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The role of microorganisms in the phenomenon of hemlock brownstain Kreber, Bernhard 1995

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THE ROLE OF MICROORGANISMS IN THE PHENOMENON OF HEMLOCK BROWNSTAIN by BERNHARD KREBER B.Sc.(Wood Science), University of Hamburg, Germany M.Sc.(Forest Products), Oregon State University, U.S.A. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE FACULTY OF GRADUATE STUDIES (Department of Forestry) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA March, 1995 ©BERNHARD KREBER  In presenting this  thesis  in  degree at the University of  partial fulfilment  of  the  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  I Jft ft IL  KA N'  M  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Y£_  LLLAS  ii Abstract  Hemlock  brownstain,  intensity  and occurs  a discoloration in amabilis  which  fir  varies  in  type and  (Abies amabilis  Forbes) and western hemlock (Tsuga heterophylla  (Dougl.)  (Raf) Sarg.) was  investigated because it is a serious problem in the high-value lumber markets.  The objective of this study was to understand the  causes of hemlock brownstain, more specifically with emphasis on the role of microorganisms, and to suggest means for its control.  While  hemlock  brownstain  can  vary  macroscopically,  a  similar  microscopic distribution of the brown coloration was demonstrated to be mainly associated with parenchyma cells and to a lesser degree with longitudinal tracheids.  The brown deposits which were  frequently associated with hyphae and bacteria, contained catechin as shown histochemically.  Inoculation of western hemlock sap and  wood with fungi and bacteria produced brownstain in vitro.  The brown colorations which can develop in western hemlock during seasoning, were then investigated in field studies on logs and lumber.  Extensive log storage time was demonstrated to promote  brownstain as was salt water storage of logs, the latter producing more brownstain than logs stored on land.  Fungi were isolated from  freshly felled logs and from sawn lumber and they were believed to represent an endemic wood microflora. A link was suggested between fungi and brownstain.  Low solubility phenols were associated with  iii brownstained  regions  when  compared  to  non-stained  areas  and  migration of phenols to the wood surface was indicated.  Infection of western hemlock lumber with Ophiostoma piceae produced brown deposits but subsequent antibody proved unsuccessful fungus.  However,  immunolabeling with a monoclonal in linking the brownstain to the  laboratory  experiments  demonstrated  that  sapstaining fungi can produce browning in western hemlock sap as they shifted the pH from 5 to near 7, which caused ionization and oxidation of phenols. Browning induced by fungi was inhibited when the sap was buffered in the acidic range. While browning did occur at neutral pH, it did not occur in the absence of oxygen.  Browning was also demonstrated in 15 /xm sections when infected with 0. piceae, suggesting that browning can occur in the presence of very  small  amounts  of  colour precursors.  Furthermore,  light  microscopy demonstrated a lack of pigmentation of the 0. piceae hyphae when grown on western hemlock which contrasted with the formation of pigmented hyphae (sapstain) when grown on lodgepole pine.  This  observation  suggested  that  physiological  factors  associated with the presence of the fungus triggered brownstain in western  hemlock  and  that  hemlock  brownstain  pigment formation by the fungus itself.  was  unrelated  to  iv Catechin was demonstrated to play a major role in brownstain of western hemlock but involvement of other, unknown, sap constituents was also indicated.  Information gained in this study suggested that a faster processing of western hemlock logs into sawn lumber may lessen the extent of brownstain problems.  In addition, biocides  supplemented  with  reducing agents and/or chelating agents and the use of a buffer to stabilize the pH of the wood surface, should be investigated.  As a spin-off of this research, the physiological factors which greatly reduced pigmentation of the sapstaining fungus 0. piceae in western hemlock, should be further investigated.  V  TABLE OF CONTENTS  Abstract  ii  Table of Contents  v  List of Figures  x  List of Tables  xii  Acknowledgement  xiv  Preface  xvi  1.0  INTRODUCTION  2.0  DISCOLORATIONS MECHANISMS  1 OF  HEM-FIR  WOOD:  A  REVIEW  OF  THE 3  2.1  Background of Non-microbial Stains  3  2.2  Economic Significance of Discolorations in Western Hemlock and Amabilis-Fir  5  2.3  Investigations of Hem-Fir Discolorations  6  2.4  Extractive Chemistry of Western Hemlock  13  2.5  Factors Possibly Contributing to Discolorations in Hem-Fir  18  2.5.1 2.5.2 2.5.3 2.5.4 2.5.5  Factors Inherent in Living Trees Felling Season Post-Mortem Factors Log Age Log Storage  19 21 22 23 25  2.5.6  S t o r a g e of L u m b e r  26  2.6  Summary  28  3.0  ADVANCES IN THE UNDERSTANDING OF HEMLOCK BROWNSTAIN  29  3.1  Objectives  29  3.2  Material and Methods  29  3.2.1 Microscopic Examinations  29  VI  3  3.2.2 Histochemical Examinations 3.2.3 Sap Experiments 3.2.4 Solid Wood Experiment  30 30 32  Results  34  3.3.1 3.3.2 3.3.3 3.3.4  34 40 45  Microscopic observations on discoloured hem-fir Histochemical observations Colour changes in inoculated sap Microscopic production of brownstain in inoculated western hemlock  47  4  Discussion  51  5  Conclusions  60  0  MONITORING PRODUCTION OF BROWNSTAIN IN WESTERN HEMLOCK LOGS AND LUMBER DURING STORAGE  62  1  Objectives  62  2  Materials and Methods  62  4.2.1 Field work at Chamiss Bay  62  Logging site description Selection of western hemlock trees Initial sampling of western hemlock logs Second sampling of western hemlock logs Inspection of western hemlock logs after 9 months storage Sawing of western hemlock logs Inspection of sawn lumber after storage 4.2.2 Laboratory work using Chamiss Bays samples Assessment of susceptibility to brownstain in disks Selection of logs susceptible to brownstain Sample preparation from selected disks Initial isolation of fungi Harvesting of sap pH measurement HPLC analysis of pressate Total soluble phenol determination Sample preparation from disks after 2 months log storage Isolation of fungi and quantification of bacterial forming colonies Sap analysis  62 63 63 64 65 65 66 66  66 66 67 69 70 70 70 70 71 72 73  Vll  Results and Discussion  74  4.3.1 Production of brownstain in fresh and 2 month-old logs  74  4.3.2  Inspection of cross-cut ends after 9 month storage  4.3.3 Inspection of lumber after 2 months of outdoor storage 4.3.4 Isolation of microorganisms 4.3.5 Sap analysis  78  84 88 95  Conclusions  103  MICROFLORA AND TOTAL SOLUBLE PHENOLS ASSOCIATED WITH BROWNSTAIN IN WESTERN HEMLOCK LUMBER  105  1  Objective  105  2  Materials and Methods  105  5.2.1 5.2.2 5.2.3 5.2.4  105 106 108 108  0  3  Sampling at CIPA sawmill Sample preparation and isolation of fungi Determination of TSP content Assessment of antisapstain treatment  Results and Discussion  109  5.3.1 5.3.2 5.3.3  109 110 113  Observations on brownstained lumber Isolation of fungi Sap analysis  4  Conclusions  117  0  IN VITRO PRODUCTION OF HEMLOCK BROWNSTAIN  118  1  Immunogold-silver staining of O. piceae when grown in western hemlock  118  6.1.1 Objective  118  6.1.2 Materials and Methods  118  Sample preparation Preparation of fungal inoculum Infection of western hemlock Visual and microscopic examination of wood samples Immunolabeling of 0. piceae # 3871 in western hemlock Sap analysis  118 120 120 121 121 123  Vlll  2  3  4  6.1.3 Results and Discussion  124  6.1.4 Conclusions  13 0  Production of brownstain in western hemlock sap  131  6.2.1 Objective  131  6.2.2 Materials and methods  131 Sample preparation Infection of sap  131 132 Sap analysis  132  6.2.3 Results and Discussion  135  6.2.4 Conclusions  147  Infection of wood sections with 0. piceae on a glass-slide  148  6.3.1 Objective  148  6.3.2 Materials and Methods  148 Slide preparation Wood sections used Infection of wood sections Microscopic examination of sections 6.3.3 Results and Discussion  148 14 8 149 149 150  6.3.4 Conclusions  154  Infection of western hemlock and lodgepole pine with 0. piceae  155  6.4.1 Objective  155  6.4.2 Materials and Methods  155 Wood used Preparation of inoculum and infection of wood 6.4.3 Results and Discussion  155  6.4.5 Conclusion  161  155 157  ix 7.0  ELUCIDATION OF THE MECHANISMS OF SAP BROWNING  162  7.1  Objective  162  7.2  Materials and Methods  162  7.2.1 7.2.2 7.2.3 7.2.4  Effect of pH on sap browning Effect of oxygen on sap browning Effect of heat on sap browning Production of sap browning by heat and by pH alteration 7.2.5 Amendment of water with phenols 7.2.6 Amendments of sap with known phenols 7.2.7 Effect of buffer on sap browning  162 162 163  Results and Discussion  167  7.3.1 7.3.2 7.3.3 7.3.4  Effect of pH on browning Effect of oxygen on browning Effect of heat on browning Production of sap browning by heat and by pH alteration 7.3.5 Amendment of water and sap with known phenols 7.3.5 Buffer experiment  167 169 172  7.4  Conclusions  183  8.0  Summary and Recommendations  184  9.0  Literature Cited  190  7.3  164 164 165 165  174 177 180  X  List of F i g u r e s Figure l a : R e p r e s e n t a t i v e specimen s h o w i n g g r e y stain on the w o o d surface  35  Figure l b : R e p r e s e n t a t i v e s p e c i m e n s h o w i n g b r o w n s t a i n o n the b o a r d e n d and o n the w o o d surface  36  Figure l c : R e p r e s e n t a t i v e s p e c i m e n s h o w i n g zebra stain on the w o o d surface  37  Figure Figure  Figure Figure Figure  Figure Figure  Figure  2: A b i e s a m a b i l i s . Radial s e c t i o n ( 6 3 x ) , r a y p a r e n c h y m a cells w i t h b r o w n d e p o s i t s  38  3: T s u g a h e t e r o p h y l l a . T r a n s v e r s e s e c t i o n ( l O O x ) , i s o l a t e d t r a c h e i d s w i t h a filled lumen among stain-free tracheids  39  4: T s u g a h e t e r o p h y l l a . R a d i a l s e c t i o n ( 2 0 0 x ) , h y p h a e e n c l o s e d in c o l o u r e d d e p o s i t s of a tracheid  41  5: T s u g a h e t e r o p h y l l a . R a d i a l s e c t i o n b a c t e r i a l i n f e c t i o n of t r a c h e i d s  42  (lOOx),  6: Tsuga heterophylla. Radial section (80x), red DMB reaction products observed in ray parenchyma cells  43  7: Tsuga heterophylla. Radial section (80x), red DMB reaction products observed in axial tracheids  44  8: Tsuga heterophylla. Radial section (320x), colourless globules observed in clean ray parenchyma cells  48  9: Tsuga heterophylla. Radial section (160x), presence of brown deposits in 0. piceae #3871 inoculated wood after 6 weeks  50  Figure 10: Tsuga heterophylla. Transverse section (250x), brown deposits in a pit connecting discoloured tracheids  53  Figure 11: Tsuga heterophylla. Transverse section (400x), hyphae surrounded by a brown sheath in an otherwise unstained tracheid  54  Figure 12: Flowchart showing sample regime of log #1 from brownstained and non-stained regions in fresh and two month old disks  68  XI  Figure 13: Representative, water-stored log showing dark colorations after 9 months of storage  81  Figure 14: Representative, land-stored log showing brownstain in sapwood after 9 months of storage  82  Figure 15: Production of severe brownstain on the boards edges and faces of lumber sawn from log #4  86  Figure 16: Establishment of a standard calibration using gallic acid  98  Figure 17: Total soluble phenols measured in brownstained regions of test logs after 0 (A) and 2 (B) months of storage  99  Figure 18: Total soluble phenols measured in two non-stained regions of control logs after 0 (A) and 2 (B) months of storage  101  Figure 19: Total soluble phenols measured in four different regions in test and control logs after months of storage  102  Figure 20: Flowchart showing sample regime of 5 cm x 10 cm western hemlock lumber  107  Figure 21: Frequency of fungi isolated from western hemlock lumber  111  Figure 22: Total soluble phenols measured in three different regions within a board  114  Figure 23: Total soluble phenols measured in three different regions within a board  115  Figure 24: Flowchart showing experimental design  119  Figure 25: Colour changes in western hemlock sap (8A; 5A) incubated with different fungi for 10 days at room temperature  13 6  Figure 26: Tsuqa heterophylla. Radial section (63x), Brown deposits associated with hyphae of O. piceae in a 15 /xm section  151  Figure 27: Production of sap browning under oxygen  170  Xll  List of Tables Table  1: Changes in pH of sap after three weeks of incubation  Table  2: Classification of selected western hemlock logs  Table  3: Production of brownstain on log ends after 9 month of outdoors storage 4: Fungi isolated from western hemlock logs selected at Chamiss Bay 5: Moisture content (MC %) and colony-forming units (CFU) in brownstained (BS) and non-stained (NS) logs  Table Table  Table Table  6: Moisture content (MC %) and pH in brownstained (BS) and non-stained (NS) sample regions  46 75  79 89  94 96  7: Total soluble phenol content (jug/mL) measured in sap from western hemlock incubated with 0. piceae for 6 weeks  127  8: Fungi evaluated for their potential to cause browning in western hemlock sap  13 3  9: Changes in western hemlock sap (8A) incubated with different microorganisms for 12 days at room temperature  137  Table 10: Changes in western hemlock sap (5A) incubated with different microorganisms for 12 days at room temperature  13 8  Table 11: Changes in western hemlock sap (4A) incubated with different microorganisms for 12 days at room temperature  14 0  Table 12: Changes in western hemlock sap (7D) incubated with different microorganisms for 12 days at room temperature  14 0  Table 13: Repeated assessment of changes in western hemlock sap (8A) incubated with different microorganisms for 12 days at room temperature  141  Table 14: Changes in pH 7 adjusted and non-adjusted western hemlock sap (8A) following incubation for 12 days at room temperature  168  Table Table  xiii Table 15: Effect of oxygen on colour and TSP (/xg/mL) changes in western hemlock sap  171  Table 16: Changes in heated sap-8A after incubation with different microorganisms for 12 days at room temperature  173  Table 17: Changes in heated sap and in pH modified sap  175  Table 18: Colour and TSP in pH adjusted sap-5A amended with phenols after 12 days of incubation at room temperature  178  Table 19: Changes in buffered and non-buffered western hemlock sap (9A) incubated with different fungi for 12 days at room temperature  181  xiv Acknowledgements  Foremost, I wish to thank my research supervisor Dr Roger Smith for his advice and guidance during the course of my graduate study. Apart from his invaluable experience in wood products research Roger passed on two things which I greatly appreciated: first he taught me to always thoroughly question my results before drawing any conclusions; second he made me understand that as a scientist you may have one or two highlights  (advancements) in your whole  career and therefore science means taking little steps at a time and often without making headway. Thanks Roger!  