to  sapwood bioassay Beta-, introduced pieces TLC  to  Sporothrix  Section  pre-extracted  found n e i t h e r  WRC  sp.  but  originate  the  It i s  from  other  under i n f e c t i o n  and  sapwood  by and  by  the  acid  pieces, sp.  were  and  f o r 15  showed that  in p r e - e x t r a c t e d  lacking  proven  plicatic  with Sporothrix  in p r e - e x t r a c t e d  be  3.8).  with the e x t r a c t i v e s  nor  compound.  unknown compound was  were then t r e a t e d  compound was  and  r e l a t i o n s h i p between t h u j a p l i c i n s  the  (see  to  there i s no d i r e c t  such as l i g n a n s ,  gamma-thujaplicins  results  samples,  The  sp.  i s an organism proven  evidence r e l a t i n g t h u j a p l i c i n s to t h i s new possible  Sporothrix  and  by  the  the days.  unknown  sapwood c o n t r o l  sapwood samples t r e a t e d addition  136  of  thujaplicins  with or  plicatic  acid.  T h i s r e s u l t c l e a r l y demonstrated that  pre-extracted  sapwood  compound.  also  It  does  not  eliminated  contain  the  pre-extracted  Sporothrix  sp.  sp.  wood complex or  itself.  unknown  the p o s s i b i l i t y  unknown compound was produced by Sporothrix with  this  The p o s s i b i l i t y  was  that  the  interacting produced  that  the  the  by  unknown  compound o r i g i n a t e d from l i g n a n s was excluded, s i n c e the new compound  was not found i n e x t r a c t i v e s obtained  containing p l i c a t i c unknown  from sapwood  a c i d and exposed to Sporothrix  compound was only  found i n sapwood  pieces  with Sporothrix  sp. and c o n t a i n i n g  thujaplicins.  obvious  the  both  that  Sporothrix formation The  sp.  of  the  treated  I t becomes  microorganism  and t h u j a p l i c i n s i s a p r e c o n d i t i o n  quantity was  reduced by Sporothrix isolated  in  of t h u j a p l i c i n s reduced by smaller sp. the  when compared  to  In the l i v i n g t r e e ,  reddish  and  f o r the  K.  thuji na  the  quantity  K.  t huji na  light-brown  heartwood, i n which the t h u j a p l i c i n content it  The  of t h i s unknown compound.  individually  was  presence  sp.  colored  was very  low, so  seems that the p r o b a b i l i t y that K. t huji na f u n c t i o n s as a  destroyer  of t h u j a p l i c i n s i s very low.  I t i s i n t e r e s t i n g to f i n d that though K. t huji na i s not a  t h u j a p l i c i n destroyer  in living  1 37  trees,  i t has  a  higher  capacity  to decrease the BE  Sporothrix  and Phialophora  sp.  WRC  heartwood  the  end  BE  samples  Sporothrix the  weight  na  of BE  (Groups 2,  sp.  sp.  and  followed  components other  may  some n a t u r a l t o x i c i t y  could  thuji  o b s e r v a t i o n s suggest that  heartwood  Changes  K.  l o s s caused  then  t h u j a p l i c i n s i n WRC possess  samples.  compared  to  higher than the weight  These  by  to  I t i s a l s o true that a f t e r the  might be a c t i v e against  also  colored  4, 6 and 7) had  (2:1) e x t r a c t i v e s  Sporothrix  with  straw  3 and 5  t r e a t e d b l o c k s were exposed  l o s s was  Phialophora  t huji na  (Table 14).  sp.  treatment  thuji  The l i g h t  of the treatments f o r Groups 1,  obvious r e d u c t i o n  other  sp.  than  (2:1) e x t r a c t i v e s were almost the same  Samples t r e a t e d with K. an  (2:1) e x t r a c t i v e s amounts  extractives.  These to  change t h e i r s o l u b i l i t y  i n a BE  by with  the than  K. the  components  decay  i n the chemical s t r u c t u r e of e x t r a c t i v e  na,  fungi.  components  (2:1) s o l v e n t  system,  as w e l l . The minor No  t r e a t e d with Phialophora  samples  l o s s of  t h u j a p l i c i n content from 0.587%  Phialophora  doubt  thujaplicins  sp. alone showed a  directly  sp.  would  not  to  0.509%.  interact  i n the l i g h t - s t r a w c o l o r e d  with  heartwood  so i t would not f u n c t i o n as a p i o n e e r . The  results  of  Analysis  of V a r i a n c e of  1 38  weight  loss  (Appendix  6) i n d i c a t e that there i s  between treatment percentage are  means.  O.-D.  In other words,  different  at a  Range T e s t s (Appendix  differences.  the means of the  Results  95%  caused  7) f u r t h e r  weight  loss  significant  illustrate  means.  Treatment  weight l o s s ,  5  which was  also  K.  sp.  also  individually  thujina  sp. , K.  (Groups 1,  d i d not show severe weight l o s s .  s t a r t e d with Sporothrix or with Phialophora that  Sporothrix  sp.  (Table  14)  thujina  and  2, and 3) d i d not  f i r s t and then with Phialophora  demonstrated  greater  Those b l o c k s t r e a t e d with  l o s s e s occurred only i n three treatments all  in a  2, 3, and 6.  a very severe weight l o s s . thujina  loss equal  resulted  significantly  that wood b l o c k s t r e a t e d with Sporothrix  show  Weight  and 7 are s t a t i s t i c a l l y  i s i n t e r e s t i n g to f i n d from the r e s u l t s  Phialophora  these  and are s i g n i f i c a n t l y g r e a t e r than a l l other  than Treatments 1, It  level.  show that there are s i x homogeneous  by Treatments 4  to each o t h e r ,  groups  confidence  subsets among the nine O.-D. weight l o s s means. means  variation  weight l o s s e s f o r nine wood block  significantly  Multiple  significant  sp. The  (Group  higher  weight  (Groups 4,5 and  and then f o l l o w e d with  sp. sp.  7) K.  This observation strongly can  not  cause  weight l o s s of the wood block d i r e c t l y , but t h a t  1 39  6)  serious  interaction  between Sporothrix  sp. and sound heartwood  for  s i g n i f i c a n t weight  In  other  words,  heartwood or  l o s s caused by l a t e r a t t a c k i n g  natural  decay  resistance  which prevents advanced  decay  removed by the a c t i o n of Sporothrix Thereby,  a  i s a precondition  sound  lessened  sp. i s shown between  Sporothrix  sp.  and  Phialophora  sp.  R e s u l t s with Groups 4, 5, and 7 (Table 14)  indicate  t huj i na  the  i s somehow  strong s y n e r g i s t i c e f f e c t K.  in  fungi.  percentage  weight  (Group 5 ) .  loss  sp.  of Sporothrix  sp.  t huji na caused a much more  severe  Phialophora  than f o l l o w e d by  and K. and  t huji na  t huji na  Thus,  Phialophora  steps  attacks  i n l i v i n g WRC for  heartwood  destroying  the  natural  and a l l o w i n g f u r t h e r advanced  van  sp.  der  Kamp  than  f o l l o w e d by  in l i v i n g  sp.  t r e e s appear to be one  The r o l e of Phialophora far.  by Sporothrix  effect  sp.  i s the same as occurs n a t u r a l l y  sequential  t huji na  sp.  i s much higher  The order of treatment with Sporothrix K.  and  spp  Thus, i t i s obvious that the s y n e r g i s t i c  between Sporothrix that  sp.  Sporothrix  that the treatments s t a r t i n g with  f o l l o w i n g with K.  then  Sporothrix  or  toxin  and  then  of of  WRC.  essential WRC  sound  decay.  sp. i s not w e l l demonstrated (1975)  a n t a g o n i s t i c e f f e c t between K.  proved  that  there  t huji na and Phialophora  140  K.  is  so no sp.  The  wood  blocks  K.  t r e a t e d with  thujina  followed  with  Phialophora  sp. (Group 6) had a weight l o s s of 1.2500% about  half  by  that  K.  thujina  (0.896%) i n d i v i d u a l l y . an  ( 1 .552%)  T h i s suggests that there  Based  on  Phialophora  the  f a c t s observed  sp.  i n l i v i n g WRC  sp.  acts  heartwood and d e s t r o y i n g provided  as  in  these  by t h u j a p l i c i n s .  K.  sp. ,  t r e e s can be  thujina  summarized  unknown  assumed  a  pioneer  This attack  sound  then prepares  wood  The phenomenon of s i g n i f i c a n t  new compound during  that  the  converted t o other  Sporothrix  chemical is  sp.  attack  heartwood i s of i n t e r e s t .  missing  thujaplicins  were  of of  I t may be  degraded  or  compounds l i k e t h i s new unknown compound,  which may not be t o x i c t o decay f u n g i .  compound  attacking  of t h u j a p l i c i n content accompanying appearance  l i g h t - s t r a w c o l o r e d WRC  the  experiments,  the n a t u r a l decay r e s i s t a n c e f a c t o r  f u r t h e r fungal a t t a c k s .  reduction an  thujina  follows. Sporothrix  for  i s neither  sp.  b i o l o g i c a l and chemical r o l e s of Sporothrix  as  sp.  K.  a n t a g o n i s t i c nor s y n e r g i s t i c e f f e c t between  and Phialophora  and  Phialophora  and  structure  and  The determination  toxicity  a key t o s o l v i n g the  d e t o x i f i c a t i o n mechanisms during  mystery  Sporothrix  141  of  this  of  of  unknown  thujaplicin  sp. i n v a s i o n .  K. spp.  has  thujina  attacks  provided  Sporothrix  the WRC heartwood a f t e r  favorable conditions.  The  K.  thujina  infection  may i n t e r a c t with other  t o x i c components  in  heartwood  e x t r a c t i v e s and f u r t h e r reduce the n a t u r a l  WRC  decay  r e s i s t a n c e of WRC heartwood with d i f f e r e n t mechanisms. Phialophora  spp.,  present  i n the dark-brown  heartwood, may act as an intermediate  between  these  colored pioneers  and the i n v a s i o n of decay f u n g i . The  whole  microorganisms heartwood  (by  process which attack pioneers),  involves sterile  a  succession  (light-straw  gradually  turning  of  colored) it  into  d i s c o l o r e d wood and f i n a l l y decayed wood (by decay f u n g i ) .  1 42  4.4  Elucidation  of  the  chemical s t r u c t u r e  for  the  new  unknown compound  Separation aided  by  preliminary  properties heartwood  of components i n complex mixtures i s  of  separations  the components.  extractives  are  were  The  a  very  strong  organic  (Barton and  acid,  40% of the WRC  very  in water compared with  extractives, et  (Gardner insoluble strongly  acetate the  al,  1959).  polar  insoluble  Removal  of  from the e x t r a c t i v e s  the  ether i s s l i g h t l y  Separation solubles  was  of  acid,  1971),  comprises It i s  components  acetate  ethyl  acetate more  a c i d , from other  less polar  e t h y l ether s o l u b l e s  performed f o r f u r t h e r  in  in e t h y l  separates the  components, such as p l i c a t i c  Ethyl  ethanol.  