A special thanks to Dr Colette Breuil for guiding me through the bureaucracies of my PhD program and also for her advice concerning my research.  I also wish to extend my thanks to Drs Bruce Bohm,  Simon Ellis and Bart van der Kamp for their interest in hemlock brownstain which resulted in many useful discussions.  I also owe a lot of gratitude to friends and research colleagues in the Treated Wood Department at Forintek Canada Corp.  I sincerely  thank Tony Byrne for his advice and guidance throughout my study and for his great editorial skills.  Tony reviewed numerous papers  of mine and his last words after reviewing my thesis were "Deo gratias" .  Tony, there will be more to come!  I also thank Paul  Morris for his scientific advice and also for many enjoyable hours while hiking or skiing.  Bob Daniels assistance with the HPLC  XV  analysis is greatly appreciated.  Futong Cui, Glenn Weigel, Maria  Chan, Jean Clark and Rob Scott are also thanked for their advice and help during my studies.  I also acknowledge the help of K.A.  Seifert who identified some of the fungi isolated in this study.  I must thank Rob Scheel and Bill Gilpin from Interfor Ltd. and the Interfor crew at Chamiss Bay.  Without their support the field  study on hemlock brownstain would have been impossible and I would not have been introduced to the adventurous world of lumberjacks. I also acknowledge Mr. L o m e Holman of CIPA Lumber Co. Ltd. who provided lumber whenever needed.  My studies at U.B.C. were supported by a VanDusen Fellowship and an University Graduate fellowship (MacMillan Bloedel) made available through  the  Department  of Wood  Science.  Last  but  not  least  Forintek Canada Corp. and the Canadian Forest Service financially assisted my studies.  xv i Preface Some of the material contained in the thesis has appeared previously in publications produced during the course of the research: 1.  Kreber, B. and A. Byrne. 1994. Discolorations of hem-fir wood: a review of the mechanisms. Forest Products Journal 44 (5) :35-42. Permission was granted from Forest Products Society (Madison, WI) to reproduce the above article in whole or in part in this thesis.  2.  B. Kreber. 1993/94. Advances in the understanding of hemlock brownstain. Material and Organismen 28 Bd. , Heft 1:17-37. Permission was granted from Material and Organismen (Berlin) to reproduce the above article in whole or in part in this thesis.  1.0  INTRODUCTION  Variation  in  colour  properties of wood.  is  one  of  the  most  distinctive,  natural  While the colour of wood is a very important  characteristic in terms of its market value colour descriptions such as dark brown or terms.  "whitewood" are relative and subjective  The colour of woods is due mainly to extraneous compounds  rather than structural cell wall components. Coloured extractives, found as deposits in the cell lumina or within the cell wall, give a characteristic colour to a species (Kuo and Arganbright, 1980; Kai and Swan, 1990; Kucera and Katuscak, 1992).  Generally, wood  colours can range from various shades of white to yellow, reddish to brown and gray to black.  Some wood species display splendid and  much-prized  for  colorations,  instance  mahogany  (Swietenia  macrophylla King) , black walnut (Juglans nigra L. ) , cherry (Prunus serotina Ehrh.), ebony (Diospyros ebenum Koenig), eastern redcedar (Juniperus virginiana L.) and they have been used for furniture and fancy goods since ancient times.  The coloration varies not only among different wood species but also within a species and often in the same piece of wood.  The  latter  and  colour  variation  can  often  be  caused  heartwood content, knots, or by grain orientation. interlocked  grain,  as  is  common  influences the colour intensity.  in  many  by  sapwood  For instance an  tropical  timbers,  Furthermore wood colours can  change with time, for instance true mahogany has a pinkish cast when freshly cut and turns into a rich reddish brown with age and  2 exposure to light (Panshin and de Zeeuw, 1980).  In fact most wood  species change their colour with exposure to light, heat and other environmental factors, becoming either lighter or darker depending on the factors involved (Fengel and Wegener, 1984) .  When changes in the coloration of woods cause an uneven or unwanted appearance, thereby decreasing their decorative market value, the problem  is commonly defined  discolorations  affecting  as discoloration.  light-coloured  wood  Generally wood species  are  very  detrimental because the natural light colour of these woods is readily disfigured.  In this context it is unfortunate that current  market trends favour the natural appearance of light-coloured woods for decorative purpose. artificially  In contrast dark-coloured woods including  darkened woods, which were highly regarded a few  decades ago, are less in demand for decorative purpose.  Western hemlock (Tsuga heterophylla (Raf) Sarg.) and amabilis fir (Abies amabilis  (Dougl.) Forbes) are both light-coloured woods.  They are highly regarded for their decorative appearance but they are  also  very  susceptible  to  abnormal  colorations.  The  discolorations affecting these wood species can vary from shades of grey and brown to black. In the current study brown discolorations were investigated in western hemlock with emphasis on the microbial involvement in the staining phenomenon.  Understanding the causes  of brownstains in western hemlock may enable some means for their control to be devised.  3 2.0  DISCOLORATIONS OF HEM-FIR WOOD:  A REVIEW OF THE MECHANISMS  2.1  Background of Non-microbial Stains  Wood discoloration problems have been known to lumber producers and customers worldwide for many years and have caused large economic losses to the wood industry (Hubert, 1926; Scheffer and Lindgren, 1940; Scheffer, 1973).  Discolorations can be divided roughly into  microbial ("biological") and non-microbial ("chemical") types.  The most obvious form of wood discoloration is sapstain caused by fungi. Numerous investigations have been conducted on microbial stains since Robert Hartig's first description in 1878, resulting in  an  understanding  prevention  of  biological  discolorations  and  their  (Munch, 1907; Lagerberg et al. 1927; Findlay, 1959;  Liese and Schmid, 1961,1964; Schmid and Liese, 1965; Zink and Fengel, 1988, 1989, 1990).  Non-microbial  wood  discolorations  are  not  understood  despite their common occurrence in both hardwoods maple, oak) and  as  well  (e.g., alder,  softwoods (e.g., pine, western hemlock).  The lack  of knowledge about chemical staining reflects the fact that these wood discolorations have long been considered to be less important because  of  their  generally  superficial  nature.  Thus  colour  disfigurations were commonly planed off, which normally removes the problem and restores the natural wood appearance.  4 However, advanced sawing technology such as thin-kerf sawing, which produces a dimension rather than oversized product, has renewed interest in non-microbial discolorations. buyers are  increasing  their demands  prefer light-coloured wood.  Furthermore, overseas  for kiln-dried  lumber and  These trends have led to increasing  concern about non-microbial wood discoloration.  Unfortunately, non-microbial wood discolorations are generally not understood and literature on the subject is limited.  This type of  stain commonly develops as wood dries and involves the formation of coloured polymers, the chemical structures of which are not clearly understood.  Chemical constituents of the particular wood species  and the role of uncertain factors microbial  infestation)  (e.g., temperature, humidity,  have to be understood  to determine the  cause(s) of non-microbial discolorations.  The complexity of non-microbial staining is reflected in confusing terminology; it is also termed chemical or oxidative stain. classification  was  recently  microbial wood discoloration  proposed  for  microbial  (Bauch, 1986).  and  A  non-  While most fungal  staining can be easily recognized, a full understanding of the causes  of  other  classification.  discolorations  is  required  to  Furthermore, it is not a trivial  use  Bauch's  task to be  certain that microorganisms or their enzymes are not involved in a chemical essential  stain.  A thorough knowledge of the discoloration is  to develop protective means to maintain the natural  5 appearance of the species.  2.2  Economic Significance of Discolorations in Western Hemlock and Amabilis Fir  Western hemlock and amabilis fir have only relatively recently been recognized as high value products of the British Columbia wood industry (COFI, 1983).  Historically, these species have had minor  end uses, for instance the production of tannin from hemlock bark for the booming leather industry in the 19th century 1989).  (Hergert,  The hemlock logs were often left in the woods (until 1900-  1910) because there was a lack of technology to convert them into lumber.  Competition from other wood species, for instance Chestnut  (Castanea sp.) or Quebracho (Schinopsis sp.), led to a decline in hemlock tannin production.  Today commodity lumber production in British Columbia economically  very  important,  worth  approximately  annually, of which about $5 billion is exported  (B.C.) is  $6.5  billion  (Goudie, 1992).  Western hemlock and amabilis fir are coastal whitewoods and sold as "hem-fir".  About 0.5 billion board feet of shop and better grade  hem-fir, worth approximately $800 million, are marketed annually from British Columbia (Byrne and Smith, 1991).  Hem-fir lumber is  successfully marketed in Europe and Japan where it is widely used for windows, doors, mouldings and other millwork.  Hem-fir lumber  is highly regarded for its wood quality, for instance fine grain,  6 strength, and ease of working (COFI, 1983).  Both species (western  hemlock and amabilis fir) present in Canadian hem-fir display an overall similar, whitish or tan, appearance, with only a slight difference between heartwood and sapwood.  Wood of the two species  can only be separated with certainty by microscopic examinations. Unfortunately,  the  bright,  particularly prone to darker  whitish  colour  of  hem-fir  is  (brown, black) disfigurations and  downgrade from shop and better to lower grades can result in an approximate 30% loss in value  (Byrne and Smith, 1991) .  sapstain fungi can be controlled  in hem-fir lumber  While  (Byrne and  Smith, 1991) brown discolorations, for which there are no known controls, are cyclically a market issue.  In 1990 a major problem  in the market for Canadian hem-fir in Europe led to industrial requests for additional information (Byrne, 1992).  2.3  Investigations of Hem-Fir Discolorations  Brown discolorations in clear-grade hemlock were first reported by Eades (1932).  Concerns were highlighted during the World War II  years as a result of the search for wood suitable for airplane manufacture (Eades, 1943; Englerth and Hansborough, 1945).  Western  hemlock was investigated for this application but discolorations found in the wood  led to speculation that  mechanical weakness was present.  incipient decay or  However, it was concluded that  discolorations did not indicate wood degradation or sapstain fungi and  no  significant  reduction  in  strength  properties  resulted  7 (Englerth and Hansborough, 1945) .  More  extensive  scientific  work  on  brownstain  in  hemlock  was  initiated in 1960 when mill operators from the Pacific coast of North America, from Oregon to B.C., reported a high incidence of brown discolorations (Evans and Halvorson, 1962).  A light or dark  brownstain was found to develop during both air and kiln drying but it affected the wood surface only, penetrating to a depth of no more than 0.5 mm.  In addition discoloration was noticed most often  in lumber from the sapwood-heartwood boundary region and was most apparent on the end or edge grain of affected lumber. colonization  in  stained  hemlock  sapwood  suggested  Bacterial a  relationship between bacteria and brownstain development.  possible Further  bacteriological studies revealed that discoloured hemlock contained sap constituents which could be oxidized to coloured polymers. Evans and Halvorson (1962) speculated that the oxidation involved a  bacterial  phenoloxidase,  which  triggered  polymerization  of  leucoanthocyanidins carried to the wood surface under favourable drying conditions.  Chemical screening trials demonstrated that  thiourea, an antioxidant, prevented surface browning and it was also assumed that brownstain was caused by atmospheric oxidation.  About the same time intensive research was conducted at the Western Forest Products Laboratory in Vancouver to elucidate the chemistry and biology of the brownstain  (Barton, 1962; Whittaker, 1962a).  Bacterial infection was found in some apparently healthy hemlock  8 trees and in felled logs but bacteria were frequently encountered in brownstained logs. bacteria  to  inconclusive  the  other  production  of  (Whittaker, 1962a).  the extractive Among  However, experiments to link the presence of the  brown  compounds  were  Barton (1962) did much work on  chemistry of amabilis  phenolic  discoloration  the  fir and western hemlock. presence  of  water-soluble  phenolics such as catechin, epicatechin and leucoanthocyanidin, which are known to polymerize under certain circumstances (Haslam, 1989), were identified in sapwood extracts. brownstain  in  expressed  hemlock  Barton, reproducing a  juice,  concluded  from  chromatographic examination that leucoanthocyanidin is probably not involved in brownstain formation. Attempts were made to chemically inhibit  brownstain  formation  with  pH  modifiers,  chelating  or  reducing agents, and it was reported that pH reducing components showed potential to moderate discoloration (Barton, 1962).  Cross-sectional distributions and seasonal changes of catechin, epicatechin and leucoanthocyanidin were demonstrated in hemlock sapwood  extractives  (Barton, 1963; Barton  and Gardner,  1966).  Subsequent work employing synthetic catechin produced a brownstain on a papergram when reacted with juice expressed  from hemlock  sapwood but only a slight discoloration developed with synthetic leucoanthocyanidin. The authors suggested interaction of an enzyme system with catechin, both constituents of sound hemlock sapwood, and subsequent oxidation, as wood dries, produces a brown polymer at  the  wood  surface.  However,  contribution  of  additional  9 compounds, for instance epicatechin or leucocanthocyanidin, to the final colour was not excluded. by  chemical  means  failed  Inhibition of brown stain formation  in  field  trials  although  chemicals  evaluated had shown promising results under laboratory conditions.  Several  serious  cases  of  an  intense,  black-brown  surface  discoloration found in kiln dried amabilis fir initiated chemical and anatomical research (Barton and Smith, 1971). on  discoloured  specimens  demonstrated  the  Microscopic work  presence  of  high  concentrations of dark-brown extractives in parenchyma cells and bacterial infection in longitudinal tracheids.  While cell wall  degradation of parenchyma cells and longitudinal tracheids was not significant, pit breakdown was observed.  Sections of discoloured  wood further revealed a change in fluorescence, when compared with sections of wood which was not discoloured,  suggesting  lignin  degradation. Chemical analysis indicated that 3,3'-dimethoxy-4,4'dihydroxystilbene (DDS) was responsible for the deep brown colour of kiln-stain in amabilis fir.  It was further hypothesized that  bacterial degradation of lignin might have produced DDS.  Chemical treatments, with 8 different compounds, did not prevent brown discolorations on stored hem-fir (Swan, 1984a).  However, it  was verified that brownstain developed in amabilis fir as well as in western hemlock.  The author concluded that basic research on  the biological and chemical aspects of brownstain was needed before a solution to this problem could be found.  10 A  recently  reported  discoloration  on  exported  hemlock  lumber  appeared as grey streaks along the wood surface (Smith and Spence, 1987) . Although macroscopically the discoloration did not resemble the  traditionalbrown  examination parenchyma  showed  stain brown  in  hem-fir  globular  lumber,  deposits  cells in the grey-streaked  wood.  authors saw fungal hyphae within the rays.  microscopic  within  the  Additionally  ray the  Isolation of fungi from  discoloured wood, which interestingly showed non-pigmented hyphae, yielded Ophiostoma piceae  (Munch) H. and P. Syd. and Sporothrix  sp. , a possible  stage  asexual  of  Ophiostoma  sp.  Subsequent  laboratory inoculations of wood with these fungi resulted in a brown discoloration. The authors suggested that oxidative activity of fungi growing within the rays caused polymerization of catechin, abundantly present in hemlock sapwood, and thus produced a grey or brown disfiguration in the lumber.  Other hem-fir samples sent to Forintek from European customers also displayed a grey or brown disfiguration as previously described by Smith  and  Spence  (1987)  and  brown,  globular  deposits  were  microscopically demonstrated  in ray parenchyma and to a lesser  extent  and  in  tracheids  (Byrne  Smith,  1991).  Unfortunately,  studies on the presence of fungi were not conducted but preliminary bacterial investigations showed the presence of bacteria in the pits of some samples (Byrne, 1992).  These grey discolorations of  hem-fir microscopically resembled grey stain found in oak (Clark, 1957; Forsyth and Amburgey, 1991), but in trials sodium bisulfite,  11 which can control grey oak stain (Forsyth, 1988), did not prevent colour formations in hem-fir (Byrne and Smith, 1991).  Further samples returned from a French customer showed a different discoloration presumably developing when hem-fir is kiln dried (Byrne and Smith, 1991) .  Preliminary studies indicated a light  red/brown coloration, occurring just under the wood surface.  This  discoloration was concentrated in earlywood giving a striped wood appearance which led to the term "zebra stain".  During  investigation  of  very  severe  cases  of  almost  black  discoloured samples, iron and/or manganese were detected using Xray spectroscopy  (Byrne and Smith, 1991).  The intensity of this  black colour increased with the amount of these elements present. Dilute phosphoric acid removed the black component and the metal ions, leaving behind the brown component of the discoloration. origin or source unknown.  of  these metals  in stained  samples  The  remained  Microbial investigations were not conducted, although  both iron and manganese play a role in the metabolism of some microorganisms.  Biogeochemical studies have also indicated that  western hemlock trees can accumulate high contents of copper and zinc (Warren and Howatson, 1947) and iron and manganese (Warren et al, 1952).  Furthermore these elements varied between different  parts of the same tree and appeared related to the age of the tree as  well  as  to  soil  type  and  climate.  High  accumulation  of  manganese was also recently demonstrated in the xylem of western  12 hemlock needles (Ballard, 1992).  Numerous agents have been evaluated suitable (Byrne  chemical  and  Smith,  treatments 1991).  in other attempts to find  to prevent The  brownstain  chemicals  tested  in hem-fir included  pH  reducers, chelating/sequestering agents, and reducing agents, but only  a  quaternary  ammonium  compound  (didecyldimethylammonium  chloride - DDAC) controlled brownstain in small specimens while drying under ambient laboratory  (2 0° C) conditions.  action of this chemical was not understood.  The mode of  However, field tests  using DDAC on commodity hem-fir lumber gave disappointing results and it was hypothesized that the quantity of extractives moving (longitudinally), as wood dries, through the end grain to the surface, might have exceeded the potential of the DDAC to inhibit brown stain formation.  Recently, a sample of amabilis fir with brown stain resulting from "kiln-burn" was  investigated  for causal  agents  (Sutcliffe  and  Miller, 1991) . A conversion of DDS to a highly coloured compound was demonstrated but it was hypothesized that other polyphenols could contribute to the discolorations.  In addition high amounts  of calcium indicated by energy dispersive X-ray analysis  (EDXA)  were observed on the surface of the stained specimen but manganese or iron were not present.  The effects of some kiln drying variables were investigated on the  13 development of brownstain in hem-fir lumber 1993) .  (Avramidis et al. ,  The authors concluded that low drying temperatures and a  more gradual drying reduced the incidence of brownstain during drying.  Furthermore,  Avramidis et al. (1993) reported that the  presence of less oxygen resulted in less stain development.  2.4  Extractive Chemistry of Western Hemlock  Undoubtedly  a  thorough  knowledge  of  the  composition  of  wood  extractives is required to fully understand the cause of brown discoloration in hem-fir products.  Many extractive compounds of  western hemlock were isolated and identified in the 1960s.  The  significance of these extractives as potential colour precursors is discussed in this section, providing examples from pulp and paper research.  In the U.S. Pacific Northwest large quantities of sawmill residues used in producing pulp are subject to discoloration during storage (Springer, 1983) .  Thus large losses in wood brightness occur and  in some instances pulp cannot be bleached to satisfactory levels (Springer, 1983).  Considerable research has therefore attempted to  elucidate compounds responsible for loss in wood brightness of chips or pulp during storage, as well as searching for ways of prevention.  14 In western hemlock, which is widely used for production of pulp, a variety of phenolic compounds give rise to undesirable chromophores (Barton, 1973a).  Extracts from western hemlock developed a red-  brown colour upon treatment with mineral acids in the presence of alcohol (Pigman et al. , 1953) . Flavan-3, 4-diols, which belong to the proanthocyanidins, compounds  appeared to be colour precursors.  could be either water-soluble  These  or water-insoluble  sapwood but heartwood contained largely the insoluble type.  in The  richest source of water-soluble flavan-3, 4-diols, however, was the inner  bark  coloured,  (including  cambium).  water-insoluble  reaction  Purified  phlobaphenes  products  of  tannin  (red-  extract  treated with mineral acid) were also suggested to be rich in flavan-3, 4-diols.  Research on the chemical composition of tannins and polyphenols from  conifer  woods  and  bark  has  demonstrated  that  monomeric  catechins and leucoanthocyanidins were commonly present in sapwood and cambium of all species studied (including western hemlock) but absent in heartwood (Hergert, 1960).  In place of these compounds  polymeric proanthocyanidins (flavan-3,4-diol type) were present in the  heartwood.  The  author  intensely coloured heartwood  further  noted  that  species  with  (e.g., Douglas-fir) also exhibited  high amounts of flavan-3,4-diols in the sapwood but species with light coloured heartwood displayed low flavan-3,4-diol contents. Polymeric tannins in hemlock, however, were suggested to be built from  compounds  such  as  catechin,  epicatechin,  gallocatechin,  15 epigallocatechin and leucoanthocyanidin (Hergert, 1960).  Cambium  and  presence  sapwood of western hemlock were  of  low  molecular-weight  examined  constituents  as  for the potential  intermediates in lignin formation (Goldschmid and Hergert, 1961). Cambium  and  glycosides  sapwood of  contained  lignan-like  alicyclic  compounds,  acids,  sugars  lignans  and  and  catechin,  epicatechin and leucoanthocyanidin but depsides of cinnamic acid derivatives were present in cambium only.  Phenolic extracts of western hemlock, possibly involved in brown discoloration of hemlock lumber and low brightness of hemlock pulp, were  reviewed  extractives,  by  such  Barton as  (1968) .  catechin  and  Flavonoid-type leucoanthocyanidin,  phenolic known  precursors of highly coloured tannins and polymeric phenol, were strongly suggested as producing brown stain.  Lignans such as  coniferin, hydroxymatairesinol or matairesinol were thought not to contribute to colour formation. Lignans, colourless or pale lemoncoloured substances, lack vicinal hydroxyl groups and their stable ring  system  products. suggested  was  believed  to  Guaiacylglycerol, to  form  colour  preclude a  simple  precursors  coloured  oxidation  by-  phenolic  compound,  was  under  acid  or  alkaline  conditions but the relatively small amount present precluded its significant participation. lignin dimer attached  (phenyl  through  A glycoside of a recently detected  coumaran),  an alcoholic  in which  the sugar moiety  linkage, was  also  suggested  is as  16 contributing to coloured products in pulp and lumber.  Another lignan, liovil, was identified after its isolation from western hemlock sapwood (Barton, 1970).  In addition, the structure  of a recently detected new lignin dimer in western hemlock was determined.  The  significance  of  these  compounds  in  colour  disfiguration was described (Barton, 1968).  The chemical composition of the extractives and their effect on optical properties of western hemlock pulp was investigated by performing  three  treatments:  a  sequestering  (ethylenediaminetetracetic acid - EDTA) treatment, ethanol-benzene extraction and acetone-water extraction  (Polcin et al., 1969).  Brightness of pulp was improved by EDTA treatment and removal of copper and manganese cations, previously reported to be present in relatively high amounts in western hemlock groundwood  (Wayman et  al. , 1968), was suggested as one possible explanation for this observation. Ethanol-benzene extraction did not improve the colour of  pulp.  polyphenols  However, and  low  extraction molecular  with  weight  acetone-water phenolic  removed  compounds  and  improved pulp brightness.  A subsequent study examined the heat stability (105°C for 18 hours) of three (EDTA, ethanol-benzene, acetone-water) extractions from western hemlock and also model compounds absorbed onto sheets of pure bleached cotton  (Polcin and Rapson, 1971).  Acetone-water  17 extracts  of  western  hemlock  sapwood  developed  a  yellow-brown  colour, presumably by air oxidation, soon after evaporation of the solvent.  Investigating the contribution of model compounds to the  discoloration indicated the importance of the flavan-3ols such as catechin. brown  Heat treatment experiments with d-catechin produced a  colour  but  addition  of  unsaturated  fat  to  d-catechin  substantially intensified the discoloration. The discoloration was even more intense when d-catechin was heated in the presence of water. The authors concluded that oxidation of d-catechin occurred at  elevated  temperatures,  forming  simple  and  complex  quinoid  structures.  It was further suggested that peroxi-radicals were  involved  the  in  oxidation  of  d-catechin  in  the  presence  of  unsaturated fats and water producing an increase in discoloration. While  formation of  free radicals  is known to increase  in the  presence of some metallic (e.g., copper, manganese) cations it was concluded that removal of these cations by EDTA treatment prevented heat discolorations of groundwood pulp.  