than  ethyl  from  ethyl  f r a c t i o n a t i o n of  extractives. The  in  is particularly  material  compounds. acetate.  and  and  heartwood e x t r a c t i v e s . other  a  preliminary  MacDonald,  plicatic  WRC  having  used i n  e t h y l acetate  approximately soluble  chemical  compounds  solvents  e t h y l ether,  According to a previous study  on  Most of components in  phenolic  c e r t a i n range of p o l a r i t y . separation  based  often  the  unknown compound was  found by TLC  e t h y l acetate solubles.  1 43  A f t e r the  to occur  mainly  ethyl  acetate  solubles column,  went  through a g r a d i e n t  the unknown compound was  elution  chromatographic  found to be present  i n the  f r a c t i o n e l u t e d by the the BE  (85:15) s o l v e n t mixture.  observation  this  indicates  that  new  compound  This  does  not  possess a very strong p o l a r f u n c t i o n a l group. The  extent  of a b s o r p t i o n of a s i n g l e component on a  p l a t e depends on the p o l a r i t y of the molecule, of  the  absorbent,  phase.  The  dependent  in  the  the p o l a r i t y of the  the  relative  values  e q u i l i b r i u m constants  mixture.  developing  activity  mobile  liquid  a c t u a l s e p a r a t i o n of components i n a mixture i s on  desorption  and  the  In g e n e r a l ,  solvent  of  the  absorption-  f o r each of the  components  when the absorbent and  are f i x e d ,  the more  polar  (Peters et  s u r f a c e of the s o l i d phase of TLC words,  solvent  and  developed by BE 0.79  can  be  compound WRC  a higher R^  (9:1),  value.  t h i s new  and  1974).  In  with  the  plates,  compound has an R^  value at  deduced  l i g n a n s in WRC  On  the  the  TLC  which i s c o n s i d e r a b l y higher  for t r o p o l o n e s  al,  a l e s s p o l a r compound flows b e t t e r has  the  functional  groups i n the compound w i l l be more s t r o n g l y absorbed on  other  TLC  than most known R^  values  heartwood e x t r a c t i v e s .  that the chemical  structure  of  this  i s l e s s p o l a r than most of the known compounds  heartwood e x t r a c t i v e s as shown by  144  i t s higher R^  It new in  value.  Since  the R^ values f o r a l l the known l i g n a n s  heartwood Swan,  e x t r a c t i v e s are from 0.00 t o 0.30  1970)  i n BE (9:1) s o l v e n t ,  in  WRC  (MacDonald  and  i t i s u n l i k e l y that  this  compound has a l i g n a n l i k e s t r u c t u r e . When  the TLC p l a t e was examined under  unknown compound was s t r o n g l y f l u o r e s c e n t . indicates  that the chemical  UV  light,  the  This observation  s t r u c t u r e of t h i s compound  possess  some chromophores or auxochromes, such as  double  bonds  and c a r b o n y l groups,  may  conjugated  or -OH and -OR  groups,  respectively. These unsaturated groups can undergo p i * * pi and n t o p i t r a n s i t i o n s i n UV l i g h t energy range. After (UV),  obtaining  infrared  (IR),  the  purified  proton  compound,  ultraviolet  and carbon-13 nuclear magnetic  resonance (NMR),  low and high r e s o l u t i o n mass s p e c t r a  were  the compound.  taken  methods  for  These  four  s t r u c t u r e of an organic molecule, There  (MS)  spectroscopic  provide an immense amount of information about  structure.  to  the  o f t e n l e a d i n g t o a unique  i s no u n i v e r s a l way of c o n s i d e r i n g  each  p i e c e of i n f o r m a t i o n .  In t h i s case, the best p l a c e to begin  i s with the molecular  ion i n the mass spectrum, from which a  molecular  formula  During spectrometry  can be deduced.  recent  years,  extensive a p p l i c a t i o n  of  mass  as an a n a l y t i c a l t o o l i n organic chemistry  has  1 45  continued  to  spectrometer many It  This  is  has the c a p a c i t y to produce  fragment then  proliferate.  because  a molecular  ions from the molecules under  separates these ions a c c o r d i n g to  charge  ratio,  ion.  If  a  the  mass  ion and  investigation. their  mass-to-  and measures the r e l a t i v e abundances of molecular  ion  has  sufficient  each  stability,  a  -5 lifetime  of  accelerated its  approximately  seconds,  i t w i l l be  fully  and recorded at i t s corresponding m/e value  mass spectrum.  mass  10  Thus,  f o r the m a j o r i t y of  in  compounds,  spectrometry p r o v i d e s an exact and unambiguous method  f o r determining molecular weight  of a molecule  (Howe et  al,  1981). In  t h i s study,  two low r e s o l u t i o n  unknown compound were recorded. by  two  spectra ratios  separate (Figure of  sample 21).  mass s p e c t r a of the  These two samples,  purifications, The  gave  corresponding  the important peaks i n the  obtained identical  mass-to-charge  spectrum  and  their  abundance data are l i s t e d  i n Table 15.  The h i g h e s t measured  mass i n the two i d e n t i c a l  s p e c t r a was 301 (Peak 1) next to a  more i n t e n s e peak with mass 300 (Peak 2 ) . and  related  isotopic  s p e c i e s correspond t o the peaks of the  h i g h e s t mass i n the mass spectrum In a number of cases,  The molecular ion  (Pasto and Johnson, 1969).  compounds g i v e molecular ions of very 146  100 Figure  90 80  21.  Low  Resolution  Mass S p e c t r u m o f t h e Unknown  Compound,  70 60 60 40 30  223  206  20  286  10  13 L-JH  n.» ' ' • • 'H " I. .'.IiT I'I'I'ITI'I i 200  100  236 iii  I ' I ' I Y I ' I I'l i'i l j i i i i ' I T i i I |'i r  T  240  220  300  ,269  260  I  I'l I I'l | I I I 1 I I I I I | I I'l I I I I I I | I I I I I  280  340  320  300  149  _  •5 90  170  80 70 60 60 40 30 1|63 20  77 69  10 0  • III,., ...I, I I l ' l ' l ' | I I'l' 60  ?6  4  80  105  119 181 j 11 • 111111 j 1 111 1 j 111 1 |.l I [ 1 j 111  100  120  140  160  180  200  Table 15. Data of the Low R e s o l u t i o n Mass Spectrum of the Unknown Compound. MEASURED MASS  301 300 266 ZB5  21B  269 257 255 251 23? 236 235 225 224 223 222 213 211 206 205 204 193 192 1S1 162 181 180 179 171 170 169 166 165 164 163 162 150 149 148 146 143 142 141 121 120 11S 118 108 107 106 105 104 103  NO. POINTS  25 35 25 43 14 29 25 25 29 25 35 21 35 35 43 17 35 21 35 43 35 35 29 35 29 51 35 43 43 51  El  35 43 51 51 43 51 59 43 43 SI  43 51 51 43 51 43 43 43 43 43 43 43  ABSOLUTE INTENSITY  71 1 . 3695. 2275. 8134. 596. 2219. 2162. 1881 . 161 1 . 1705. 4129. 1064. Z676 . 3311 . 17811 . 833. 4628. 955. 4681 . 15466. 3195. 4394 . 1998. 3239. 1971 . 6665. 2492. 4825. 6770. 52476. 10134. 7 307. 12466. 45579. 14700. 7952. 33188. 314064 . 11365. 19927. 10177. 6409. 51652. 45747. 10047. 56109. 8417. 9547. 19969. 14456. 37611. 20947. 11759.  14B  X  INT. BASE  0.5 1.2  B.l  2.6 0.2  B.l B.l  0.6 0.6 0.5 1 .3 0.3 0.9 1 , 5, 0. 1 . 0. 1 . 4. 1 .0 1 .4 0.6 1 .0 0.6 2.1 0.6 1 . 2. 16. 3. 2. 4.0 14 .6 4.7 2.5 10.6 100.0 3.6 6.3 3.2 2.0 16.4 14.6 3.2 17.9 2.7 3.0 6.4 4.6 12.0 6.7 3.7  X  INT. NREF  0.5 1.2 0.7 2.6 0.2 0.7 0.7 0.6 0.6 0.5 1.3 0.3 0.9 1. 1 5.7 0.3 1.5 0.3 1.5 4.9 1 .0 1 .4 0. 1 . 0. 2. 0.8 1.5 2, 16. 3, 2. 4 .0 14.5 4.7 2.5 10.6 100.0 3.6 C.3 3.2 2.0 16.4 14. 3. 17. 2. 3. 6 4, 12.0 6.7 3.7  X TOT.  1 ON  0.0 0.2 0.1 0.4 0.0 0.1 0. 1 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.9 0.0 0.2 0.0 0.2 0.6 0.2 0.2 0.1 0.2 0.1 0.3 0.1 0.2 0.3 2.6 0.5 0.4 0.6 2.3 0.7 0.4 1.6 15.6 0.6 1.0 0.5 0.3 2.6 2.3 0.5 2.8 0.4 0.5 1.0 0.7 1.9 1 .0 0.6  Table  15. (continued)  MEASURED MASS 97 96 95 94 93 92 91 85 84 83 83 82 81 60 79 76 77 71 70 69 68 67 66 65 59 58 57 56 55  NO. POINTS 43 35 43 43 43 35 43 71 51 35 35 43 43 43 43 43 43 43 35 51 35 43 21 35 35 29 43 35 43  ABSOLUTE INTENSITY 15345. 8963. 5100S. 13940. 49294. 10166. 39061. 14686. 6155. 12709. 10708. 21964. 64014. 9485. 22000. 12036. 50115. 10028. 63*4. 35524. 4659. 18514. 954. 9325. 4573. 1992. 8099. 5544. 9906.  149  X INT. BASE  X INT.  4.9 2.9 16.2 4.4 15.7 3.2 12.4 4.7 2.0 4.0 3.4 7.0 20.4 3.0 7.0 3.8 16.0 3.2 2.0 11.3 1.5 5.9 0.3 3.0 1.5 0.6 2.6 1.8 3.2  4.9 2.9 16.2 4.4 15.7 3.2 12.4 4.7 2.0 4.0 3.4 7.0 20.4 3.0 7.0 3.8 16.0 3.2 2.0 11.3 1.5 5.9 0.3 3.0 1.5 0. 6 2.6 1. B 3.2  NREF  X TOT  ION  0.8 0.4 2.5 0.7 2.4 0.5 1.9 0.7 0.3 0.6 0.5 1.1 3.2 0.5 1.1 0.6 2.5 0.5 0.3 1.8 0.2 0.9 0.0 0.5 0.2 0.1 0.4 0.3 0.5  low  intensity.  The  i n t e n s i t y of Peak 1 was  (711/3565).  The  peak.  the n a t u r a l  From  peak  next  and 285,  i s o t o p i c abundance  entire  18  group.  spectrum  was  carbon-13,  intense peaks were at  with 285 being l i k e l y generated 3  of  parent  carbons are p r e s e n t .  set of r e l a t i v e l y  by l o s s of a CH  of Peak 2  at 300 would appear to be a  t h i s i n d i c a t e s approximately The  19.3%  from the parent ion  The h i g h e s t i n t e n s i t y peak in at  mass 149.  This  peak  parent i o n ,  with the a d d i t i o n a l l o s s of two hydrogens. the  fragmentation  pattern  of  i n d i c a t e d that t h i s unknown compound appeared fragments fragments M-2X44,  of the same mass. such as 300  peak with mass of M-2X15 may CH  3  groups,  assigned In mass weight  this  M),  M-15,  of  the The  spectrum  to c o n t a i n two  The presence of the  (parent i o n ,  M-2X14-2X44, and M/2  that  the  rather  because  of  i t s mass i s e x a c t l y h a l f  is  interesting  study  286  distinct  M-2X15,  M-44,  support t h i s o b s e r v a t i o n .  The  be a s s i g n e d to the l o s s of  two  while the peak with the mass of M-2X44 may  to the l o s s of two -CO-O- groups i n the summary,  spectrum  i n f o r m a t i o n p r o v i d e d by a low  of t h i s compound showed that  the  molecule. resolution molecular  of t h i s unknown compound i s 300 with approximately  carbons p r e s e n t .  be  18  The chemical s t r u c t u r e of t h i s compound i s  p o s s i b l y a symmetric  dimer.  150  The  information  finalize  the  magnetic  resonance  gained  molecular  information.  so f a r  formula.  (NMR) spectrum  Pulse  Fourier  is  not  adequate  A n a l y s i s of provided  Transform  a  nuclear  more  proton  to  valuable  and  proton  decoupling carbon-13 spectra were obtained i n t h i s study  on  the unknown compound. The the  proton  NMR  spectrum  of the unknown compound  present study i s shown i n F i g u r e 22.  spectrum,  the  how  different  many  number of s i g n a l s i n the spectrum types of protons are  molecule.  In t h i s case the spectrum  only  s i n g l e t peaks.  four  1.28, to  2.3, and 7.3 ppm.  the  12  indicates  present  i n the  i s r a t h e r simple  of  the  internal  protons  present i n the molecule.  of the protons p r e s e n t ,  structural  and the c o u p l i n g of the  s i g n a l s i n d i c a t e s the r e l a t i o n s h i p s between d i f f e r e n t i n the molecule.  methyl,  methylene,  Appendix  2  factors  and  (Nakanishi,  which  influence  0.0,  standard  T h e r e f o r e , there are only three type  The chemical s h i f t s of the s i g n a l s show the  of protons  with  The sharp s i g n a l at 0.0 ppm i s due  with d i f f e r e n t environments  environment  NMR  T h e i r chemical s h i f t s are  e q u i v a l e n t protons  s o l u t i o n TMS.  In a proton  of  types  The chemical s h i f t s of standard methine  1962).  protons In d i l u t e  chemical s h i f t s  151  are  of  listed  in  solution,  the  protons  are  Figure 22. Proton NMR 1% 11  SO 10  •0  Spectrum of the Unknown Compound, IS 10 s  IS  10  $  io  10  ie  it  SIO S  predominantly anisotropic factors. due  intramolecular.  Inductive  effects  e f f e c t s of chemical bonds are the two I f the e l e c t r o n d e n s i t y around  dominant  an atom i s reduced  to the i n d u c t i v e e f f e c t of an a t t a c h e d  atom, resonance  and  electronegative  occurs at a lower value of the a p p l i e d  field  and the nucleus experiences a g r e a t e r d e s h i e l d i n g . The  chemical s h i f t s of the methyl hydrogens of  compounds  are  substituent  X.  dependent on the  electronegativity  From Appendix 8,  methyl  proton change from 0.9  methylene and methine protons, will  be  1.4  and  1.5 ppm  the  protons  from  to 1.1  respectively.  ppm.  to a s u b s t i t u e n t l i k e -C-0-.  suggests  the presence of a CH^-Ar or  t h i s peak i s a s i n g l e t ,  ppm  ppm  shifts  which  are  possibly  The peak at 2.3 a  of  can be assigned  -C-CH C=C-  2  ppm  group.  t h i s means there are not  neighbouring protons a t t a c h e d to the adjacent carbon Thus, the s i n g l e t at 2.3  X  For  In the spectrum  groups  attached  Since  to 1.3  the s m a l l e s t chemical  methyl  the  the chemical s h i f t s of  the unknown compound, the s i g n a l at 1.28 to  of  i t i s c l e a r that when  changes from C to -C-C=C- and to C-0, the  CH^-X  any  atoms.  can only be from a methyl  group  in CH ~Ar. 3  Approximate aromatic  chemical  shifts  l i n k a g e s are i n the 6.0  of protons  to 9.0  1 53  ppm  attached  range  to  (Appendix  8).  The  f o u r t h peak i n the unknown compound i s a t 7.3  ppm  and can be a s s i g n e d t o the aromatic protons i n the molecule, or  more  precisely  involving There  i t may come from  aromatic  i s a small shoulder i n the f o u r t h peak  ppm).  system  a c a r b o n y l group l i k e -CH=C-CO (6.5 t o 8.0 ppm).  residual  due  to the  protons i n the deuterated c h l o r o f o r m s o l v e n t (7.25 Other very small peaks appearing i n the spectrum are  p o s s i b l y due to sample  impurities.  In a proton NMR spectrum, the  an  the area of the s i g n a l s  r e l a t i v e numbers of these d i f f e r e n t types of  Integration  of  numbers  the three  of  the peak areas  shows  types of  that  protons  compound are 1:3:6 f o r the s i g n a l s a t 7.3,  tell  protons.  the  relative  present  i n the  2.3 and 1.25 ppm  respectively. In NMR only  spectrum three  protons 7.3,  summary,  types  and  environments  proton  of t h i s compound i n d i c a t e s that i t possesses of d i f f e r e n t  protons.  has any neighbouring p r o t o n s .  2.3  1.25  None  of  these  The three peaks  ppm can be assigned  as  of -CH=C-CO (within an aromatic  and CH^-C-O-, The  the i n f o r m a t i o n obtained from the  protons  at from  system), CH^-Ar  with the number r a t i o s of 1:3:6 r e s p e c t i v e l y .  proton NMR spectrum  g i v e s only  the protons present i n the molecule.  1 54  information  about  Information about the  carbons present i n the molecule can be recorded i n a carbon13 NMR  spectrum.  The chemical s h i f t  organic  compounds i s 0-230 ppm  roughly  20  shifts, be can  times  e a s i l y recognized. due  ( r e l a t i v e to i n t e r n a l  the range observed f o r  so the d i f f e r e n t  arise  range f o r carbon-13 i n  to  carbon-13  spectra,  decoupling  (or  proton  TMS),  chemical  type of carbons i n the molecule can Complexity  carbon-proton a  i n a carbon-13  spectrum  coupling.  simplify  technique  called  To  heteronuclear  proton noise decoupling) i s normally  Carbon-13 s p e c t r a obtained in t h i s way  used.  contain several  sharp  l i n e s , each of which r e p r e s e n t s a unique type of carbon atom in the molecule. Carbon-13 Broad Band (BB) proton decoupled and Attached Proton  Test  (APT)  s p e c t r a are shown i n  computer a n a l y s i s of the s p e c t r a l The that  carbon-13  BB proton decoupled spectrum  clearly  there are at l e a s t e i g h t carbon atoms i n the  present resonate absence  in in  the 0-60  ppm  16.  shows  molecule  types of carbon atoms 3  In g e n e r a l , range  The  sp  ( r e l a t i v e to  carbon TMS)  atoms in  the  of e l e c t r o n e g a t i v e atoms such as oxygen or halogens.  alkenes  normally  the molecule.  23.  data i s shown i n Table  or that there are only eight d i f f e r e n t  In  Figure  and  resonate  benzenoid a r o m a t i c s , in  the  100-160 1 55  these ppm  carbon  region.  atoms Extreme  F i g u r e 23. Broad Band and Attached Proton of the Unknown Compound.  C 1 3  B B  AND  A P 11  T 80 1  160  Spectra  E X P E R I M E N T S  ' I ' '  APT  Test Carbon-13  100  ' I ' 60  I  40  I  I  I  I  I  I  I  20 PPM  I  I  I  i  i |  0  i  Table  16. Broad Band and Attached Proton Test Spectra Data of the Unknown Compound.  #  CURSOR  FREQ  PPM  1 2 3 4 5 6 7 8 9 10 1 1 12 13  5072 701 7 7203 7998 8040 1 1 046 1 1066 1 1087 1 271 6 1 3829 1 441 7 1 61 36 1 61 46  16897.696 13930.131 13646.385 12432.486 12368.493 7782.546 7750.746 7718.990 5233.955 3535.788 2638.261 15.389 .206  167 .9457 1 38.451 1 1 35.631 0 123 .5661 1 22.9301 77 .3505 77 .0344 76 .7188 62 .0201 35 .1421 26 .221 6 . 1 530 .0021  INTEGRAL  INTENS  .362 2.640 2.528 2.338 2.364 2.222 2.806 2.530 1 .373 1 .322 7.829 .677 10.324  5.717 15.700 16.112 22.892 16.903 16.903 18.086 18.068 16.886 16.893 81.081 6.716 78.630  APT SPECTRUM ANALYSIS: INDEX 1 2 3 4  D Q Q Q  FREQ 10476.4 3937.6 1981.5 12.7  PPM  INTENSITY  138.89 35.20 26.27 .17  NO OF PROTONATED CARBONS: 4 CH CH„ CH,  1 0 3  157  Carbon-13  260.055 17.174 65.493 44.315  polarization  of  the pi-system of a double bond  resonance o u t s i d e these l i m i t s . appear at a very low f i e l d , Voelter,  i . e . 155-230 ppm  cause  resonances  (Breitmaier  and  1974).  The  chemical  resonances 1980). fall  Carbonyl carbon  may  shifts  of  are shown i n Appendix  The  some 9  carbon-13  (Williams and  Fleming,  e i g h t types of carbon i n the unknown  compound  i n t o the three ranges mentioned 3  belong t o sp  typical  carbon atoms.  above.  Three of  them  Four types of carbon are i n the  alkene  and  carbon  i s present i n the c a r b o n y l carbon atom region of the  spectrum. valuable  benzenoid aromatic r e g i o n .  Carbonyl in  Only one  carbon atom resonances  structure  e l u c i d a t i o n since  are they  nature of c a r b o n y l f u n c t i o n to be deduced. and  amines  aldehydes  (ppm  and ketones  g r e a t e r than 180)  1980).  carbonyl  carbon atom i n the molecule  i n an e s t e r ,  In  (ppm  t h i s case,  of  extremely allow  Acids,  the  esters,  l e s s than 180) can be d i f f e r e n t i a t e d  Fleming,  be  type  from  (Williams  the s i n g l e s i g n a l  and  for  (at 169.945 ppm)  a  could  l a c t o n e or a c i d but not i n a ketone or  an  aldehyde. APT and  carbon-13  s p e c t r a are s u p e r i o r to normal  proton decoupled carbon-13  numbers  carbon-13  s p e c t r a f o r determining  of protons bonded to a carbon i n a molecule. 158  the In a  carbon-13  APT  spectrum,  methylene carbons appear carbons  the  and  while the methine and methyl  APT spectrum of the unknown  the s i g n a l s at 76.72,  the  deuterated  normal  i n the  of the data  split  compound  spectrum.  According  (Table 16),  i n t o a t r i p l e t form.  There are f i v e  no carbon Thus,  in  quarternary  the  spectrum  carbons.  are  due  According  to  to  to  other  computer  i n the molecule  there are  methylene carbons present i n the molecule. signals  (Figure  77.03 and 77.35 ppm are due  chloroform solvent.  signals  analysis is  normal  quarternary  are i n v e r t e d .  In 23),  the s i g n a l s f o r  not  A l l five the  any  normal  presence  of  shifts,  the  to chemical 3  s i g n a l a t 62.02 can be assigned as a sp  quarternary  atom.  Three types of carbons with ppm at  122.93,  135.63  are  the  the  quarternary  benzenoid aromatic r e g i o n .  carbons  in  carbon 123.57,  alkene  and  The q u a r t e r n a r y carbon at 167.95  ppm i s a c a r b o n y l type carbon. The  computer a n a l y s i s i n d i c a t e s one carbon  i n t o a doublet and i s found at 138.89 ppm. inverted, the  so  molecule.  benzenoid carbons  is  split  This signal  is  i t i s a s s i g n e d as the only methine carbon  in  This  aromatic  methine  carbon.  carbon Three  is  types  an of  alkene methyl  are assigned a c c o r d i n g to the q u a r t e t s p l i t t i n g 159  or 3 sp in  the s p e c t r a l  data.  26.27 and 0.17 TMS  They are the i n v e r t e d s i g n a l s  ppm.  