Changes in western hemlock wood extractives during refining and reduction of chromophore production during and after refining by chemical applications have been studied (Barton, 1973b).  During  refining, high temperatures (exceeding 8 0° C) were recommended to modify flavonoid and lignan materials as well as lignin precursors and lignin.  Several chemical additives such as acids, reducing  agents and oxidising agents improved brightness of western hemlock mechanical pulp.  18 Chemical agents for maintaining the brightness of stored western hemlock  wood  (Springer,  chips have been evaluated  1983).  Dilute  (2%)  in a laboratory  aqueous  solutions  of  trial sodium  bisulfite enhanced and maintained the brightness of chips during 12 weeks storage.  Bisulfite  ions, effective biocides and enzyme  inhibitors, were demonstrated to oxidize in solution to bisulphate ions causing a large decrease in pH; liquors squeezed from western hemlock sapwood dropped from 5.4 to 2.6.  Loss of brightness in mechanical pulp as a function of storage time of  western  hemlock  chips  was  correlated  with  a  decline  in  concentration of d-catechin monomers (Hrutfiord et al., 1985).  It  was hypothesized that oxidation of d-catechin produced a brown polymer, which was retained in the wood fibres of freshly chipped western hemlock.  2.5  Factors Which May Contribute to Discolorations in Hem-Fir  Although this section has summarized the most significant research on discolorations in hem-fir it is useful to speculate on factors which may play a role in these colour formations.  Examples will be  given from research on stains found in other wood species.  19 2.5.1.  The  Factors Inherent in Living Trees  occurrence  of  discolorations  recognized for many years 1984; Shortle, 1984).  in  living  trees  has  been  (Sachs et al. , 1966; Bauch and Baas,  It is possible that conditions leading to  discoloration of felled logs and wood products may have already been  initiated  in  the  living  tree.  The  following  examples  illustrate links between the living tree and discolorations in wood products  where  predisposition  of  the  wood  substrate  to  discoloration may have originated in the tree.  Discolorations wounding.  in living trees have been  shown  to arise  from  For instance, broken branches, pruning, severe logging  damage (Shigo and Hillis, 1973; Phelps and McGinnes, 1984), and/or dying branches  (Aufsees, 1984)  have been  shown  to cause wood  discolorations. Minor wounds may produce slight colour changes due to the formation of chemical protection barriers. wounding  can  lead  to  interactions  between  However, severe microorganisms  (sapstainers, decay fungi) and living cells, producing  intense  colour disfigurations in the living tree (Shigo and Hillis, 1973).  Discolorations have also been found in living trees attacked by insects, for instance bark maggots (Cheilosia alaskensis Hunter) causing black streaks in western hemlock (Englerth and Hansborough, 1945; Moeck, 1968) or by the symbiotic fungi of ambrosia beetles which cause localized sapstain  (Funk, 1965) .  In a recent study  20 discolorations induced by larvae of Sermanotus japonicus Lacordaire were demonstrated in living trees of Cryptomeria japonica D. Don. (Yamada et al., 1987).  In this study cation (K, Mg) accumulation  appeared to explain an increase in pH over a five year time period. Although microorganisms (bacteria and non-hymenomycetes) were found in the discoloured areas, cation concentration, but not composition of the microflora, has been proposed to explain the advance in discoloration.  Thus a shift in pH may have triggered polyphenolic  reactions of accessory compounds. manganese  Interestingly, accumulation of  (Warren et al., 1952; Ballard, 1992) or copper (Warren  and Howatson, 1947; Wayman et al. , 1968) has been detected in western hemlock trees.  The role of bacteria in standing trees as a factor in discoloration also  requires  consideration.  Bacteria  apparently healthy western hemlock trees  have  been  shown  in  (Whittaker, 1962a) and  have also been associated with wetwood present in softwoods, for instances in western hemlock (Bauch et al. , 1975; Ward and Zeikus, 1980) and white fir  (Abies concolor  (Wilcox and Oldham, 1972).  (Gord. & Glendl.) Lendl.)  Bacteria have also been associated with  wetwood in hardwoods such as Populus sp. (Knutson, 1973; Sachs et al.,1974; van der Kamp et al. , 1979; Scott, 1984; van der Kamp, 1992) and Fagus sylvatica L.  (Walter, 1993) .  In addition to  participation in wetwood formation bacteria have been linked to the pH  of  the  wood  and  to polymerization  producing discolorations  of  phenolic  compounds,  (Schink and Ward, 1984; Schmidt, 1986;  21 Schmidt and Mehringer, 1989).  Bacterial infestation of western  hemlock trees has been specifically suggested as a causal factor in discoloration  of  lumber  produced  from  such  trees  (Whittaker,  1962a).  2.5.2  Felling Season  In temperate zones trees undergo seasonal changes which are most obvious in deciduous wood species because of defoliation in fall. Metabolism of trees is reduced during the winter season and this is reflected  in a decrease  seasonal  changes  also  in water uptake and sap flow. influence  the  composition  of  These certain  compounds in living trees.  Extractives possibly involved in discolorations of hem-fir lumber were also shown to undergo seasonal changes in living western hemlock trees (Barton and Gardner, 1966) .  For instance maximum  catechin levels were detected in April, May and June, declining to lower but still significant levels in July - October.  In kiri wood  (Paulownia  tomentosa  Steud.) peroxidase  activity  triggering discoloration was demonstrated to occur in September and October at levels 12 fold those of June or November (Ota et al. , 1991) .  The authors suggest that harvesting Kiri trees should be  avoided during this time.  22 2.5.3  Post-Mortem Factors  Post-mortem changes causing discolourations in the sap of trees upon exposure to air have been reported in hardwoods and softwoods (Bailey, 1911).  For instance freshly cut Alnus sp. or Liquidambar  sp. can develop discolorations within hours after sawing under favourable conditions of temperature and humidity.  Bailey (1911)  demonstrated that heating of the woods in boiling water controlled discolorations thus indicating a plant enzyme as the cause of the problem.  An orange coloured polymer, oregonin has been identified developing in red alder Hrutfiord  (Alnus rubra Bong.) after sawing  and  Luthi  (1981)  demonstrated  (Karchesy, 1992).  that  phenoloxidases  reacting with accessory organic compounds after oxygen penetrated the wood tissue produced the observed colour formations in red alder.  A similar reaction causing discolorations in freshly sawn Kiri wood has  been  shown  (Ota  et  al. , 1991).  Disruption,  or  spatial  separation of enzymes (peroxidases) and wood extractives (Ota and Taneda,  1989),  caused  by the  sawing  action, was  suggested  as  producing the colour reaction.  Peroxidase activity on phenolic extractives has been demonstrated to cause brownstain in sugar pine (Pinus lambertiana Dougl.), in  23 eastern white pine (P. strobus L.) and in western white pine (P. monticola  Dougl.)  (Stutz, 1959; Stutz et al. , 1961).  Unlike  brownstain in white pines which was shown to also develop under air seasoning  conditions,  kiln  discolorations in sugar pine. pines  and  sugar pine  drying  was  required  to  produce  The control of brownstain in white  has been  shown  in  laboratory  and  field  experiments (Stutz, 1959; Stutz et al. , 1961; Arganbright, 1972; Shields et al., 1973; Oldham and Wilcox, 1981).  2.5.4  Log Age  The time elapsed important  from stump to saw has been suspected as one  factor affecting the formation of hemlock brownstain  (Evans and Halvorson, 1962) . The significance of log age was also underscored  in a survey on brown discolorations  in sugar pine  developing during kiln-drying (Herman, 1937) . Subsequent research on  sugar pine  indicated  that  nine month old  sugar pine  logs  discoloured three times as much as fresh ones (Rasmussen, 1940). It was speculated that higher oxygen tensions or higher levels of insect damage could cause the "log age effect" observed in pine species (Stutz, 1959).  Brown discolourations in maple (Acer pseudoplatanus L.) logs which intensified  with  time  have been reported  (Koltzenburg,  1974).  These discolorations can appear without microbial interaction at a given ratio of oxygen, temperature and wood moisture.  However,  24 bacteria were demonstrated to produce a similar brown discoloration in the same wood species (Zimmermann, 1974).  In a detailed study  (Starck et al. , 1984; Bauch et al. , 1985;  Yazaki et al. , 1985) a reddish-brown discoloration found on the end grain of freshly cut logs or on the surface of freshly cut lumber of Ilomba (Pycnanthus angolensis Excell) was traced to the presence of bacteria.  The authors suggested that some bacterial strains,  for instance Pseudomonas fragi Hussong et al. , can alter the pH of the  wood  from  about  5.5  to  7.5  reactions of accessory compounds. wood  surface  with  formic  discolorations (Bauch, 1986).  acid  thereby  triggering  chemical  Stabilization of the pH of the was  reported  to  inhibit  In addition, (+)-catechin and  (-)-epicatechin were suggested as possible contributors to colour formation in Ilomba (Yazaki et al.,1985).  It should be emphasized that changes within the logs, for instance in moisture content and wood temperature, and chemical changes of accessory compounds, are more likely to be responsible for colour formation than log age per se.  The role of microbial populations  interacting with certain wood extractives must also be considered in producing discolorations.  In this context, however, it is  interesting to mention that an aging process has traditionally been used on Kiri logs to prevent a discoloration, which develops when lumber is immediately sawn from fresh-cut logs (Ota et al., 1991) . Thus Kiri logs are exposed to the weathering action for 6-9 months  25 followed by an outdoor exposure of the sawn boards for another two years to enhance the appearance.  2.5.5  Log Storage  Delay between felling of logs and processing into kiln-dried lumber probably increases the risk of potential discolorations (including sapstaining)  in most wood  species.  Unfortunately,  it is not  possible to totally avoid delays and thus western hemlock and amabilis fir logs are commonly stored from several months to 1.5 years before sawing (Kreber and Byrne, 1993). In most cases short term storage of logs is in dry decks but for longer periods water storage is employed.  Microbial infestation can be controlled for  long time periods by keeping wood in water saturated condition (Liese and Peek, 1984).  However, logs stored in ponds or sprinkled  are known to develop high bacterial populations  (Smith, 1975).  Bacteria may cause changes in wood permeability  (Unligil, 1972;  Johnson, 1979) due to decomposition of pit structures (Liese and Karnop, 1968; Greaves, 1969).  Several species of Bacillus (Ellwood  and Ecklund, 1959) and Pseudomonas (Grosu et al., 1973) have been reported  in ponded  logs.  Discolorations  of  lumber  sawn from  sprinkled hardwood and softwood logs have been attributed to the presence of bacteria (Stout, 1959; Stutz, 1961; Lane and Scheffer, 1969; Hedley and Meder, 1992) .  26 Water  storage/ponding  of  logs was  also  beech  (Hoster,  1974)  discolorations  in  accelerate  development  (Forsyth  the and  Amburgey,  of  1992).  gray  reported and  to  to  promote  discolorations  The  influence  effectiveness  and  in red oak of  sodium  bisulfite in preventing discoloration of lumber sawn from freshlycut oak, decreased dramatically with increasing storage time of the sawlogs (Forsyth and Amburgey, 1992) .  Water storage or floating of logs has been suggested as a means to redistribute accessory compounds  (flavonoids) from the phloem of  unpeeled spruce logs into the xylem (Adler, 1951). logs are often floated to mill yards.  In B.C. hem-fir  Interestingly it has been  claimed, that western hemlock trees felled and immediately salt water floated produced less discolorations than fresh water floated logs (Barton, 1962).  2.5.6  Storage of Lumber  Storage of unseasoned lumber in close piles, the common practice for exported lumber, decreases air drying rates significantly. This may influence colour formation in hem-fir.  Storage of lumber in bulk piles was demonstrated to influence brown discolorations in sugar pine lumber  (Stout, 1950) .  The author  showed that pine lumber stacked in bulk piles between sawing and stickering for more than 5 days developed severe discolorations.  27 These observations were also confirmed for close-piled, white pine lumber (Cech, 1966) .  In a recent study developing scleroxylon  (Hansen, 1988) green and red discolorations  on the surface of K.  Schum.)  formation was most  boards  noticeable  freshly were  sawn Samba  investigated.  in the  centre  of  (Triplochiton The  colour  stacks.  The  presence of a bacterium Pseudomonas aerucrinosa Migula was suggested to have produced the discolorations.  The author proposed faster  drying of the wood and treatment with a bactericide to prevent discolorations in this white wood species.  A bright yellow discoloration has been reported in heartwood of green  oak  lumber  when  stored  with  insufficient  ventilation,  particularly when thin stickers did not permit drying  (Bauch et  al. , 1991) . The mould Paecilomyces variotii Bain, was suggested as causing the colour formation from hydrolyzable tannins. (yellow) discoloration was also found  The same  in kiln dried oak.  P.  variottii, however, was shown to tolerate acidic substrates as well as high (50° C) temperatures, conditions which are prevalent during the first stage of kiln drying.  The authors concluded that growth  of P. variotii was probably not inhibited during the initial stage of kiln drying thus explaining yellow discolorations found during drying.  28 2.6  Summary  Discolorations of hem-fir, other than those caused by sapstain fungi, have become an economically important problem.  With the  move towards increased kiln-drying of the wood species mixture and to  more  added-value  products,  such  discolorations  are  less  tolerable.  Although discoloration of hem-fir lumber has been a puzzle for many years,  knowledge  of  the  cause(s)  remains  rudimentary.  Most  research into hem-fir discolorations has been conducted on only a few wood samples.  While polymerization of wood extractives has  been proposed as the probable cause, involvement by bacteria and fungi has also been suggested in the literature.  Factors involved  in discolorations of other wood species, such as factors inherent in the living tree, season of tree felling, post mortem changes, and  log  age  understanding  and of  storage, may the  cause(s)  also be of  involved.  hem-fir  A  thorough  discolorations  is  necessary before recommendations or preventive treatments can be devised to maintain the natural colour in hem-fir products.  29 3.0  ADVANCES IN THE UNDERSTANDING OF HEMLOCK BROWNSTAIN  3.1  Objectives  The objectives of this study were to: a) describe the location of the stain in the wood tissue; b) elucidate the composition of brownstain deposits histochemically; c) evaluate the potential of three Ophiostoma piceae (Munch) H. & P. Syd. strains and a mixed bacterial culture to produce brownstain in sap of western hemlock, amabilis fir and lodgepole pine and in western hemlock wood.  3.2  Material and Methods  3.2.1 Microscopic Examinations  Representative samples showing various types and intensities of colorations,  were  selected  brownstained specimens.  from  Forintek's  collection  of  The specimens came from edge-grain stock  and had been sent from Europe to illustrate the market problem. Transverse and radial sections (15-20 /xm) were prepared from twenty five discoloured samples of western hemlock and amabilis fir using a sliding microtome.  Sections were dehydrated through a series of  ethanol solutions, and finally passed through xylene and mounted using Permount resin (Fisher Scientific, Nepean, Ontario). Light microscopy and phase contrast microscopy were employed to examine specimens using a Zeiss photomicroscope.  30 3.2.2 Histochemical Examinations  Radial sections were also sampled from freshly sawn hem-fir boards and from small (66 x 18 x 6 mm) sapwood beams of western hemlock as outlined (see section 3.2.1).  Additional hem-fir sections showing  microscopical brown deposits, were extracted either in 10 mL of methanol or in 10 mL of acetone/water bath  for  two hours  at  room  (7:3) using an ultrasonic  temperature.  Every  section  (non  extracted or extracted) was placed in 1% 2,4 dimethoxybenzaldehyde (DMB) reagent  (Aldrich Chemical Co., Milwaukee, WI) prepared as  described by Mace and Howell (1974) mounted on a glass slide in a drop of DMB and then stored at room temperature to yield a red coloured  product  when  reacting  with  catechin  and  its  derived  tannins (Halloin, 1982) . Control sections were prepared according to the procedure outlined but DMB was omitted from the reagent. Wood  species  microscopically lactophenol.  and  presence  verified Microscopic  on  of  brownstain  additional  examination  of  deposits  sections  mounted  treated  samples  was in was  performed using light microscopy.  3.2.3 Sap Experiments  Sampling and sterilization of sap: Two freshly sawn boards of western hemlock  (hereafter referred to as Hem-1, Hem-2), one of  amabilis fir and one of lodgepole pine (Pinus contorta (Dougl. ex. Loud.)) were used as sources of sap.  Visual examination of the  31 boards indicated no signs of brownstain or sapstain but ambrosia beetle  infestation  was  noticed  in  some  regions  of  Hem-2.  Microscopic examinations verified the presence of brown deposits in the amabilis fir and western hemlock.  Sap was pressed from each board using a hydraulic press and was filtered twice through Whatman # 1 filter paper followed by filter-sterilization using a 0.2 pirn cellulose acetate membrane (Nalge Company, Rochester, N Y ) . The sterilized sap was stored in a refrigerator (4°C) overnight.  Microorganisms used: Ophiostoma piceae strains WFPL # 3871, WFPL # 387K, WFPL # 387T were cultured on 1.5% malt agar plates for approximately 5 weeks at 25°C. mycelial  fragments  was  A suspension containing spores and  prepared  scraping the culture surface.  from  each  culture  by  gently  Mycelial fragments and spores were  then washed from the Petri plates using sterile, distilled water. The crude fungal suspensions were individually blended in a Waring blender for 15 seconds and then drained into sterile 16 oz glass jars.  Sterile, distilled water was added to give approximately 300  mL of each fungal suspension.  A mixed bacterial culture (Mix-B) isolated from the sap of a board of western hemlock which showed brownstain, was cultured in a 250 mL Erlenmeyer flask containing 50 mL of nutrient broth (Difco Laboratories,  Detroit,  MI)  for  approximately  24  hours.  A  32 suspension was prepared by adding about 5 mL of the Mix-B nutrient broth culture to approximately 300 mL of distilled, sterile water.  Inoculation  of  filter-sterilized  sap: Approximately  20 mL  of  filter-sterilized sap was added to a series of autoclave-sterilized (121°C, 30 minutes) 125 mL Erlenmeyer flasks and inoculated with 0.5 mL of either bacterial or fungal suspension using a sterile pipette tip.  One replicate was set up for each treatment and for  each control containing no inoculum.  Flasks were stored in a  laminar flow hood at room temperature (22°C) for three weeks.  Evaluation  of  changes  in  inoculated  sap:  Colour  inoculated sap was visually recorded over time.  changes  in  In addition, the  pH of filtered saps was measured prior to inoculation and again after a three week incubation period. Readings were taken while stirring using a pH-meter. Qualitative HPLC analysis, as described by Kreber and Daniels (1993), was conducted on inoculated Hem-2 sap after  incubation  and  was  compared  to  HPLC  analysis  of  the  uninoculated control and freshly pressed Hem-2 sap.  3.2.4 Solid Wood Experiment  Inoculation of solid wood: Small (66 x 18 x 6 mm) sapwood beams of western hemlock, showing no brownstain (verified microscopically) , were used in this study.  The beams had been cut from a freshly-  felled tree within 24 hours of felling and they had been kept in a  33 freezer for 3 years.  Four beams were employed for each treatment,  placed on a polypropylene mesh in an aluminum tray containing 3 sheets of cotton.  To keep the chamber moist, distilled water  (90 mL) was added to each tray and trays were then autoclaved (30 min, 121°C) prior to inoculation.  Each wood sample was dipped  in inoculum (fungal or bacterial suspension prepared as described above) for three seconds prior to being placed in the sterile tray. Each tray was sealed in a polyethylene bag to maintain a moist environment and stored at room temperature  for six weeks.  In  addition, four control beams were dipped in sterile, distilled water only.  Assessment of brownstain in inoculated wood beams: Two wood beams selected at random were removed from each treatment after 4 and 6 weeks.  Radial sections (15 /xm) were prepared as described above,  mounted  in  lactophenol  and  the  determined with a photomicroscope. specimens  were  compared  to  temperature or in a freezer.  extent  of  brownstaining  was  Sections from treated wood  untreated  controls  kept  at  room  34 3.3  Results  3.3.1 Microscopic observations on discoloured hem-fir  Generally, microscopic examination indicated similar distribution of brown deposits for all discoloured samples investigated.  This  observation was rather surprising since specimens were sampled from boards showing different macroscopic  patterns of  discoloration  (Figure la-c).  Brown deposits were most noticeable in rays but they varied in colour intensity  (pale yellow to dark brown),  spherical, pillow-like) apparently  random  and size  distribution  (Figure 2 ) .  in shape  Furthermore, an  of brown deposits was  across and between growth rings.  (e.g.,  observed  In severely stained samples the  lumina of ray parenchyma cells were seen to be filled with chestnut brown deposits.  Ray tracheids in western hemlock were never found  to contain such deposits.  Although brown chromophores entered the  half-bordered pit from parenchyma cells there was no penetration through the margo into the ray tracheids.  Axial tracheids with deposit-filled lumina were rare in sections examined.  Tracheids with a filled lumen were often isolated among  stain-free tracheids  (Figure 3) .  In most cases the lumina of  discoloured tracheids were lined with a thin deposit ranging from yellow to brown in colour.  35  JiJ  Figure la: Representative specimen showing grey stain on the wood surface.  36  Figure lb: Representative specimen showing brownstain on the board end and on the wood surface.  37  I h r \n  Figure lc: Representative specimen showing zebra stain on the wood surface.  38  1*  i  .ill  Figure 2: Abies amabilis. Radial section (63x), ray parenchyma cells with brown deposits.  9& .j.  2  f  ct  #  Figure 3: Tsuaa heterophylla. Transverse section (lOOx), tracheids with a filled lumen among stain-free tracheids.  40 Fungal hyphae were frequently seen in ray parenchyma and tracheid cells and they were most often hyaline. rays  and within  the tracheids were  Some hyphae within the  seen enclosed  in coloured  deposits or surrounded by a brown sheath (Figure 4 ) . Hyphae were also detected in some stain-free cells.  Bacteria were frequently observed in bordered pits in otherwise unstained  tracheids  but  bacteria  were  also  seen  enclosed  in  coloured deposits in the lumena of tracheids (Figure 5 ) .  3.3.2 Histochemical observations  Sections  prepared  from  sapwood  beams  of  clean,  fresh  western  hemlock showed no signs of brown deposits in either parenchyma cells or axial tracheids, as verified microscopically. sections  mounted  in DMB  developed  a  red  reaction  However,  product  in  parenchyma cells and axial tracheids within six hours suggesting presence of catechin/epicatechin (Figure 6, 7 ) . The intensity of DMB reaction varied between and among both parenchyma and tracheid cells but the colour change was not observed when specimens were mounted in the absence of DMB.  Every section prepared from each of the hem-fir boards showed brown deposits.  The presence and spatial distribution of brown deposits  within sections confirmed microscopic observations as described above.  DMB also demonstrated a red reaction product in brown  41  Figure 4: Tsuqa heterophylla. Radial section enclosed in coloured deposits of a tracheid.  (200x),  hyphae  42  f»  Figure 5: Tsucra heterophylla. Radial section infection of tracheids.  (lOOx) , bacterial  43  Figure 6: Tsuqa heterophylla. Radial section (80x), red DMB reaction products observed in ray parenchyma cells.  44  I  " L  J  *  mJ  Figure 7: Tsuaa heterophylla. Radial reaction products in axial tracheids.  section  w1  (80x), red DMB  45 deposits  within  6 hours  in the  sections  including  additional  sections which had been extracted in methanol or acetone/water prior to treating with DMB.  Sections mounted in the absence of DMB  produced no colour change.  3.3.3 Colour changes in inoculated sap  Microbial inoculation gradually produced a distinct colour change only in sap of Hem-2 when compared to the control.  After three  weeks a brown colour was recorded in all samples of inoculated Hem2 sap irrespective of the inoculum used.  Unlike  uninoculated,  uncontaminated  sap,  the  pH  changed  in  inoculated sap of Hem-2 from pH 5 to above pH 8 over the three weeks; interestingly a similar pH shift was recorded in each of the treated Hem-2 sap samples independent of the inoculum (Table 1 ) .  Qualitative HPLC analysis (data not shown) of inoculated compared to uninoculated  sap of Hem-2,  indicated  that higher molecular  weight compounds were no longer present, while the proportion of lower molecular weight compounds considerably increased. Also many fewer compounds were detected in inoculated sap of Hem-2 after incubation using HPLC analysis.  These observations were recorded  for all treatments and independent of inoculum type. control  sample  (uninoculated)  Although the  of Hem-2 was not discoloured, a  slight turbulence (possible contamination) was noted.  This may  46  TABLE 1: CHANGES IN pH OF SAP AFTER THREE WEEKS OF INCUBATION. WOOD SPECIES Treatment  Western hemlock (1)  Western hemlock (2)  Lodgepole pine  Control/frozen  5.7  5.9  5.2  7.0  Control/22°C  8.41  8.62  4.72  7.0  0.p_.3 3871  8.7  8.4  8.2  8.7  0.£.  387K  8.6  8.1  8.5  8.3  0.p_.  387T  8.7  8.2  8.4  8.4  8.7  7.5  8.5  8.7  Mix-B4 1 2 3 4  Amabilis fir  = = = =  sap indicated heavy contamination sap indicated slight contamination Ophiostoma piceae (Munch) H. & P. Syd. mixed bacterial culture isolated from a western hemlock board showing brownstain  47 have caused the minor changes observed in the phenolic composition.  Treated sap of amabilis fir, which already showed a light brown colour at the time it was pressed, demonstrated some darkening over time but controls (uninoculated) became contaminated and developed brownstain. of  colour  Therefore the effect of inoculation on the production changes  in  sap  of  amabilis  fir  was  inconclusive.  However, a brown precipitate was observed in amabilis fir sap independent of treatment.  Measurements of pH indicated shifts in  pH similar to those recorded in sap samples of Hem-2 (Table 1 ) .  Neither the sap of Hem-1 nor of lodgepole pine incubated with microorganisms changed colour.  Interestingly an increase in the pH  of Hem-1 and lodgepole pine sap was also recorded after incubation and again this pH shift was independent of treatment (Table 1 ) .  3.3.4 Production of brownstain in inoculated western hemlock  Microscopic examination of frozen, clean western hemlock sapwood beams showed no brown deposits.  However, ray parenchyma cells  contained oval and rounded, colourless globules (Figure 8) instead of the brown deposits previously described.  These  colourless  structures were accumulated in particular ray parenchyma cells but they were not necessarily present in adjacent cells. Ray tracheids or axial tracheids did not contain colourless globules.  