The  s i g n a l at 0.17  ppm  at 35.20,  i s caused by the  i n t e r n a l standard. One  is  of the most s t r i k i n g f e a t u r e s of carbon-13  the  intensity  intensities  are  d i r e c t l y bonded In NMR  quite  characteristic  resonances. of  carbons  Low without  the i n f o r m a t i o n p r o v i d e d by the carbon-13  makes  i t clear  there are e i g h t d i f f e r e n t three  between  hydrogens.  summary,  spectra  variation  spectra  that i n the  unknown  compound  types of carbons present, but only  types have protons bonded to them.  The  other  five  types of carbons are a l l q u a r t e r n a r y carbons. Gathering the evidence o b t a i n e d from proton and 13 NMR of  spectra,  the  1:1:2  methine  because  i t i s not d i f f i c u l t  to f i n d that the  carbon to two types of methyl  t h e i r proton r a t i o i s 1:3:6.  ten  protons present i n the molecule.  compound  i s symmetric,  fragments  involve  at  least  Evidence from the low shows that t h i s  unknown  and c o u l d t h e r e f o r e be a dimer  compound present d u r i n g i t s formation, such as It  is  Therefore i t can  s a i d that there are at l e a s t nine carbons and  mass s p e c t r a l  ratio  carbons  be  resolution  carbon-  of a  thujaplicins.  i s reasonable to assume the molecule should at l e a s t 2X9  carbons  and  2X10  160  protons.  The  carbon  at  167.9457 ppm acid, or  i n the carbon-13 NMR  spectrum i s a s s i g n e d to an  e s t e r , or l a c t o n e c a r b o n y l carbon but not an aldehyde  ketone c a r b o n y l carbon,  which means that t h i s  carbon bonds d i r e c t l y to two oxygens the compound has a symmetric at l e a s t  four oxygen  carbonyl  i n the molecule.  dimer s t r u c t u r e ,  Since  i t will  have  atoms.  The combination of a l l the above f a c t s give a molecular formula  for  molecular weight  the  weight  unknown of  compound  300.1362  ^i8 20 4  organic  used  in  compounds  1980.  The  (a.m.u.).  mass  high-resolution (Table 17) from  mass deduced Two  This  w  ^ ^ f c  mass  molecular  300,  mass  s p e c t r a were  for of  Fleming,  collected  determine the molecular weight of the  exactly  resolution  and  which i s the same as  of these s p e c t r a i s shown i n F i g u r e 24.  resolution.  spectrometry  Williams  a  low the  from the above s t r u c t u r a l d a t a .  high-resolution  accurately  wieght  O  of the molecular ion o b t a i n e d i n the  r e s o l u t i o n mass spectrum was  has  H  i s c a l c u l a t e d based on a t a b l e of atomic weights  isotopes  One  of  the The  same  pattern  molecular  as  those  The done  weights given by the  s p e c t r a are s l i g h t l y d i f f e r e n t .  One  to  compound. spectrum at  low  two  high  molecular  i s 300.1368 and the other i s 300.2075, which i s 0.02%  higher than the expected molecular weight.  161  The composition  Table 17. Atomic Weight of Isotopes Used Spectroscopy.  Isotope  Atomic Weight (C =12.000000) 12  i n High R e s o l u t i o n Mass  N a t u r a l Abundance (percent)  H  1.007825  99.985  H  2.011402  0.015  1 2  C  12.000000  98.9  1 3  C  13.003354  1.1  1 4  N  14.003074  99.64  1 5  N  15.000108  0.36  1 6  0  15.994915  1 7  0  16.999133  0.04  1 8  0  17.999160  0.2  1  2  99.8  Source: Willams and'Fleming, (1980).  1 62  100  9B  F i g u r e 24.  BB 7B  High R e s o l u t i o n Mass Spectrum of t h e Unknown Compound.  SB 50 40 30 20 10 0  I I I I I | M'l  260  " ... TW rrr n.f i f111111 280  m  II I I I I J II IT T T  I | I II  300  320  340  II I I  | I M I I I I I I | I I I I II I I I | I I I I I I I I I | I I M I I I I I [ I I I  360  380  400  420  440  TT  from The  exact i s o t o p i c weights  ^18 20°4" H  fragments appearing i n the two s p e c t r a at 149.0594  149.0609  best  composition  same  match  a  CgHgO,,.  symmetric,  mass of This  149.0602  clearly  which  has  and the  demonstrates  the  d i m e r i c s t r u c t u r e of the unknown compound.  The  fragments p r e v i o u s l y d i s c u s s e d with respect to the low  resolution  mass  the  high  If the i n f o r m a t i o n obtained from both mass s p e c t r a  and  resolution  NMR  f o r mass 300.1368 i s  were a l s o observed  in  spectra.  spectra  compound  spectra  is  correct,  the  molecular  weight  of  this  i s 300.1368 and the molecular formula i s ^13 20^4" H  According t o the molecular formula, the r a t i o of the numbers of  carbons  amount  to hydrogens  i n the  of carbon and hydrogen  expected  to  molecule  contained  is  18:20.  i n the molecule i s  be 71.89% and 6.71% of the  molecular  weight.  R e s u l t s of micro-elemental a n a l y s i s on a c r y s t a l i z e d the and  form of  unknown compound showed that the percentages of hydrogen  respectively.  in  the  The  molecule  are  The  72.14%  carbon  and  d i f f e r e n c e beteween found and  6.78%, expected  values are +0.16% f o r carbon and +0.07% f o r hydrogen, are  both  analysis  acceptable (maximum  according to  2%).  This result  previous conclusions.  164  the  precision  which of  the  s t r o n g l y confirmed the  The  information  unknown mass  compound  units  structure  so  f a r proves  that  possesses a molecular weight of  with of  outlined  a molecular  formula  as  t h i s compound appears as a  From sapwood bioassay r e s u l t s ,  C  300.1362  18 20°4" H  i t has been demonstrated  reasonable  to assume that the symmetric  structure  p o s s i b l y c o n t a i n s the dimer  e  that It i s  of  of  ^  dimer.  i s a p r e c u r s o r of t h i s unknown compound.  compound  T  symmetric  thujaplicin  unknown  this  this  modified  thujaplic ins. The number of double bonds and r i n g s i n the molecule i s given by the equation: C^^O  double bond e q u i v a l e n t s (DBE) (2a  + 2) - b  DBE =  . (Williams and Fleming, 1980)  2 The  (2a  saturated or the is  +  2) term i s the number of  hydrogens  hydrocarbon having 'a' carbon atoms.  double bond means two fewer hydrogen  atoms,  in a  Every while  a c t u a l number of hydrogen atoms present from (2a + subtracted.  double  D i v i d i n g by two g i v e s the t o t a l number  bonds and r i n g s i n the molecule.  compound,  DBE = 9 ,  In  this  ring 'b', 2), of  unknown  so the t o t a l number of double bonds p l u s  rings i s nine. There  are  dozens of s t r u c t u r a l 165  isomers  of  compounds  with  the  molecular  formula  i n f o r m a t i o n obtained,  C  ig 20°4*  Based on  H  the most reasonable  a l l the  s t r u c t u r e so f a r  i s shown i n Figure 25. UV,  visible  and  IR s p e c t r a were obtained  further  examine  accuracy  UV  and  visible  s p e c t r a of the compound are shown i n F i g u r e 26  and  27.  of the proposed s t r u c t u r e .  to The  The p o s i t i o n (285 nm) of the strong UV a b s o r p t i o n peak  indicates  an  extended system of  carbonyl  group.  This  proposed  from other  usually  gives  rise  to  compounds  occurs  appears  at  probably  due to t h i s  The  415 nm  structure *  The n t o p i peak  i n the v i s i b l e 340-440 nm region  and  color  and Fleming, i n the  unknown  of  1980).  some  of  these  The peak  which  compound  spectrum  is  transition.  FTIR spectrum of t h i s unknown compound i s shown i n  F i g u r e 28.  The s p e c t r a l data are l i s t e d  evidence  provided  Selected  standard  absorption  a  or quinones w i l l give a second  the l i g h t yellow  (Williams  including  i s i n agreement with the  s p e c t r o s c o p i c evidence.  t r a n s i t i o n of diketones which  conjugation  i n Table  by the IR spectrum i s q u i t e  18.  The  conclusive.  carbon-hydrogen a b s o r p t i o n band and  band data are given  i n Appendix 10 and 11  X-H  (Pasto  and Johnson, 1969). The  oxygen-hydrogen s t r e t c h i n g a b s o r p t i o n 166  of a l c o h o l s ,  Figure  25.  Chemical  S t r u c t u r e of " T h u j i n " .  molecular  foumula:  m o l e c u l a r weight:  c  s 20°4 H  1  300.1362 (a.m.u.)  3,3,4,7,7,8-hexamethy1-2,6,dioxa-1,5-anthracenedione  167  168  F i g u r e 28. F o u r i e r Transform IR Spectrum of the Unknown Compound.  3800. Q  3200. O  26QO. O  2QOO. D  1700. O  WAVENUMQERS  140Q. O <CM-l>  1 IOO. Q  BOO.OO  SOO. OO  200. OO  Table 18. Fourier Transform Compound.  Peak no.  IR  S p e c t r a l Data  Wavenumbers  1  3016  2  2962  3  2878  4  1733  5  1599  6  1574  7  1500  8  1459  9  1381  10  1261  11  1192  12  1126  13  1076  14 15  745 721  171  of  (cm  )  the  Unknown  phenols, appears 3650  and as  to  i n t e r - or  hydrogen  bonding  a broad band with a maximum a b s o r p t i o n  i n the  3200  cm  intra-molecular  range.  1  Only i n the case  of  oxygen-  hydrogen s t r e t c h i n g of c a r b o x y l i c a c i d s w i l l the be  out of t h i s r e g i o n ,  maximum down  nearly  intermolecular The  appearing as a very broad band with  a b s o r p t i o n at approximately to  2500  absorption  cm  hydrogen  owing  1  2940 to  cm ,  extending  1  the  bonding between  very  carbonyl  strong groups.  lack of any a b s o r p t i o n peaks i n the IR spectrum of the  unknown compound i n t h i s region emphasizes the absence of an OH group i n the s t r u c t u r e . Carbon-hydrogen  a b s o r p t i o n occurs i n two r e g i o n s ,  C-H s t r e t c h i n g region from 3300 to 2500 cm region from 1550 to 650 cm . 1  to  and the bonding  The methyl group g i v e s  rise  two s t r e t c h i n g bands which g e n e r a l l y occur at 2960  and  2870  cm . 1  of  2962 cm  and 2878 cm .  the  1  the  unknown 1  asymmetric  and  compound  to  symmetric  a  stretching  region  1  1  FTIR  wavelength  of to  deformation  Methyl groups a l s o give  two peaks i n the asymmetric and symmetric  deformation region near 1460 cm 1  at  These two bands a r e a s s i g n e d  i n v o l v i n g the e n t i r e methyl group. rise  cm  These two peaks a r e observed i n the  spectrum  cm  1  the  and 1380 cm . 