48  Figure 8: Tsuga heterophylla. Radial section (320x), colourless globules observed in clean ray parenchyma cells.  49 Examination of control (autoclaved, non inoculated) specimens demonstrated a few small, yellow to light brown deposits in ray parenchyma cells but these had increased in number after 4 and 6 weeks.  Light-coloured  deposits  were  recorded  in  a  few  parenchyma cells following autoclaving (121°C, 30 minutes).  ray Thus  heating alone produced some coloured globules, however, atmospheric oxidation was shown to form additional internal discolorations.  Specimens inoculated with # 3871 strain of 0. piceae showed hyphae in the tracheids and in ray parenchyma cells after 4 and 6 weeks of incubation.  Brown deposits were seen in ray parenchyma cells  containing hyphae and also in cells apparently free of hyphae but they were not seen in ray tracheids or in axial tracheids (Figure 9) .  Several  discolorations.  axial  parenchyma  cells  also  demonstrated  brown  A considerable increase in brown deposits and  numerous small brownish particles was seen in ray parenchyma cells when compared to controls after 4 and 6 weeks.  In some cases light  brown deposits completely filled the lumen of ray parenchyma cells.  Hyphae and brown deposits were less frequent in wood inoculated with # 387K or # 387T when compared to specimens inoculated with # 3871.  Wood inoculated with Mix-B demonstrated a few small, light brown deposits in ray parenchyma cells but smaller, light brown particles were more frequent than in controls.  Surprisingly, bacteria were  50  I  m •  HPNI  i l l tfmMLm**tt  Figure 9: Tsuga heterophylla. Radial section (160x), presence of brown deposits in O. piceae #3871 inoculated wood after 6 weeks.  51 rarely observed in either ray parenchyma or tracheid cells of the inoculated wood.  3.4  Discussion  The pronounced occurrence of brown deposits in ray parenchyma cells indicates their central role in the process of hemlock brownstain. Biosynthesis of phenolic substances occurs in ray parenchyma cells as they undergo cytological  changes upon aging  Parameswaran and Bauch, 1975). cells  throughout  distribution  of  the  It is also known that parenchyma  sapwood  nutrients  but  (Fengel, 1970;  are are  engaged also  in  involved  storage in  and  heartwood  formation (Panshin and de Zeeuw, 1980) .  Proanthocyanidins  (PAs), condensed tannin precursors  (Hemingway,  1989) are colourless but they undergo chemical changes, coincident with disintegration of cytoplasm as wood matures (Stafford, 1988) . In some cases PAs may come into contact with enzymes such as polyphenoloxidases  or  peroxidases,  forming  condensed  tannins  (Stafford, 1988).  The localized nature of the distribution of brown deposits is quite striking. PAs  Possible explanations are the variable distribution of  (Stafford,  1988)  and  the  high  degree  of  physiological  specialisation of ray cells even within an apparently homogeneous ray (Sauter and van Cleve, 1989).  52 The occurrence of discoloured tracheids surrounded by stain-free tracheids was seen in this study, and agreed with a recent report (Ellis  and  Avramidis,  1993) .  These  authors  suggested  that  physical-chemical differences between tracheids may create critical conditions which are necessary for formation of stain.  They also  reported that the lumina of some tracheids were lined with thin deposits.  However, in the current study brown chromophores were  also seen in bordered pits (Figure 10) and in intercellular spaces. Thus for the first time this microscopic study has provided strong evidence that precursors of hemlock brownstain are mobile.  The presence of microorganisms in wood specimens examined is of particular interest.  Bacteria were frequently present in bordered  pits but bacterial degradation of cell wall substance was rarely seen, unlike the observations recorded for kiln burned amabilis fir (Barton and Smith, 1971) .  Bacteria have been demonstrated to  induce discolorations in other wood species (Bauch et al. , 1985; Hedley and Meder, 1992).  In this study fungi, rather than bacteria  were frequently seen in discoloured hem-fir samples, the hyphae being observed in both rays and tracheids.  Hyphae were usually  hyaline and were frequently enclosed in a coloured deposit. Of particular interest was an observation showing hyphae entering the lumen of a stain-free tracheid via a bordered pit but the hyphae were enclosed in brown sheaths (Figure 11).  53  Figure 10: Tsuga heterophylla. Transverse section (250x), brown deposits in a pit connecting discoloured tracheids.  54  Figure 11: Tsuga heterophylla. Transverse section (400x), hyphae surrounded by a brown sheath in an otherwise unstained tracheid.  55 This observation agreed with a recent study in which the presence of  non-pigmented  0.  piceae  or  Sporothrix  sp.  was  strongly  correlated with brown deposits in hem-fir lumber (Smith and Spence, 1987).  Yellow discolorations recorded in green oak (Quercus sp.)  heartwood were explained as being caused by a mold fungus altering the cell contents (Bauch et al., 1991).  In the current study the  fungus appeared to modify the cell contents and produce a brown polymer locally around the hyphae.  In this study DMB reacted to catechin and epicatechin when spotted on filter paper at low (50/xg/mL) concentrations. also  showed  microscopic  that  catechin  sections  distribution  of  free  and/or of  catechin  histochemically demonstrated specific  sensitivity  of  DMB  epicatechin  brown  and/or  The DMB reagent were  deposits. epicatechin  present  The was  localized therefore  for the first time in wood. to  catechin  type  units  in  has  The been  demonstrated on condensed tannin precursors (catechin) in sections of one week old roots of cotton seedlings (Mace and Howell, 1974) . To the best of my knowledge, however, the use of DMB on wood has not previously been reported.  In  addition  the  DMB  reagent  provided  strong  evidence  that  catechin/epicatechin were not only present in brown chromophores but also in brown deposits of methanol or acetone/water extracted  hem-fir  sections  (15ptm) .  The  latter  (7:3)  observation  indicates the polymeric nature of brown chromophores agreeing with  56 a similar study (Halloin, 1982) in which DMB was used to locate catechin  and  their  derived  condensed  tannins  in  cottonseeds.  Catechin is thought to be the main tannin precursor in cotton plants  and  cottonseeds  histochemical  (Halloin,  1982).  study the brown deposits  In  the  in hem-fir  current  lumber were  demonstrated to contain units of catechin/epicatechin.  Inoculation of sap used in this study showed that bacteria and fungi were capable of growing in sap without additional nutrients. This observation agrees with a recent study (Schmidt, 1986; Schmidt and Mehringer, 1989) . Furthermore the increasing pH of inoculated sap indicated  that proteins were utilized  independent both of  microorganisms and the source of sap evaluated. Schmidt  (1986)  reported that sap with a high glucose content promoted acidity while a low glucose inoculation  with  containing  bacteria.  sap encouraged  In the  current  arabinose were not indicated in sap of Hem-2  alkalinity upon  study  glucose  or  (uninoculated and  inoculated) but they may have been present below the detection levels used (Sutcliffe, 1992).  Since proteins are known to bind to phenols and PAs  (Hagerman,  1989) a release of phenolics upon microbial utilization of proteins may have contributed to observed colour changes in Hem-2 sap. Solubility encouraging  and  reactivity  rearrangements  (Daniels, 1993a).  of and  phenols  increase  oxidation  of  at  these  higher  pH  compounds  In the current study qualitative changes in  57 lower molecular weight compounds in inoculated Hem-2 sap and the decrease of detectable were oxidized.  (HPLC) compounds suggested that phenolics  Thus oxidized compounds may have become insoluble  in inoculated sap and did not elute from the column used for HPLC analysis.  Bacteria have been shown to discolour solutions of  polyphenols by a peroxidase system (Shortle et al. , 1978) .  Also  sapwood sawdust, in the presence of hydrogen peroxide could be discoloured Halvorson  by  bacteria  (Shortle  et  al. , 1978) .  Evans  and  (1962) have speculated that bacterial phenol oxidase  caused hemlock brownstain.  Brown colorations were produced in the  cambial region of freshly debarked western hemlock when mushroom peroxidase and hydrogen peroxide were added 1985) .  (Hrutfiord et al. ,  In the present study no attempt was made to verify phenol  oxidizing enzymes in inoculated sap.  The pH change associated with colour change of inoculated sap was not continuously measured over time and thus the precise pH at which the colour change started remains unknown.  In a recent study  (Schmidt and Mehringer, 1989) in vitro brown discolorations were recorded  in sap of beech  (Fagus sylvatica L.)  different bacteria at pH 7.3.  inoculated with  The authors, however, prevented in  vitro discoloration of beech sap when glucose and fructose were added thus keeping the pH below 7.  In another study stabilization  of the pH of the wood surface of Ilomba  (Pycnanthus angolensis  Exell.)  discoloration  wood with  formic  acid  inhibited  (Bauch,  1986) . This was linked to a pH shift caused by bacteria (Starck et  58 al.,  1984; Bauch et al. , 1985; Yazaki et al. , 1985).  context  acidic  chemicals  have  been  shown  to  In this  inhibit  hemlock  brownstain in laboratory trials (Barton and Gardner, 1966) while alkaline  treatments  have  been  thought  to  intensify  hemlock  brownstain (Swan, 1984b).  The reasons for colour changes observed in sap of Hem-2 and in amabilis fir but not in Hem-1 are not yet understood.  Ambrosia  beetle infestation, indicated by pinholes and the accompanying staining  fungus in some regions of the Hem-2 board, may have  predisposed this wood substrate to discolorations. of  fungi  and bacteria  The presence  in wood of Hem-2 was not verified but  Whittaker (1962a) has reported bacteria in insect-damaged trees of western hemlock.  Changes in the total phenol content may have  occurred in Hem-2 as has been reported in cricket bat willow wood infected with a bacterium (Wong and Preece, 1978), in wounded red maple  (Shevenell and Shortle, 1986) and in wetwood of western  hemlock (Schroeder and Kozlik, 1972).  0. piceae isolates produced in vitro brownstain in western hemlock sapwood but the discoloration seemed to be less intense than found in a  similar  study  (Smith and  Spence,  1987).  These  authors  demonstrated a dark brown discoloration 6 weeks after inoculation with 0. piceae isolates. 10  cm  boards  inoculation.  which  Smith and Spence (1987) employed 5 cm x  had  produced  brown  endstain  prior  to  Thus the authors believed that their material was  59 highly prone to brownstain. However, heating (103°C) commonly used to identify hem-fir lumber susceptible to brownstain, produced no macroscopic brownstain and indicated that the substrate was not highly susceptible to brownstain.  It is possible that insufficient  precursors to cause hemlock brownstain are present in small size samples (Ellis, 1993) or surface drying is too fast to enable mass flow of sap to the surface for oxidation reactions to occur.  The  Mix-B  Brownstain  inoculated has  wood  been  provided  transmitted  inconclusive  results.  bacterial  infected,  from  discoloured western hemlock slabs to sound wood when placed in a pond but variable results were observed (Evans and Halvorson, 1962; Whittaker,  1962a).  It  is possible  that  moisture  content  of  autoclaved wood specimens used in the current study was too low to allow prolific growth of bacteria.  Autoclaving  of  wood  prior  to  inoculation  may  have  altered  composition of wood extractives and thereby reduced the effect of microbial infection on in vitro production of brownstain.  Heating  of sap of western hemlock has been indicated to cause darkening from light yellow to brown (Evans and Halvorson, 1962) and a slight discoloration was also microscopically recorded in the present study.  Wood used by Smith and Spence  prior to inoculation.  (1987) was not autoclaved  60 In the present study it was not possible to determine whether enzymes present in living cells were able to discolour parenchyma cells since autoclaving would almost certainly have inactivated them.  Enzymes of living cells have been reported to react with  accessory Stutz,  compounds causing wood discolorations  1959;  However,  Hrutfiord  atmospheric  and  Luthi,  oxidation  has  1981; also  (Bailey, 1911;  Ota been  et  al. ,  1991).  demonstrated  to  produce less discoloration than microbial oxidation for inoculated beech wood (Schmidt and Mehringer, 1989) .  3.5  Conclusions  Microscopically  observed,  the  spatial  distribution  of  brown  deposits in hem-fir lumber was irregular in ray parenchyma cells and even more so in tracheids.  This is possibly a result of a  localized microbial infections.  For the first time in situ histochemical examinations provided evidence that brownstain chromophores in hem-fir lumber are at least  partially  composed  Catechin/epicatechin brownstain.  The  were  of  also  localized  catechin demonstrated  nature  of  the  or in  epicatechin. wood  free  distribution  of of  catechin/epicatechin was further demonstrated and this observation was found to resemble the variable distribution of brown deposits seen in discoloured wood.  61 In  vitro  production  of  brownstain  was  demonstrated  inoculated wood and inoculated sap of western hemlock.  in  both  A range of  microflora were found to shift the pH of sap from slightly acidic to  slightly  alkaline.  Alkaline  conditions  promoted  brown  discoloration in sap but other, unknown factors (e.g., composition of extractives) were also implicated.  62 4.0  MONITORING PRODUCTION OF BROWNSTAIN IN WESTERN HEMLOCK LOGS AND LUMBER DURING STORAGE.  4.1  Objectives  The objectives of this study were to: a) evaluate the effect of storage time and storage condition on the production of brownstain in freshly felled trees and in lumber sawn from the trees; b) to determine  the  microflora  associated  with  brownstain;  c)  to  determine changes in the gross phenolic composition associated with brownstain.  