1  The  bending 1380  i s extremely v a l u a b l e f o r d e t e c t i n g presence of  1 72  methyl groups because t h i s r e g i o n i s almost  devoid of  types  of a b s o r p t i o n bands (Pasto and Johnson,  peaks  are observed  at  1459  cm  i n the spectrum  and  1  deformations.  The  1381  1  1969).  of the unknown  corresponding  Two  compound  to  these  t y p i c a l a b s o r p t i o n bands corresponding to  methylene ( C r ^ )  alkyl  cm  other  and a l k y l methine (CH) were not  found  in the spectrum. Carbon-carbon extremely  variable  intensity. than  1400  single  bond  stretching  1  (Fessenden,  i n p o s i t i o n and are u s u a l l y q u i t e weak in  ,  which i s a l s o c a l l e d the f i n g e r p r i n t region  1982).  In the f i n g e r p r i n t  r e g i o n , the  i s o f t e n q u i t e complex and c o r r e l a t i o n of an with a s p e c i f i c  the  1969).  determinations.  cm  1  r e g i o n (Appendix  In the spectrum  peaks with wavelengths of 1599, this  region.  vibrations  It  little  double bond s t r e t c h i n g a b s o r p t i o n occurs  2000 to 1430  Johnson,  be made with  these a b s o r p t i o n bands are of  in s t r u c t u r a l  Carbon-carbon  spectrum  i n d i v i d u a l band  f u n c t i o n a l group u s u a l l y cannot  Therefore,  p r a c t i c a l use  in  are  They u s u a l l y appear i n the wavelength range l e s s cm  accuracy.  bands  is  obtained,  1574,  w e l l known  12)  1500,  that  (Pasto  and  there are four and  the  1459  cm  skeletal  1  in C=C  of aromatics g i v e r i s e to a s e r i e s of four bands  in t h i s r e g i o n .  These bands occur very c l o s e to 1600,  173  1580,  1500 in  and  1450  cm  (Appendix 13).  intensity.  pattern  The  observed  -C=C-  presence  conjugated  1  .  of a  a b s o r p t i o n data.  closely  These peaks prove the  s t r u c t u r e i n the  molecule.  a r i s i n g from the peak i s observed  Appendix  for  somewhere  group. between  This 1640  of compounds that c o n t a i n the  peak 1733  appears cm  1  in  the  unknown  usually and  13).-  Other  cm  aldehydes  OO  1820  nearby and  1710  peaks cm  1  group.  compound  , which i s a c h a r a c t e r i s t i c  an a c y c l i c e s t e r or a six-membered  (Appendix for  carbonyl  13. l i s t s the band p o s i t i o n s f o r a number of  types  spectrum at  1  absorption  one  carbonyl  peak  the  of the most d i s t i n c t i v e bands i n an IR spectrum i s  different The  and  One  strong cm  IR data  of the unknown compound i n t h i s region very  match aromatic  the  These bands vary g r e a t l y  carbonyl  ring  lactone  i n t h i s area are for  carboxylic  Since there are no OH a b s o r p t i o n s  1725  acids  (Appendix  13).  spectrum,  the p o s s i b i l i t y of the c a r b o n y l peak being due  a carboxylic acid i s eliminated.  The  carbon-13 NMR  IR  in  the to  spectrum  of the unknown compound has c l e a r l y demonstrated the l a c k of aldehyde and all,  the  ketone c a r b o n y l groups i n the s t r u c t u r e .  1733  cm  lactone carbonyl, c a r b o n y l i n the  1  Above  a b s o r p t i o n band i s c l o s e s t to that f o r a so t h i s band can be assigned to a l a c t o n e  molecule.  174  In of  summary,  evidence o b t a i n e d from UV and FTIR  the unknown compound f u l l y  structure.  s u p p o r t s the proposed chemical  E l u c i d a t i n g chemical s t r u c t u r e of t h i s  by j o i n t a p p l i c a t i o n MS,  NMR  spectra  compound  (proton and carbon-13),  FTIR,  and  UV s p e c t r o s c o p y p r o v i d e s c o n c l u s i v e evidence l e a d i n g to  the  final  proposed A compound •through previous Two  of  i d e n t i f i c a t i o n of the unknown  compound  as  the  structure. literature was  search f o r any p r e v i o u s r e p o r t  c a r r i e d out by s e a r c h i n g  the  period  from  information the c l o s e s t  Chemical  1920 t o the  end  of  about t h i s compound has structurally  on  related  this  Abstracts 1985.  been  No  found.  compounds  found  were:  (Tamura According Applied of  the  al ,  1974)  International  Union  of  Pure  and  Chemistry (IUPAC) d e f i n i t i v e r u l e s f o r nomenclature  organic  named  to  et  compounds (Weast,  1978) t h i s compound  can  be  3,3,4,7,7,8-hexamethyl-2,6-dioxa-1,5-anthracenedione.  175  A  trivial  name of 'Thujin' was given to t h i s  compound  (as  suggested by Drs. Swan and Wilson) based on i t s source (from Thuja  plicata  Donn. and t h u j a p l i c i n )  176  (Figure 25).  4.5.  Mechanisms of T h u j a p l i c i n ' s  It  is  well  extractives  recognized  exhibit  Poria  common  decay  coastal  area  to  Poria  al bi pel  I uci da  (Buckland,  Baxter.  I uci da  was  tested  to  the  Obviously, t h u j a p l i c i n  functional toxicity.  beta-thujaplicin experiment (Raa  been  tested  and  hypothesis, concentration test  i s toxic  was  is  Activity has  to t h i s  reactive  of  the  for  the  1965).  (5  thujaplicin  methylated by  etheral  study.  thujaplicin these s i x t e e n Figure  responsible  keto-enolic  before,  in  in  an  thujaplicin  to  with  the  never  At  this  highest  thujaplicin  diazomethane.  is for  group  In order to t e s t  solution  1 77  16.  k e t o - e n o l i c group  T h i s assumption has  mg/ml) used i n the  the  fungus.  t o x i c i t y of  for wood decay f u n g i .  most  thujaplicin this  in  only been t e s t e d once  Goksoyr,  the  shown  group in t h u j a p l i c i n s  conducted  yeast  t h u j a p l i c i n at  fungus  i s b e l i e v e d that the  in  c o n c e n t r a t i o n s of  fungal i n h i b i t i o n of  fungi  t r e e s in  Baxter.  The  fungal  from l i v i n g WRC  t o x i c i t y of  used.  key  WRC  i s c o n s i d e r e d the  The  were s i x t e e n d i f f e r e n t  the  in  (Rennerfelt,  1946).  There  concentrations  thujaplicins  timber i n s e r v i c e  fungus i s o l a t e d  al bi pel  It  that  a strong t o x i c i t y to f i n a l decay  which a t t a c k l i v i n g t r e e s and 1948).  Toxicity  toxicity 5  mg/ml,  thujaplicin's Baxter.  inhibition  was  very  strong.  methylated t h u j a p l i c i n 17C.  The  results  to the  of  methylated t h u j a p l i c i n it  can  be  effect  The  positively  no  al bipel  inhibiting  test  indicate  concluded  that  certain  groups  presence  the  reactive  k e t o - e n o l i c group.  disappear i f the  group i s blocked, as As  would  a c t i v i t y of  i s done by  heartwood by  convert the  which has to Poria was  of  fungi  the  thujaplicins  I uci da  Baxter.  and  thujin,  first  to a new  was  The  which was  were  t e s t e d by  17A,  it  but  thujin  of t h u j i n with much  the  the  keto-enolic  same  The  At  higher  the  amount  178  of sp.  (thujin) thujin  experiment  obtained  present s i g n i f i c a n t l y i n h i b i t e d show any  infection  t o x i c i t y of  from  of the  same time,  concentrations  same method (Figure 17B).  present d i d not  of  toxicity  inhibiting effects  solutions  thujaplicin  The  tested.  four  found that i n the  to  compound  amount of wood sample (Figure 17A).  is  due  fungus Sporothrix  same  the  is  trees,  based on a comparison between the  beta-thujaplicin  that  methylation.  s t r u c t u r e as demonstrated. al bipel  of  toxicity  this reactive  discussed e a r l i e r , in l i v i n g  straw-colored  clearly  the  to  will  effect  da  t o x i c i t y to t h i s fungus, thus,  thujaplicin of  I uci  same fungus i s shown i n F i g u r e  this has  Poria  to  From of  wood,  fungal  inhibition.  Figure the  growth,  It might  be  argued, based on the r e s u l t of F i g u r e i s not t o x i c at t h i s c o n c e n t r a t i o n fungus  at  Figure  17B  of  higher  used  concentrations.  in F i g u r e  17B  c o n c e n t r a t i o n of t h u j a p l i c i n  can be concluded  from  The  results The  were much  is  l o s t when the t h u j a p l i c i n  the Sporothrix the  sp.  in  concentrations  higher  thujaplicin thujaplicin No  of  than  the  keto-enolic  were  a c t u a l biochemical thujaplicin  involves group  in  isomerization  made i n t h i s study  to  in t h i s c o n v e r s i o n  isolate process,  pathway f o r the formation  during  the Sporothrix  sp.  unknown.  Based  products,  the p r o p e r t i e s of the compounds and  biochemical  clear the the of  to a l a c t o n e type compound.  the intermediates  of  during  i t is  detoxification  reactive  Baxter,  to t h u j i n  by o x i d a t i v e d i m e r i z a t i o n and  attempts  identify  I uci da  Structurally,  of t h i s  the  al bipel  i s converted  infection.  mechanism  deactivation  nature  given  t h i s set of experiments that  to Poria  t o x i c i t y of t h u j a p l i c i n  from  thujin  used i n the experiment shown in  the  the  that  17A.  It  that  alone,  but might be t o x i c to the  e l i m i n a t e d t h i s assumption.  thujin  Figure  17A  on s t r u c t u r e s of the s t a r t i n g  these processes, pathway can be  however,  suggested.  179  one  of  so  thujin  infection and  and  is  ending  the enzymatic  of the  possible  When  t h e s t a r t i n g compound i s b e t a - t h u j a p l i c i n ,  the  f o l l o w i n g may o c c u r .  When  t h e s t a r t i n g compound i s g a m m a - t h u j a p l i c i n ,  f o l l o w i n g may o c c u r .  180  Cl,  the  The  postulated  formation  biochemical processes, unlike under c o n t r o l l e d feature  of  substrate  synthetic  activity  specificity,  in  is  catalytic  specificity  which  that  their  high  further  and  of  is  processes its  enzymatic  of  only  the concomitant  necessary  the only way  to  and  specific  p r o d u c t i o n of  i d e n t i f i c a t i o n of  involved steps  is  biological  ensure s y n t h e s i s  in  the  before  the n a t u r a l processes can  research  outstanding  substrate  intermediates be  An  of  T h e r e f o r e , the i s o l a t i o n and  knowledge  reactions  degree  byproducts.  