4.2  Materials and Methods  4.2.1 Field work at Chamiss Bay Logging site description  A  logging  (Interfor),  site belonging was  selected  to International at  Chamiss  Bay  Forest  Products LTD  on Vancouver  Island.  According to Interfor's preharvest silvicultural prescription the site  (Forest classification HB: 951, hemlock-"balsam"  over 260  years old, Site Class I) is on East to South-East aspect, 350-370 metre elevation above sea level and was located within the coastal western hemlock wet biogeoclimatic subzone.  The soil depth was  estimated to be 55 cm and to be well drained.  Deer fern (Blechnum  63 spicant  (L.) Roth),  sword  fern  (Polystichum  muniturn  (Kaulf.)  Underw. (Sward F.)) and huckleberry (Vaccinium parvifolium Smith) were the main indicator species associated with the mature western hemlock and amabilis fir trees.  Medium western hemlock dwarf  mistletoe (Arceuthobium camplyopodum Engelm. forma tsucrensis Gill) infection was scattered through the site. Selection of western hemlock trees  Twenty eight western hemlock trees were selected in Interfor's logging site 211A on August 5, 1993. The trees were classified by two Interfor foresters for basal diameter and stem degrade (e.g. frost scars, dead branches, mistletoe, sweep) and they were felled the  following  morning  (August  6,  1993).  On August  7, 1993,  thirteen logs were transported from the logging site to the sorting yard at Chamiss Bay.  The other 15 logs were left at the site  because the grapple yarder was unable to reach them. Initial sampling of western hemlock logs  At the sorting yard the logs were bucked (10-15 cm) at the butt end prior  to  cutting  off  two  consecutive,  10-15  cm  thick  disks  (hereafter referred to as outer disk and inner disk) from the butt end.  The  polyethylene  disks and  were they  labelled were  then  and  individually  stored  outdoors  wrapped  in  overnight.  Additional western hemlock logs felled on August 4, 1993 and other  64 western hemlock logs which had been logged approximately 3-4 weeks prior to this date at site 211A, and which had been on the ground since then, were also sampled as described previously.  The logs  were cut to 3 metre long sections following the sampling of disks and they were then oriented on dry-land in a North-South direction. The  disks  were  transported  back  to  Forintek  Canada  Corp.,  Vancouver, B.C. on August 9, 1993. Second sampling of western hemlock logs  Logs stored at Chamiss Bay were visited again after two months. The ends of the logs were examined visually for brown endstain. Two disks (10-15 cm) were then sawed from the same butt end of the logs, labelled and wrapped as described above (see . Logs were then selected at random to be put in ocean water storage (#'s 2, 3, 4, 19, 20, 25, 27, 33, 36, 40, 43, 46, 47, 48, 50; 51, 54, 55) or to be placed on dry land (#'s 1, 5, 6, 16, 17, 24, 26, 32, 35, 37, 41, 42, 44, 45, 49, 52, 53, 56).  The dry-land logs were  stacked in one pile whereas all water-stored logs were bundled together prior to placing in the water.  Disks sawn from 17 logs  (#'s 1, 3, 4, 6, 16, 17, 24, 25, 26, 32, 33, 40, 41, 42, 43, 44, 48) were transported back to the laboratory while the others were left  behind  vehicle.  due  to  a  restricted  load  capacity  of  Forintek's  65 Inspection of western hemlock logs after 9 months storage  The  western  hemlock  logs  were  evaluated  for  production  of  brownstain at the log endcuts following approximately 9 months of storage.  The logs stored in ocean water had been taken out after  5-6 months and subsequently stored exclusively on land. visually examined on both cross-cut ends.  Logs were  Wood chips were then  taken at random from discoloured and non-discoloured regions and they were analysed quantitatively for calcium, manganese and iron using X-ray spectroscopy.  Ten logs (five stored in water and five  stored on land) were then chosen to be sawn up into lumber.  The 10  logs included the six logs (#'s 1, 16, 40, 42, 44, 48) evaluated for microorganisms and wood extractives plus 4 additional logs (#'s 4, 6, 35, 43) which were selected at random. Sawing of western hemlock logs  The ten selected logs were sawn up into lumber 9-10 months after felling.  A bandsaw was used to produce 5 cm thick boards at  Chamiss Bay. produce  Each log was rotated after the first opening cuts to  edge grain material.  Board  surfaces were  sawdust and a fungicide, F2 concentrate  cleared of  (11.4% didecyldimethyl  ammonium chloride + 16.8% disodium octaborate tetrahydrate) diluted 12:1, water:F2 concentrate, was brush-applied to the wood surface to control mould fungi and sapstaining fungi. taken of representative boards from all logs.  Photographs were  Boards produced from  66 each log were closed-piled following normal industrial practice. Inspection of sawn lumber after storage  Visual  examination  of boards was undertaken  approximately two  months after sawing and about 12 months after felling of the trees. The boards were evaluated for surface brownstain and photographs were taken of representative examples from each log.  4.2.2 Laboratory work using Chamiss Bay samples Assessment of susceptibility to brownstain in disks  Susceptibility  to brownstain  was assessed  in the outer  disks  collected at the initial sampling. To determine brownstain samples were kiln-dried  (48 hours at 60°C followed by 48 hours at 80°C)  using a laboratory kiln immediately following the arrival at the Forintek laboratory.  The samples were then visually examined and  brownstained regions were delineated.  The inner disks were stored  in a cold room (3-5°C) until use.  After  the  Selection of logs susceptible to brownstain  initial  brownstain  assessment  six  disks  from  six  different logs were selected for further laboratory studies. Three  67 disks were from logs # 1, 40 and 44 which produced brownstain and three disks, from logs # 16, 42, and 48 which remained stain-free, were selected as controls.  Sample preparation from selected disks  For logs which developed brownstain on the outer disks during kilndrying the outlines of six stained regions were transferred onto the inner disks (Figure 12).  The first area was selected around  the centre of the brown discoloration, the second from a nonstained region, the third from the edge of a discoloured region and the fourth from a non-stained area.  These four regions were coded  (e.g. 1-1; 1-2; 1-3; 1-4) and they were then sawn out using a bandsaw.  Each sample, approximately 4 cm x 4 cm x 15 cm, was then  divided into three equal sized (4 cm x 4 cm x 5cm) pieces, a centre piece and two outer samples.  Each centre piece was immediately  sealed in a plastic bag and was stored in a refrigerator (4°C) for 24 hours prior to undertaking fungal isolations.  The outer pieces  prepared from each sample region were used to determine moisture content  (MC).  Two  additional  samples  were  prepared  from  a  brownstained and a stain-free region, coded (e.g., 1-BS; 1-NS) and placed in a freezer prior to collection of sap (Figure 12).  Sample preparation  from non-stained  disks  (# 16, 42, 48) was  undertaken at random from similar cross-sectional regions of the inner disks to correspond with the sampling from stained disks.  68  1-NS  PRESSATE * ISOLATION OF FUNGI * BACTERIAL COLONIES * MOISTURE CONTENT  *pH *TSP *HPLC  Figure 12: Flowchart showing sampling regime of log #1 from brownstained and non-stained regions in fresh and two month-old disks.  69  Fungal  Initial isolation of fungi  isolation  attempts  were  performed  by  splitting  each  centrepiece (4 cm x 4 cm x 5 cm) in half using a flame-sterilized chisel.  Eight small (approximately 8 mm x 2 mm) chips were removed  from the freshly exposed wood surface using a flame-sterilized chisel and forceps. individually  placed  Four flame-sterilized wood chips were then in the  centre of  a Petri dish  containing  tetracycline malt agar (2% malt extract, 1% bacto-agar plus 100 ppm tetracycline to control bacterial growth)  (MA-T) while the other  four flame-sterilized chips were plated on a starch-casein-nitrate agar (SCN - containing starch 10g; casein 0.3g; potassium nitrate 2.0g; sodium chloride 2.0g; potassium monophosphate 2.0g; magnesium sulfate 0.05g; calcium carbonate 0.03g; ferrous sulfate O.Olg; rose bengal 0.35g; 1% bacto agar, distilled water 1 litre). were incubated in the dark at 25°C for 8 weeks.  The dishes  Petri dishes were  inspected after 1, 2, 3, 4 and 8 weeks and colonies growing out of the  wood  into  the  media  were  containing either MA-T or SCN.  sub-cultured  into  petri  dishes  The purified cultures were then  incubated on malt agar (2% malt extract, 1% bacto agar) prior to culturing on slant agar tubes. at 4°C.  The purified cultures were stored  70  Harvesting of sap  The two samples prepared from each disk  (e.g., 1-BS; 1-NS) for  collection of sap were individually placed in plastic bags and then put into a hydraulic press.  The pressate, decanted into 2 0 mL  vials with teflon seal caps, was frozen until use.  pH measurement  A pH meter was used to determine hydrogen ion concentration in the stirred sap following thawing.  HPLC analysis of pressate  Thawed sap was injected onto the column of a HPLC and the effluent was  qualitatively  monitored  to  determine  the  gross  phenolic  composition at a wavelength of 280 nm as described by Daniels (1993b).  In addition, the amount of catechin and epicatechin was  quantified in the sap using standards purchased from Sigma (St. Louis, M O ) .  Total soluble phenol determination  Western hemlock sap was analysed for total soluble phenol (TSP) content using the Folin-Ciocalteau reagent (Sigma, St. Louis, M O ) . Sap was diluted 25 times by adding 0.02 mL of the sap to 0.480 mL  71 of distilled water followed by 2.5 mL of Folin-Ciocalteau reagent, diluted 10 times with distilled water, and then 2 mL of sodium carbonate solution (8.25 g sodium carbonate monohydrate in 100 mL of distilled water).  The solution was first placed in a water bath  at 50°C for 5 minutes and then in a cool water bath (10- 15°C) for approximately 5 minutes. Samples were transferred into polystyrene cuvettes  (1 cm path length) and absorbance was read at 76 0 nm  against a blank  (distilled water).  TSP content was calculated  using the average absorbance of three readings for each sample. Gallic acid (Sigma, St. Louis, MO) was used to establish a standard curve  (5-80  /zg/mL) .  The  measurements  were  performed  with  a  Shimadzu spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, M D ) .  Sample preparation from disks after 2 months log storage  The outer disks sampled after two months of outdoor log storage, were heated to induce brownstain as outlined in  The disks  that were used for these further studies, came from the same logs as previously sampled, log 1, 40, 44 with brownstain and # 1 6 , 42, 48 without brownstain.  Six regions were mapped on each inner disk  and these  then  areas were  described in  sampled  according  to the  procedure  72  Isolation  of  fungi  and  quantification  of  bacterial  forming colonies  Microorganisms were isolated from the centre of the three pieces sampled from each region according to the procedures described in, except that all wood chips were plated aseptically on MA-T and SCN, respectively.  The plates were incubated in the dark (25°,  70% RH) , monitored and sub-cultured as previously described in  In addition bacterial colony-forming units (CFU) were counted using one of the two outer pieces per sample region (e.g., 1-1) which had been sealed in a plastic bag and then stored in a refrigerator for approximately three weeks. from one viable cell.  It was assumed that each CFU was formed  For each sampling one outer piece from each  region (e.g., 1-1) was split in half using a chisel.  Both pieces  were flame-sterilized in a laminar flow hood and then individually heat-sealed in a polyethylene bag.  The sealed wood sample was then  positioned at the upper edge of the bag and placed in a hydraulic press.  As pressure was applied sap was collected in the lower,  hanging part of the bag.  The bag was removed from the press and  re-sealed below the wood sample to separate the sap from the wood. The sap was then removed to a laminar flow hood and a serial dilution plating was performed using 1.5% nutrient agar (NA) (Difco Laboratories, Detroit, MI) as growth medium.  Two plates were  employed per serial dilution and for control (sterile, distilled  73 water). All plates were incubated at room temperature for 48 hour prior to counting CFU.  Prior to using this specially developed technique a number of checks were run.  Additional NA Petri dishes were seeded with  sterile, distilled water which was transferred in a plastic bag prior to plating.  This experiment which was undertaken to verify  the methodology employed, showed that the polyethylene bags were free of contamination.  Other NA plates were incubated to check  whether the pressing procedure would assure aseptic handling of the sap.  For this purpose distilled, sterile water and a bone-dry,  flame-sterilized wood sample were sealed in a similar plastic bag, and handled according to the procedures described previously.  All wood samples were weighed prior to pressing sap and following drying (103°C, 12 hours) to determine moisture content.  Sap analysis  The  qualitative  pH,  HPLC  analysis  of  the  gross  phenolic  composition, quantitative catechin and epicatechin analyses and TSP were determined (see  74 4.3  Results and Discussions  4.3.1 Production of brownstain in fresh and 2 month-old logs.  The cross-cut ends of the freshly-felled western hemlock trees looked brownstain and defect-free.  Wet zones were noticed in the  inner heartwood and/or in patches within the sapwood.  The absence  of hemlock brownstain on the fresh log ends suggested that this type of coloration is not produced in the living tree and thus is different from such discolorations (Basham and Taylor, 1965; Sachs et al. 1966; 1974; Shigo and Sharon, 1968; Cosenza et al. , 1970; Shigo et al. 1971; Wilcox and Oldham, 1972; Shigo and Hillis, 1973; Mackay,  1975; Blanchette  et  al. , 1981; Bauch  Djuesiefken et al., 1984; Walter, 1993)).  