would  organic  f e a t u r e of  products without  processes  enzymatic  determines  biomolecular  enzymes  by  biological  Another c r i t i c a l b i o l o g i c a l  reactions  the  is  conditions in a laboratory.  enzyme  function.  of t h u j i n  be  above  a  greater  gained.  test  the  Also,  proposed  pathway. Although during  the  the exact pathway of t h i s process dimerization  thujaplicins  change  s t r u c t u r e s to the required  by  energy, the  two  most  fungi,  and  of  from  thujaplicin stressed  to  seven  more s t a b l e s i x member r i n g  s t o i c h i o m e t r y that atoms of carbon  important  in  this  are r e l e a s e d .  essential  i s unknown, thujin,  carbon  ring  form.  It i s  process,  beside  Carbon i s one  elements f o r the  growth  i s r e q u i r e d i n g r e a t e r q u a n t i t i e s than any  181  the  of of  other  essential  element by f u n g i .  rather  important,  behind  the Sporothrix  carbon  to support  Thus, t h i s r e l e a s e of carbon i s  since i t implies a sp.  infection  i t growth.  182  possible  motivation  process i s to generate  4.6.  Future  This  perspectives  study  demonstrated the b i o l o g i c a l  three e a r l y stage a t t a c k i n g fungi on WRC trees,  particularly  the  mechanism of Sporothrix by  sp.  Sporothrix  of  is  supported  measurement  evidence.  of t h u j i n showed,  however,  60% and  45%  (from L-0  to D-0  are the remaining  explanation  On  the  resulted heartwood.  from  quantitative  that  concentration  (D-O)  and  in b i o d e g r a d a t i o n  toluene  an  0.187% i n  noticable  with  One  important  unexpected of  by t h i s work.  The  These metal  time from WRC  finding  discolored  were  chelates  heartwood  WRC  chelate not were  extractives  v e r i f i c a t i o n of the metal c h e l a t i n g  183  the  processes.  i n d i s c o l o r e d heartwood which  f o r the f i r s t  of  t h u j a p l i c i n p o r t i o n s are  extraction  BE s o l v e n t .  during  to D-I r e s p e c t i v e l y ) .  There i s a c e r t a i n amount of t h u j a p l i c i n present  about  disappeared  t h u j a p l i c i n portions?  other hand,  compounds  isolated  and L-I  could be that remaining  the intermediates  l a c t o n e compound,  T h i s accounted f o r  of the t h u j a p l i c i n s which had  discoloration Where  (D-I).  The  thujaplicins  The  0.291% i n d i s c o l o r e d outer heartwood  d i s c o l o r e d inner heartwood  sp.  d e t o x i f i c a t i o n of  by  of  heartwood i n l i v i n g  c o n v e r t i n g t h u j a p l i c i n s to a dimerized  thujin,  was  role  functions  was  achieved  by c a r r y i n g out a s e r i e s of r e a c t i o n s and  finally  i d e n t i f y i n g FeS (dark black-green c o l o r e d p r e c i p i t a t e ) , and thujaplicins  by  TLC  as  products.  Another  alternative  explaination  f o r t h u j a p l i c i n l o s s i s that during  heartwood  d i s c o l o r a t i o n , accompanying the a c t i v e accumulation of metal ions  ( S a f f o r d and Shigo,  1974), a p o r t i o n of t h u j a p l i c i n s i n  WRC heartwood form c h e l a t e s with metal i o n s . the  The source of  metal ions and the i n f l u e n c e of these metal c h e l a t e s on  the e n t i r e microorganism a t t a c k i n g process and  consequently  decay r e s i s t a n c e would make an i n t e r e s t i n g research The  due  mechanism of t h u j a p l i c i n t o x i c i t y t o decay  as Poria  such  al bipel  I uci da Baxter.,  t o a c t i v i t y of the k e t o - e n o l i c  thujaplicin  properties,  several  They  involve p o s s i b i l i t i e s  with  metal  associated  ions with  thujaplicin  fungal  salts of  fungal p r o t e i n . fungal  inside  group  in thujaplicins.  with  hydrogen  modes can  f o r formation the  wood,  enzymes,  or  amino-acids bonding  be  of metal  or  with  between  the  184  chelates  metal  formation of  Based on proposed.  of  ions amine  fungi,  or  thujaplicins  and  Any or a l l of these a c t i o n s would  growth.  fungi,  has been proven t o be  D e t a i l e d modes of a c t i o n , however, remain unknown.  formation  topic.  inhibit  CONCLUSIONS  Thujaplicins resistance within  of  which  WRC,  l i v i n g WRC  attacking (Peck)  trees.  In t h i s study,  fungal  attacking  pattern.  first  fungus  attacking  sound  alone  did  new  toxic  study  can  sound  dimerization  antifungal selective.  wood d e s t r y i n g toxic  and  to decay f u n g i ,  highly  stage  t huj i na  sp.)  fungi,  in  WRC  indicated sp.  was  the  Sporothrix  heartwood. weight l o s s  of  wood  a  sp. blocks  t o x i c i t y of t h u j a p l i c i n s to decay  WRC  interaction  heartwood  and i s o m e r i z a t i o n  resulted  such as  Poria  between in  an  of t h u j a p l i c i n s to  which was proven to be no al bipel  I uci da  longer Baxter.  a c t i v i t y of t h u j a p l i c i n s are shown to On one hand,  they are very t o x i c to  on the other hand,  to some "pioneer" f u n g i ,  even d e t o x i c i f y  Sporothrix  demonstrated that  l a c t o n e compound,  Therefore, be  but d i d remove  sp.  oxidative a  not cause s e r i o u s  This  Sporothrix  three e a r l y  d i s t r i b u t i o n of the fungi  clear  fungi.  existing  Ki r s chest er ni el I a  sp. ,  decay  heartwood were i s o l a t e d and i d e n t i f i e d . frequency  directly,  fungi  & E t h e r i d g e and Phialophora  Pomerleau  The  i n t e r a c t with v a r i o u s  (Sporothrix  fungi  discolored  are r e s p o n s i b l e f o r n a t u r a l  they are not very  l i k e Sporothrix  thujaplicins.  185  sp.,  which  It has  been demonstrated that the chemical mechanism of  thujaplicin of  t o x i c i t y to decay fungi  the r e a c t i v e k e t o - e n o l i c  i s due  group.  to the  T o x i c i t y disappears i f  the a c t i v i t y of t h i s r e a c t i v e group i s blocked, in  t h i s study by methylation  also  altered  thujaplicin's Sporothrix  of the group.  Sporothrix  by  antifungal sp.  sp.,  as was  The  is  removing  mechanism  thujaplicins  s t r u c t u r a l l y , d e a c t i v a t i o n of the  done  T h i s group  thereby,  activity.  detoxifying  presence  of  involves,  reactive keto-enolic  group  of  t h u j a p l i c i n s by o x i d a t i v e d i m e r i z a t i o n  and  isomerization  of  it  to  its  to a lactone compound.  structure,  this  lactone  According  compound  is  named  hexamethyl-2,6-dioxa-1,5-anthracene-dione. of  t h u j i n i s a l s o given.  of t h u j i n i s achieved chromatographic and  The  by the  3,3,4,7,7,8-  A trivial  i s o l a t i o n and  chemical,  methods.  R e l a t i o n s h i p between the three e a r l y a t t a c k i n g established  in  t h i s study.  between Sporothrix  sp. and K.  much higher sp. other  A strong thujina  toxic  different  compounds mechanisms.  in  WRC  synergistic  sp. and  thujina heartwood  Hence,  186  fungi i s effect  i s evidenced, which i s  than that between Sporothrix  It i s a l s o observed that K.  name  identification  j o i n t a p p l i c a t i o n of  spectroscopic  chemical  may  Phialophora  i n t e r a c t with  extractives  sequential  attacks  by by  Sporothrix  sp. and then K.  essential  step  thujaplicins  thujina  i n l i v i n g WRC  for destroying  natural  t r e e s i s an  toxins  such  i n WRC heartwood and l e a d i n g t o f u r t h e r  as  attack  by decay f u n g i . 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McGraw-Hill, London, pp. 75-151.  Wise, L. New Y o r k .  E . 1 9 4 4 . Wood pp. 661.  Heather. 1983. Influence invading the stems of 37:163-166.  over  Chemistry.  of wood Eucalyptus  Second E d . Reinhold  Publ.  Wright, J . , Smith, D. G . , M c l n n e s , A . G . and L. C . V i n i n g . 1969. Use of C in biosynthetic studies. Incorporation of acetate and formate into the fungal tropolone sepedonin. Can. J . Biochem. 47:943-949. Zabel, R. A. 1948. white oak. N. Y. State  V a r i a t i o n s i n the decay r e s i s t a n c e of College F o r . Tech. Publ. No. 68:53.  Zavarin, E . , Smith, R. M. and A . B. Anderson. chromatography o f t h e t r o p o l o n e s o f Cupressaceae Chem. 24:1318-1321.  200  1959. Paper II. J .O r g .  Appendix 1. List Fomes  of Synonyrni  annosus  f o r the Fungi Used i n the Text.  (Fr.) Karst. Heterobasi di on annosum  Fomes  nigrol  Fomes  pi ni (Thore) L l o y d . Phellinus pini  Fomes  Polyporus  Poria Poria Poria  imi t at us (Romell) E g e l . Phellinus ni gr ol i mi t at us (Rom.) B. & C . (Fr.) Karst.  pi ni col a (sw) Cke. Fomitopsis pi ni col a ( F r . ) K a r s t .  Polyporus  Poria  (Fr.) Bref.  balsameus Peck. Tyromyces balsameus schweinitzii Phaeolus  Fr. schweinitzii  al bipe11 uci da Baxter. Poria rivulosa a s i a t i c a Overh. Tyromyces monticola weirii  Murr. Tyromyces  (Peck) Murr. (Fr.) Pat.  (B. & C.)  seri ceomolIis placenta  Murr. PhelI i nus w e i r i i  Cooke. Rom.  ( F r . ) Ryv. Murr.  201  Appendix 2. T h u j a p l i c i n Content from Old Growth WRC by C o l o r i m e t r i c Method. Sample  Radius from pith,mm  Ave. age, yr.  Thujaplicin (o-d wood)  Tree 1 butt height  38 76 11 4 1 52 190 228 267 305 343 381  26.0 77.0 128.0 180.0 231 .0 282.0 334.0 385.0 436.0 488.0  0.000 0.002 0.003 0. 1 30 0.440 0.620 0.830 1 . 1 30 1.150 1 .220  Tree 2 butt height  1 52 1 90 228 267 305 343 381 419 457  197.0 253.0 309.0 366.0 422.0 478.0 534.0 591 .0 647.0  0.009 0.013 0.014 0.019 0. 120 0.580 0.610 . 0.610 0.670  .  91 .0 111.0 135.0 161.0 191.0 218.0 255.0 284.0  0.110 0. 150 0.510 0.410 0.500 0.580 0.600 0.630  7.0 19.0 40.0 60.0 101.0 139.0 202.0  0.001 0.007 0.011 0.036 0.036 0.360 0.520  Tree 3 breast height  Tree 3 33% height  Source: MacLean and Gardner,  (1956b). 202  Appendix 3. T h u j a p l i c i n Content from Old Growth WRC by Gas Chromatography Method. Sample  Average rings from pith  Total thuja pi ic i %  Sample  #5  #1  20.0 70.0 1 30.0 1 90.0 235.0 262.0 280.0  0.034 0.337 0.460 0.543 0.564 0.655 0. 100  #2  62.0 184.0 289.0 379.0 469.0 544.0 587.0 615.0  0.248 0.602 0.867 0.852 0.970 0.543 0.974 0.057  40.0 1 25.0 210.0 310.0 410.0 475.0 558.0 610.0  0.068 0.377 0.352 0.763 0.865 0.747 0.919 0.051  50.0 1 25.0 295.0 385.0 475.0 565.0 640.0 683.0 703.0  0.320 0.706 0.690 0.570 0.622 0.377 0.575 0.621 0.015  #3  #4  Source: N a u l t ,  #6  #7  (1986). 203  Average rings from pith  Total thuja pi ic i  67.0 1 79.0 254.0 314.0 389.0 464.0 509.0 539.0 566.0  0.024 0.238 0.352 0.392 0.651 1 .774 1 .742 0.707 0.072  20.0 70.0 1 30.0 175.0 205.0 238.0 250.0  0.014 0.333 0.607 0.840 1 .400 1.315 0. 125  15.0 90.0 195.0 270.0 330.0 375.0 383.0 412.0  0.040 0.044 0.093 0.020 0.004 0.121 0.278 0.032  %  Appendix 4. T h u j a p l i c i n Content from Second Growth WRC by Gas Chromatography Method. Sample  Average rings from pith  Total thujaplicin, %  Sample  Average rings from pith  Total thujaplicin, %  #1-1  2.5 7.5 12.5 17.5 25.0 35.0 45.0  0.014 0.011 0.058 0.145 0.280 0.323 0.091  #2-1  12.5 27.5 32.5 37.5 42.0 50.0 60.0  0.043 0.097 0.161 0.244 0.467 0.406 0.436  #1-2  2.5 7.5 12.5 17.5 25.0 35.0  0.086 0.086 0.108 0.081 0.034 0.058  #2-2  5.0 12.5 17.5 25.0 35.0 45.0  0.099 0.134 0.186 0.249 0.415 0.315  #1-3  2.5 7.5 12.5 17.5 25.0 35.5 45.5  0.021 0.058 0.060 0.111 0.190 0.366 0.224  #2-3  12.5 27.5 32.5 37.5 42.5 50.0 60.0  0.033 0.046 0.080 0.205 0.188 0.186 0.327  #1-4  2.5 7.5 12.5 17.5 25.0 35.0 45.0  0.035 0.070 0.077 0.060 0.264 0.525 0.156  #2-4  2.5 7.5 12.5 17.5 25.0 35.0  0.058 0.007 0.030 0.130 0.154 0.169  #1-5  10.0 22.5 32.5 37.5 45.0 55.0  0.022 0.058 0.169 0.376 0.348 0.426  #2-5  30.0 62.5 67.5 72.5  0.099 0.213 0.157 0.250  Source: Nault, (1986). 204  A p p e n d i x 5.  CHECK  LIST OF F U N G I C O L L E C T E D ON W E S T E R N R E D CEDAR B R I T I S H C O L U M B I A F R O M 1943 T O 1945  Species Armillaria  Collybia  auricularis  Corlicium Corlicium Corlicium Corlicium Corlicium Corlicium Corlicium Corlicium  (Gray) M a r t i n  18*  13  betulae (Schum.) K a r s t . puteana ( S c h u m . ex F r . ) K a r s t . suffocala (Peck) Massee  13 16 16  sp. bicolor P e c k cebennense B o u r d . coronilla H o h n . livido-cacruleum Karst.. racemosum B u r t . radiosum F r . sidphureum (Pers. ex F r . ) F r . herbarum P e c k  Flammula Flammula  decorala M u r r . liqiiiritiac (Weinm.) Quel.  1,3, 3 3 3 1,3 12 12 1  7,  11  17 17 11  applanatus (Pers.) W a l l r . annosus (Fr.) C k e . nigrolimitatus (Romell) Egel. Pini (Thore) L l o y d and v a r . abielis K a r s t .  Fomes pinicola  (Swartz) C k e .  Gloeocyslidium  ochroleucum  3 1,3, 7, 11  5,  Bres.  7,9  9, 11, 12,  1,2,  13,  15  10  1  sp.  Hypholoma Hypholoma  13,  12  Crcpidolus  Helvetia  12,  sp.  Coniophora Coniophora Coniophora  Fomes Fomes Fomes Fomes  L o c a l i t y of collections  mellea ( F r . ) Q u e l .  Auricularia  IN  3  fasiculare (Fr.) Q u e l . capnoides ( F r . ) Q u e l .  3 1  IJyynenochaele fidiginosa (Pers.) B r e s . llymenochaete tabacina (Sow.) L e v .  13 1, 3, 5, 12,  Lenziles saepiaria  16  (Wulf.) Fr.  * The numbers refer lo localities on the map  205  13,  18  Appendix  5.  (continued)  Species  Locality of collections  Mnnixmius scorodonius Fr.  3  Mcmlius Jugnx Fr.  1  Mycena griseiconica KaufTm.  5  Odonlia sp. Odontic alutacea (Fr.) B. & G. Odonlia alutacea subsp. flocctisa B. & G.? Odonlia aspera (Fr.) Bourd. Odonlia lactea Karst.  1 . 3 , 11 1 16 9 12, 13  Omphalia campanella (Fr.) Quel.  4, 7, 8, 12, 15, 18  Peniophora sp. Peniophora crassa Burt. Peninphora Jlavo-ferruginea Karst. Peniophora san guinea (Fr.) Hres. Peniophora vcluiina (DC) Ckc?  j  Phlchia mellea Overh.  13  Polyporus ahietinus (Dicks.) Fr. Polyporus bnlsameus Peck Polyporus cacsius (Sclirail.) Fr. Polyporus cuneaius (Murr.) Overh. Polyporus dichrous Fr. Polyporus elcgans (Bull.) Fr. Polyporus hirsulns (Wulf.) Fr. Polyporus immitis Peck Polyporus perennis (L.) Fr. Polyporus Schweinitzii Fr. Polyporus sewipileatns Peck Polyporus undosus Peck Polyporus versicolor (L.) Fr.  13, 12, 19 5, 13, 3, 16, 10 5 5 13, 1  Poria sp. Poria alhipellucida Baxter Poria asiatica (Pilat) Overh. Poria candidissima (Schw.) Cke. Poria isabellina (Fr.) Overh. Poria lenis Karst. Poria nigrcscens Bres. Poria sericeo-tnollis (Komcll) Baxter Poria sinuosa (Fr.) Sacc. Poria subacida (Peck) Sacc. Poria Weirii Murr.  1, 13 1, 2, 3, 4, 5, 6. 7, 8, 11, 13 1, 4, 5, 6, 7, 8, 12, 14, 15, 18, 19 1 1, S, 6 1 11 1 11 1 , 2 , 3 , 5, 7, 11, 12, 13, 18 1 , 3 . 4 , 8 , 9, 10. 12, 13. 15, 17, 18  Psathyrella sp.  11  15, 18 15, 17 7, 11, 12, 13, 14, 15, 17, 18 16, 18 12 18  15, 18  5  Schizophyllum commune Fr.  1  Stereum Chailletii Pers. Stereum rugosiusculum B. & C. Stereum sanguinolenlum Alb. & Schw.  3 3 18  Tomenlella sp.  1  Trameles carbo'naria (B. & C.) Overh. Trametes mollis (Sommerf.) Fr. Trameles sepium Berk.?  1, 16, 19 13 19  206  Appendix  5.  (continued)  LOWER  BRITISH C O L U M B I A  S c a l e — 1 inch = 1 4 0 miles 132*  130*  128*  126*  124*  S T U D Y OF DECAY LOCALITIES  122"  IN  WE5TERN  1 - C o w i c h a n Lake Nixon Creek Meade Creek Copper C a n y o n , Chemainus  11121314-  Muir C r e e k , Sooke Bonell Creek, Nanoose Bay  151617-  Elsie L a k e ,  Alberni Tsable River, Fanny Bay Courtenay District Port Moody Cedar Sawmill  Localities Source: Buckland,  RED  CEDAR  INVESTIGATED  2 3 4 -  5 6789 10-  120*  1819-  S k i d e g a t e Lake Clearwater River Blue R i v e r Upper Thompson R i v e r LaForme Creek Illecillewaet R i v e r Begbie Creek Mabel L a k e Shuswap R i v e r  investigated in the study of decay in western red ceda  (1946). 207  Appendix 6, Results of A n a l y s i s of V a r i a n c e f o r the WRC Weight L o s s .  Heartwood  UNIVARIATE 1-WAY ANOVA ANALYSIS OF VARIANCE OF 4 . WL%  N= 36 OUT OF 36  SOURCE  DF SUM OF SORS  BETWEEN WITHIN TOTAL  8 27 35  ETA=  .9892  ETA -SQR=  139 .53 3.0629 142.59 .!9785  EQUALITY OF VARIANCES:  (VAR COMP= 4.3319  DF= 8. 656.10  N  MEAN  VARIANCE  (1) (2) (3) (4) (5) (6) (7) (8) (9)  4 4 4 4 4 4  1.4005 1 .5521 .89627 5.4758 3.6285 1.2500 5.5609 .63486 .21286 -1  .29684 .12223 .11459 .65591 .43461 .20537 .37924 .67357 .26849  2.2689  4.0741  4  GRAND  36  Conclusion:  F-STATISTIC SIGNIF  17.441 153.75 .0000 .11344 (RANDOM EFFECTS STATISTICS)  TREAT  A 4  MEAN SOR  '/.VAR AMONG=  F= 4.2082  97.45) .0001  STD DEV -1 -1 -1 -1  . 17229 .11056 .10705 .25611 .65925 .14331 .61582 .25953 .16386 - 1  -1 -1 -3  2.0184  The v a r i a n c e s of nine groups are not homogenous and data t r a n s f o r m a t i o n i s needed.  UNIVARIATE 1-WAY ANOVA ANALYSIS OF VARIANCE OF 7.SOWL%  N= 36 OUT OF 36  SOURCE  DF SUM OF SORS  BETWEEN WITHIN TOTAL  8 27 35  ETA=  .9919  ETA-SQR=  17.471 .28727 17.758 .9838  EQUALITY OF VARIANCES:  Conclusion:  MEAN SOR  F-STATISTIC SIGNIF  2.1838 205.25 .0000 .10640 -1 (RANDOM EFFECTS STATISTICS)  (VAR COMP = .54330  DF= 8, 656.10  '/.VAR AMONG* 98.OS)  F«= 1.2880  .2466  After square root data transformation, v a r i a n c e of nine groups are homogenous. 208  the  Appendix 6.  (continued) ANALYSIS NO. NO. NO.  OF V A R I A N C E / C O V A R I A N C E - R E P O R T  OF V A R I A T E S OF C O V A R I A T E S OF OBSERVATIONS INDEX INDEX  A  1 1 36  RANGE  9  REGRESSION COEFFICIENT  COVARIATE  STANDARD ERROR  WT1 0.05778 T E S T OF A L L COVARIATES TOGETHER  Conclusion:  The covariate signi f icant. ANALYSIS NO. NO. NO.  0.10998  F-VALUE  PROB  0.2760 0.2760  0.6096 0.6096  (original  OF V A R I A N C E / C O V A R I A N C E - R E P O R T  OF VARIATES OF COVARIATES OF OBSERVATIONS INDEX A INDEX  PHASE  weight)  IS  not  PHASE  1 0 36  RANGE 9  ANALYSIS OF VARIANCE/COVARIANCE OVERALL MEANS SRWL 1.3325 OVERALL  STANDARD SRWL  DEVIATIONS  0.7123  ANALYSIS  SRC. 1 2  N  0  3  SOURCE TREATMENT ERROR TOTAL  Conclusion:  D.F. 8 27 35  OF V A R I A N C E / C O V A R I A N C E  SUM OF SQUARES 17.470663 2.872729E-01 17.757936  FOR VARIABLE  MEAN SQUARE 2.183832 1.063973E-02  SRWLX  F VALUE 205.2525  F PROB 0.0000  There i s a s i g n i f i c a n t d i f f e r e n c e between the averages of the percentage O.-D. weight l o s s e s of the nine group. 209  TESTED AGAINST 2  Appendix 6. ( c o n t i n u e d ) . Original  Data:  (Weight Losses of WRC Heartwood Blocks C o n t a i n i n g High T h u j a p l i c i n Content A f t e r I n f e c t i o n With Three 'Early Stage' Fungi (WR1, WR2 and WR3).) Code O r i g i n a l no O.D. weight (g)  O.D. Weight after treatment (g)  Weight loss % (O.D.)  Group 1 : (8 weeks with WR1 )  Ave:  1-1 1-2 1-3 1-4  6. 1 02 5.473 5.997 6.072  6. 001 5. 403 5. 918 5. 990  1 .6552 1 .2790 1 .3173 1 .3505 1 .4005  Group 2 : (8 weeks with WR2)  Ave:  2-1 2-2 2-3 2-4  5.702 6.010 6. 1 74 6.069  5. 614 5. 918 6. 069 5. 982  1 .5433 1 .5308 1 .7007 1 .4335 1 .5521  Group 3 : (8 weeks with WR3)  Ave:  3-1 3-2 3-3 3-4  6. 1 06 5.929 6. 1 43 6.041  6. 060 5. 872 6. 082 5. 988  0 .7534 0 .961 4 0 .9930 0 .8773 0 .8963  Group 4 : (4 weeks with WR1 , then 4 weeks with  Ave:  4-1 4-2 4-3 4-4  5.928 5.978 5.738 6.023  5. 581 5. 662 5. 430 5. 698  5 .8536 5 .2860 5 .3677 5 .3960 5 .4758  210  Appendix 6. (continued) Code O r i g i n a l no O.D. weight (g)  O.D. Weight after treatment (g)  Group 5: ( 4 weeks with WR1,  Ave:  5-1 5-2 5-3 5-4  6. 1 29 6.076 5.743 6.050  Ave:  6-1 6-2 6-3 6-4  6.012 6. 104 5.876 5.903  then 4 weeks with  5.919 5.796 5.556 5.855  Group 6: (4 weeks with WR2,  Weight loss % (O.D.) WR3)  3.4263 4.6083 3.2561 3.2231 3.6285  then 4 weeks with  5.928 6.025 5.814 5.829  1.3972 1.2942 1.0551 1.2536 1.2500  Group 7: (4 weeks with WR1, then 4 weeks with 4 weeks with WR3)  Ave  7-1 7-2 7-3 7-4  5.651 6.135 5.764 6.031  Group 8: ( c o n t r o l  Ave:  8-1 8-2 8-3 8-4  9-1 9-2 9-3 9-4 Ave:  5.8043 5.0040 5.1180 6.3174 5.5609  l e v e l 2: 8 weeks with c u l t u r e  6. 137 5.820 6.048 6. 193  Group. 9: ( c o n t r o l  5.323 5.828 5.469 5.650  6.115 5.786 6.001 6.1 32  medium)  0.3583 0.5842 0.6118 0.9850 0.6349  l e v e l 1: 8 weeks i n empty P e t r i d i s h e s )  5.871 6.213 5.954 5.834  5.869 6.213 5.953 5.832  0.0341 0.0000 0.0168 0.0343 0.0213 21 1  Appendix  7.  Results of Bioassay.  Multiple  MEANS FOR SOURCE* FACTOR LEVELS A 1 2 3 4 5 6 7  Range  Tests  f o r Wood  Block  TREATMENT DIVISOR 4 4 4 4 4 4 4 4 4  B  9  RANGE TESTS FOR  FREQ 4 4 4 4 4 4 4 4 4  MEAN 1 . 182 245 945 340 899 1 17 355 784 0 . 125  SRWL7O STD DEV 0.071 0.044 0.057 0.054 0 . 167 0.065 0 . 130 0 . 161 0.087  SRWL  DUNCAN'S MULTIPLE RANGE T E S T , RANGES FOR ALPHA=0.05 2.8995 3.0469 3.1481 3.2112 3.2624 3.3029 3 3351 THERE ARE 6 HOMOGENEOUS SUBSETS(SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT S I Z E ) WHICH ARE LISTED AS FOLLOWS ( 9 ) ( 8) ( 3) ( 6, ( 5) ( 4,  1.  2)  7)  STUDENT I ZED RANGES FOR NEWMAN-KEUL'S T E S T , ALPHA=0.05 2.902 3.507 3.870 4.131 4.334 4.498 4.639 4.759 THERE ARE 6 HOMOGENEOUS SUBSETS(SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT S I Z E ) WHICH ARE LISTED AS FOLLOWS ( ( ( ( ( (  9) 8) 3) 6. 5) 4,  1.  2)  7)  STUDENTIZED RANGE FOR T U K E Y ' S T E S T . ALPHA= 0 . 0 5 4.759 THERE ARE 6 HOMOGENEOUS SUBSETS(SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT S I Z E ) WHICH ARE LISTED AS FOLLOWS  (  9)  ( (  8. 3,  3) 6.  1)  (  6.  1.  2)  (  4.  7)  (  5)  212  3 3611  8.  Appendix  Chemical S h i f t s of Some P r o t o n s . Ap?roxiroart Chemical Shifts of Protons A r u c b e d to L'nsarumed Linkages  6  Proton R-CHO Ar-CHO H-CO—0 H—CO—N -Cs=C—H Aiomatic proiont  t  oo-o*  9-4-100 97-105 8 0-8 2  -O5-03 1 8-20 I 8-20  &-0-8: 16-3 1  6-9-s:  6-0-9-0  1-0-40  Proton  i  T  —C=CH—C=CH—CO —CH=C—CO —CH==C—0 —C=CH—O —CH=C—N —C=CH—N  45-60 5-8-67 6 5-8-0 40-50 6-0-8 1 37-50 57-80  40-5-5 3-3-4-2 2-0-35 5-0-6-0 1-9-40 50-63 2-0-43  Chcmictl Shifti of Methyl, Methylene and Methine Protons Methyl Protons Proton C H j - C CH --C-C=C CH.-C--O CHj—0=C CHj-At CHj-CO— R CH,CO-Ar C H y—CO—O—R CHi—CO—O—Ar CHj—CO-N—R CH —O-R 3  3  Methylene Protons  6  t  09 11 13 1-6 2 3 22 2-6 2-0 24 2-0 33  91 6-9 87 8-4 7 7 78 7-4 80 76 8-0 67  CH -0—C=C CHj—O-Ar CKj—O—CO—R CHj-N  3-8 38 37 23  62 62 63 77  CHj-N CHj-N—Ar CH -S  3-3 3-0 21  6-7 7-0 79  CHj-C-NO, CHj—C=C—CO C-=CtCHj)-CO  16 2-0 lk  84 8-0 82  3  3  CH,—N—CO—R  2-9  Proton  Proton C—CH—C  :~o —CH—Ar —C—CH—CO—R  —C—CH —CO—N—R — C — C H j —O—R —C—CH,—O—H  22  TH,—O—Ar Ij—O—CO— R —C—CH.-N  43  2  —C—C\i\—NO, —C—CHj—C—NO, —C—CH j —C=C—C C=CtCHj)—CO C—CHj—Cl C—CH..—Br C—CHj—1 C—CH-—QsN  213  85  2-0  8-0  3-0 27 3-3  7-0 73 67  —C—CH—OH  37 3-9  63 61  2-5  57 5-9 75  —C—CH-O—CO—C—CH—N  48 28  52 72  2-4 4-4 21 2-4 2- 4 36 35 3- 2 23  7-6 56 7-9 7-6 7-6 64 65 68 7-7  32 47  68 53  43 43 27 41  57 57 73 5-9  4 1  71  N a k a n i s h i , (1962).  1- 5  7-8 6-6 64  3-4 3-6  5-9  Source:  Methine Protons  41  C—CH—Br C—CH—I C—CH—C==N C—CH-N—CO—R  Appendix 9. Chemical S h i f t of Some Carbon-13 Resonances.  220 200 ISO MO UO 120 K>0 80 Details or substituent effects on " C chemical shifts are given in Tables 3-19. 3-20 and 3-21.  Source: Willam and Fleming,  (1980).  214  Appendix 10. IR Data f o r S e l e c t e d Carbon-Hydrogen A b s o r p t i o n Bands, Frequency Functional Group  Wavelength  cm'  Assignment  M  1  Remarks t  Alkyl -CH,  - C H , -  I —C—H  1 1  —OCOCH,  2960 2870 1460 1380  3.38 3.48 6.85 7.25  2925 2850 1470 1250  3.42 3.51 6.83 -8.00  »u  s, lu  *•  m, lu s. lu m.gu s. lu  ».  scissoring twisting and wagging  m, Iu f. lu s, nu  2890  3.46  r  w, nu  1340  7.45  c  w, nu  1380-1365  7.25-7.33  «t  s, gu  —COCH,  -1360  -7.35  s, gu  —COOCH,  -1440 -1360  -6.95 -7.35  s, gu t. gu  3080 2975 - 1420 -900  3.24 3.36 -7.0-7.1 -11  Vinyl -CH,  c-< mono-substituted ru-disubstituted //•a/u-disubstituted trisubstiiuied  3020  3.31  990 900 730-675 965 840-800  10.1 11.0 13.7-14.7 10.4 11.9-12.4  a (in-plane) e (out-of-plane)  m. lu m, lu m, lu s, gu  V  m, lu  »u  e a c a a  (out-of-plane) (out-of-plane) (out-of-plane) (out-of-plane) (out-of-plane)  gu gu gu gu m-s, gu  s, s. s. s,  Acetylene *=C— H Aldehyde  •°  3300  3.0  »  m-s. gu  2820  3.55  9  m, lu  2720  -3.7  H  Source: Pasto and Johnson,  overtone or combination band  (1969). 215  m.gu  Appendix 11. IR Data f o r S e l e c t e d X-H A b s o r p t i o n Functional group  Frequency cm'  Bands.  Wavelength  Remarks  1  Alcohols (nonbonded) primary  3640  2.72  secondary tertiary  3630 3620  2.73 2.74  phenols  3610  2.75  m. gu. usually determined in dilute solution in nonpolar solvents.  intermolecularly H-bondcd  3600-3500 3400-3200  2.78-2.86 2.94-3.1  m. dimeric. rather sharp s, polymeric, usually quite broad  intramolecularly H-bonded  3600-3500  2.78-2.86  m-s. much sharper than intermolecular hydrogen bonded O H : is not concentration dependent.  m. gu. m.gu.», m-s. gu. corresponds to scissoring deformation.  Amines RNH,  -3:500 -3400 1640-1560  -2.86 - 2.94 6.1-6.4  R NH ArNHR  3500-3450 3450  2.86-2.90 2.90  2  Pvrroles. indoles  3490  2.86  w-m. gu. r w-m,gu  w-m. gu  Ammonium salts NH *  3300-3030 1430-1390  3.0-3.3 7.0-7.2  -N-Hj  3000 1600-1575 1490  — 3.0 6.25-6.35 - 6.7  2700-2250  3.7-4.4  s. gu. v and »„ usually broad or a group of bands  1600-1575 2700-2250  6.25-6.35 3.7-4.4  m. gu. a s. gu. », P N H - band is weak and of no practical utility.  2600-2550  3.85-3.92  s, gu band is often very weak and can be missed if care is not exercised.  4  ^N*H,  ;N*H  Mercaptans —SH  Source: Pasto and Johnson,  s. gu s.gu s. gu. usually quite broad S. gu. a s. gu. B, u  u  (1969). 216  Appendix 12. IR Data f o r S e l e c t e d Carbon-Carbon A b s o r p t i o n Bands. Functional group  Frequency em' 1  Wavelenglh u  Remarks  Alicyclic C—C(»c— c> monosubstituted l.l-disubstiluted ci*-1.2,-disubst. fra/u-l.2-disubst. trisubstituted tetrasubstiluied  1645 1655 1660 1675 1670 1670  6.08 6.04 6.02 5.97 5.99 5.99  m. m, rn, w, w, w,  these bands are of little utility in assigning mostitution and stereochemistry; the C — H out-of-plane bending bands in the fingerprint region are recommended for this purpose.  Conjugated C—C(»c—e) with aromatic with C—O  1650 and 1600 1625 1600  6.06 and 6.25 6.16 6.25  s. gu, s. gu, l . gu.  Cyclic C — C ( » c - c ) 6-membered ring and larger  1660-1650  6.03-6.06  m.  monosubstituted 5-m. unsubstituted monosubstituted disubstituted 4-m. unsubstituted 3-m unsubstituted monosubstituted disubstituted  1680-1665 1615 1660 1690 1660 1640 1770 1880  5.95-6.00 6.19 6.02 5.92 6.02 6.10 5.65 5.32  m. m. m. w. m. m. m. m.  Aromatic C — C  1600 1580 1500 1450  6.24 6.34 6.67 6.9  »-s. s. w-s. s,  S o u r c e : P a s t o and Johnson, ( 1 9 6 9 ) . 217  of limited utility due to closeness to the alicyclic region. can be of great utility although the ring size must be assigned first for those compounds whose absorption falls into regions consistent with other types of absorption bands (nuclear magnetic resonance spectra can be very useful in this respect). in-plane skeletal vibrations. the intensities of the 1600 and 1500 cm* may be rather weak. 1  Appendix IR  13.  Data  for  Selected Carbon-Oxygen  Functional group  Frequency cm' 1  Wavelength M  A b s o r p t i o n Ban  Remarks  C—0 Single Bonds Primary C — O — H  1050  9.52  Secondary C — O — H Tertiary C — O — H  1100 1150  9.0S 8.68  s. gu. »c—o (See discussion for substituent effects.) s. gu. » . _ S, gu, r _  Aromatic C — 0 — H  1200  8.33  S, gu. » _ o  1150-1070  8.7-9.35  Ethers-acyclic C—C—o—C  c  c  0  0  c  1270-1200 and 7.9-8.3 and 1070-1020 9.3-9.8  s. gu. an'.isymmetric VQ—O—C s. gu. antisymmetric »c—o—C s, gu. symmetric «x—O—C  1140-1070 1100-1075  s. gu. s. gu.  "yclic ethers 6-m. and larger 5-m. 4-m. Epoxides G'j-disubstituted 7>art5-disubstituted  980-970  8.77-9.35 9.1-9.3 10.2-10.3  1250 890 830  S.O 11.25 12.05  1715  5.8?  s. gu. * c — o - unstrained 0"=0 group in acyclic and 6 m ring compounds in carbon tetrachloride solution. (See discussion for effects of conjugation and ring size.)  1685  5.93  s, gu. »c—o- For the s-cis configuration O u R (C. JZ) the »c—c appear  s. gu. s. gu. s. gu. s.gu.  C — 0 Double Bonds Ketones  a. ^-unsaturated  m  a  v  above I60C c m " (below 6.26 u) with an intensity approximately that of the 1  »C—oO  The C  fflflj-configuration does not show this  enhanced intensity of absorption of theC—C. o- and 0-dikeiones  1720  5.81  s, gu. *c—O- ' * ° bands at higher frequency when in the i-cu configuration O li JO  1650  6.06  s. gu. «>c—C 'f enolic ( 0 ~ C )  c—c  OH Quinones  1675  5.97  s, gu. » c - o  218  Appendix  13. (continued) Functional group  Frequency em-'  Tropolones  •°  Aldehydes — C .  Ho tlrnglh M  I6S0 1600  6.06 6.26  *. »C-0 s. j u . »c—0- if intramolecular? hydrogen bonded as in «-tropolones.  1725  S.80  i . gu. »c—o (See discussion for effects of conjugation.)  5.84  t. gu. »c—O' usually as the dimer in nonpolar solvents, monomer absorbs at 1730 c m " (5.78 ) and may appear as a shoulder in the spectrum of a carboxylic acid: for conjugation effects see the discussion.  H Carboxylic acids and derivatives X  1710  /  Remarks  —C—OH  1  u  Esters  1735  5.76  1300-1050  7.7-9.5  s. gu, r<>.o. acyclic and 6-m. lactones:  i,  Iu. symmetric ana antisymmetric »C—o—C 8< 'ig 2 bands, indicative of type of ester, for example, formates: 1178 e m " ' (8.5 u) acetates: 1242 cm"'(8.05 n) methyl esters: 1164 c m " (8.6*) others: 1192cm" (8.4 u) but the distinction generally is not great enough to be of diagnostic value. v  1  1  Anhydrides  1820 and 1760  5.48 and 5.68  s. gu. re—O- the intensity and separation of the bands may be quite variable. (See discussion for general effects of conjugation and ring size.)  Acid halide  1800  5.56  s, gu. » c - o . *cid chlorides and fluorides absorb at slightly higher frequency while the bromides and iodides absorb at slightly lower frequency. (See discussion for effects of conjugation.)  Amides  1650  6.06  1300  7.7  1665  6.01  s. gu. » - o "Amide I" band, this frequency is for the associated amide t (see C O O H ) , free amide at 1686 cm (5.93 it) in dilute solution, cyclic amides shift to higher frequency as ring size decreases. s. gu. »c—N. "Amide III*' band, free amide at slightly higher frequency. a, gu. » - o  a . ^-unsaturated Carboxylate  to  1610-1550 and 1400  Source: Pasto and Johnson,  6.2-6.45 and 7.15  (1969).  219  c  c  i . gu  antisymmetric and symmetric  stretching o f —  

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