et  al. , 1982;  Furthermore, hemlock  brownstain, requiring storage to occur, differs from that observed for example on log ends of red alder, which develop within hours after the felling of the tree (Hrutfiord and Luthi, 1981; Terazawa et al., 1984; Kreber et al., 1994) .  Brownstain was produced in inner heartwood regions and in outer regions of some disks following kiln-drying outer  regions  brownstain  heartwood/sapwood interface.  seemed  to  a  decayed  zone  across  associated with brownstain.  associated  In the  with  the  In addition, brownstain was observed  in regions where decay was conspicuous. showed  be  (Table 2) .  the  For instance, disk # 44  section  and  this  zone  was  While wet zones, perhaps "wetwood"  75  TABLE 2 : CLASSIFICATION OF SELECTED WESTERN HEMLOCK LOGS. TREE DEGRADE1  LOG # 2  l (70)  3  Frost crack; clear 7 m; light infection  STAIN mistletoe  H4  2  (85)  frost crack grown over; large dead branch @ 5 m; moderate mistletoe infection  No  3  (40)  frost crack @ 7m; large dead branch @ 4m; heavy mistletoe on top  No  4  (45)  clear;  No  5  (30)  heavy mistletoe infection; dead branches;  No  6  (40)  heavy mistletoe infection; dead branches;  No  16  (75)  scars; sweep; moderate mistletoe; dead branches;  No  17  (80)  frost scars; heavy mistletoe;  19  (30)  scars; moderate mistletoe;  No  20  (30)  scars;  No  24  (45)  25  (40)  mistletoe; dead branches;  No  26  (30)  frost scars;  NO  27  (45)  ND  No  327 (60)  ND  No  34  (60)  ND  H  35  (70)  ND  No  37  (50)  ND  No  (45)  ND  H  41  (50)  ND  No  42  (45)  ND  No  43  (40)  ND  No  44  (50)  ND  H; H/S  45  (30)  ND  No  46  (45)  ND  No  48  (50)  ND  No  40  8  ND6  H;H/S5  No  76  1 2 3 4 5  49  (45)  ND  No  50  (30)  ND  No  51  (45)  ND  No  52  (40)  ND  No  53  (30)  ND  No  54  (30)  ND  No  55  (45)  ND  No  56  (30)  ND  No  = = = = =  refers to degrade assessed on the standing tree by Interfor. trees 1-27 were selected at site 211A. numbers in brackets refer to diameter (in cm) at the butt. brownstain observed in heartwood after kiln-drying. brownstain observed in heartwood/sapwood interface after kiln drying. 6 = no degrade was determined 7 = logs 32-37 were selected at the sorting yard at Chamiss Bay. 8 = logs 40-56 were selected at the sorting yard at Chamiss Bay and they had been harvested from site 211A 3-4 weeks prior to selection.  77 (Ward and Zeikus, 198 0) were frequently observed western  hemlock  disks  these  areas  showed  in the fresh  no  particular  susceptibility to brownstain following kiln-drying.  After two months of land-storage hemlock brownstain was not found on the original cross-end cuts of western hemlock logs.  However,  a visit to the logging site 211A to inspect some of the tree stumps sampled  two  months before  showed  pronounced  brownstain.  For  instance brownstain was found in the heartwood of stump #16 whereas disk segments removed from this tree remained stain-free both at the initial brownstain assessment and after two months. likely that sap flow from the root system continued felling of the  It is  following  tree which resulted in an accumulation of phenolic  compounds producing brownstain at the stump surface.  Heating of disks from the logs stored  for two months on land  provided similar results to those recorded in the fresh disks. Accordingly brownstain was found in the same regions within the disks of logs # 1, 40 and 44 whereas disks # 1 6 , 42 and 48 remained stain-free.  Thus  heating  of  western  hemlock  disks  produced  consistent results in determining those logs with potential for brownstaining.  The fact that the control logs  (# 16, 42, 48)  remained stain-free upon heating also suggested that the two month-old logs were not yet predisposed to brownstain.  78 4.3.2  Inspection of cross-cut ends after 9 months storage  Inspection of log ends demonstrated that most logs had produced some endstain after 9 months of outdoor storage  (Table 3 ) .  Two  distinct differences were noticed between water-stored and landstored logs.  Firstly, the water-stored logs showed a much darker  colour ranging from dark brown to black (Figure 13).  These dark  colorations were not found on the cross-end cuts of logs stored on land.  In the water-stored logs dark colorations were noticed in  the heartwood and/or sapwood regions where they were associated with groups of adjacent growth rings. When wood chips were removed from these regions the brown-black colour in the water-stored logs was found to be superficial.  X-ray spectroscopy (data not shown)  for calcium, manganese and iron in black discoloured wood chips showed  no  qualitative  difference  compared  to wood  chips  with  lighter discolorations (Ingram, 1993). Secondly, water-stored logs produced dark brown to black stain in heartwood and sapwood (Figure 13) while land-stored logs showed brownstain only in the sapwood and/or in the sapwood/heartwood interface (Figure 14).  These observations demonstrated that log storage time, independent of storage conditions, promoted development of brownstain because it was not observed on the log ends of the two month-old logs.  Log  age has been suspected to influence both production of hemlock brownstain  (Evans and Halvorson, 1962; Kreber and Byrne, 1993;  Scheel, 1993) and discolorations in other softwoods and hardwoods  79  TABLE 3: PRODUCTION OF BROWNSTAIN ON LOG ENDS AFTER 9 MONTHS OF OUTDOOR STORAGE. LOG#  STORAGE  APPEARANCE OF LOG ENDS  1  land  stain-free  2  water  HW1 darker stained than SW2; black-brown stain associated with certain growth rings  3  water  SW light brown in places; HW dark-brown w/ black patches; HW/SW3 interface brownstained  4  water  dark and wet; black stain in some growth rings; inner HW enclosed by a black band  5  land  SW light brown;  6  land  SW w/ some brownstain; HW/SW interface w/ narrow brownstained band  16  land  SW greyish; dark-brown zone around HW  17  land  SW greyish w/ brown patches  19  water  wet and dark, possibly dirt  20  water  log was missing  24  land  SW w/ brownstain  25  water  inner HW w/ dark brown stain  26  land  stain-free  27  water  stain-free  32  land  SW w/ brownstain (outer 10 cm)  33  water  log was missing  35  land  SW w/ brownstain (outer 5 cm)  37  land  SW w/ brownstain  40  water  HW w/ patches of black and brownstain  41  land  SW w/ brownstain (outer 5 cm)  42  land  stain-free  43  water  HW dark brown surrounded by a black band  44  land  HW w/ dark brown patches  45  land  SW w/ brownstain  46  water  HW grey-green; SW w/ brownstain  80 47  water  HW grey surrounded by a 2-3 mm brown band  48  water  SW w/ black patches  49  land  SW w/brownstain  50  water  SW w/ brownstain (outer 1-2 cm)  51  water  HW light brown enclosed by a black band  52  land  SW w/ light brownstain  53  land  SW w/ brownstain  54  water  HW grey w/ brownstain in some growth rings; HW enclosed by a 1-2 cm black band  55  water  SW w/ light brownstain  56  land  stain-free  1 = HW refers to heartwood 2 = SW refers to sapwood 3 = HW/SW refers to transition zone between heartwood and sapwood  81  .**  Figure 13: Representative, water-stored colorations after 9 months of storage.  \  log  *  showing  dark  82  maBm  •tffjk  '  Figure 14: Representative, land-stored log showing brownstain in sapwood after 9 months of storage.  83 (Herman, 1931;  Rasmusseen, 1940; Stout, 1950; Koltzenburg, 1975;  Forsyth, 1988).  In  the  current  study  salt  water  storage  also  demonstrated  a  striking effect on the intensity of the coloration developing on the log ends.  Several studies have shown that fresh water storage  of logs can promote discolorations on the log ends or on the lumber sawn from the logs (Hoster, 1974; Braun and Lewark, 1992; Forsyth and Amburgey, 1992; Hedley and Meder, 1992 Lubbers and GroiS, 1992; Rathke, 1991).  Some explanations have been given for this water-  storage phenomenon, for example: infection of water-stored logs by bacteria which appeared to modify glycosidic flavonoids (Hedley and Meder, 1992) ;  death of parenchyma cells causing dissolving of  phenols (Hoster, 1974); and redistribution of bark phenols into the wood (Adler, 1951; Lubbers and GroS, 1992).  Interestingly, Lubbers  and GroS (1992) showed that debarking of spruce  (Picea sp.) and  true fir logs prior to storing in water decreased brownstain on the logs ends and on the lumber surface.  Similar observations were  made when investigating brownstain in radiata pine lumber produced from logs debarked prior to water storage (Hedley and Meder, 1992).  In the present  study redistribution  of bark phenols  may have  contributed to hemlock brownstain associated with sapwood regions but it is unlikely to have influenced the dark colorations found deeper in the wood, for instance in the heartwood/sapwood interface or even in the heartwood.  However, it is possible that salt water  84 with a pH of above 7 and with trace amounts of metal ions promoted the dark colorations on the log ends of the water-stored western hemlock.  Extensive quantitative metal ion analysis of wood would  be required to determine whether these ions are associated with, or play a role, in the dark colorations occurring in stored logs in ocean water.  4.3.3  Inspection of lumber after two months of outdoor storage  Freshly cut western hemlock boards produced from logs which had been stored surface.  for 9.5  months, showed no brownstain on the wood  This observation agreed with recent studies (Lubbers and  GroS, 1992; Rathke, 1991) wherein brown discolorations were also absent in freshly sawn spruce and true fir lumber but developed with time.  Observations made in this study suggests that hemlock  brownstain, rather than being formed in the living tree, as for example with the black colorations associated with the heartwood of yellow cedar (Smith, 1970) or the discolorations associated with tree  wounds  (Shigo  and  Sharon,  brownstain  was  observed  1968),  involved  atmospheric  oxidations.  However,  inspecting  it after  2 months  of  on  the  lumber  surface  storage; this was  the  when first  inspection, so it is not known how quickly (before two months) the stain first appeared.  Staining soon after cutting into lumber has  been frequently reported by mill managers (Blake, 1992).  Lubbers  85 and GroS (1992) and Rathke (1991) have also demonstrated that brown surface colorations formed a few days after sawing spruce and true fir logs.  The boards sawn from log #4  (water-stored) developed the most  severe brownstain, darkly disfiguring the board edges (Figure 15). In addition the faces of # 4 boards were also discoloured, mostly concentrated at the end of the board and in the outer regions of the board (Figure 15) .  Boards from logs # 48 and 43 (both logs  stored in water) showed brownstain at the wood surface in a striped pattern.  The coloration was associated with the outer regions of  the boards and rarely affected the whole length of the board. Lumber from logs # 1 , 6, 16 and 35 showed a little brownstain, most noticeably on the edges whereas the faces were less affected. Lumber from logs # 4 0 , 42 and 44 looked very clean, showing only minor brownstain on the edges of some boards whereas the faces were stain-free.  In  this  study  water-stored  logs  produced  considerably  brownstain in sawn lumber than did logs stored on land. water-storage  did not always result  more  However,  in brownstained wood: the  lumber sawn from the water-stored logs #4 0 and 44 remained stain-free.  While both logs (40 and 44) developed brownstain upon  heating and thus were thought of as being susceptible to brownstain this observation indicated that brownstain produced in kiln-dried western hemlock differed from those colorations developing during  86  jfe  Figure 15: Production of severe brownstain on the boards edges faces of lumber sawn from log #4.  87 seasoning. However, the factors which prevented surface brownstain on sawn lumber of logs # 40 and 44 are not well understood, wood moisture content may have been involved.  It is possible that logs  # 4 0 and 44 were not submerged during water-storage as some logs in a bundle always float above the water surface.  Thus drying could  have occurred which then yielded lumber with a lower moisture content.  Lubbers and GroS (1992) reported that spruce and true fir  logs with a higher moisture content produced more brownstain in the sawn lumber.  In the current study another interesting observation was made, namely that brownstain did not develop on a closed-piled board or at the sticker location, but brownstain was evident on the wood surface  that  stacked.  was  exposed  to  air, where  boards  were  unevenly  This observation indicated that reduced photooxidation,  inhibition of moisture movement or reduced oxygen content, could result in reduced brownstain in the sawn lumber.  Inhibition of  moisture movement and reduced oxygen content have been proposed to explain the occurrence of less discoloration underneath a sticker in western hemlock (Evans and Halvorson, 1962) as well as in other softwoods  (Anderson  et  al. , 1960; Miller  hardwoods (Bauch et al. , 1991) .  et  al. , 1983)  and  However, photooxidation has not  been related to hemlock brownstain, but photo-labile constituents of western hemlock may well be involved as indicated in the current study.  Many wood species have been known to change colour when  88 exposed to light and for example, woods with stilbenes have been reported very photo-labile (Morgan and Orsler, 1968). Light-induced  colour  changes  of  catechin,  epicatechin  and  dihydroquercetin have recently been demonstrated in a study on chemical brownstain in Douglas-fir (Arvey, 1993) .  4.3.4  Isolation of microorganisms  Of 192 isolation attempts made from the freshly, felled western hemlock, 25 fungal cultures resulted.  From this initial sampling  the fungi cultured were categorized as: green moulds (3) , pigmented fungi (16) and basidiomycetes (6) (Table 4 ) . After two months of log storage some similar 27 cultures were isolated yielding 22 pigmented fungi and 5 yeasts but no decay fungi and green moulds. At both sampling times log # 44 yielded the most fungi and log # 42 the second most isolates.  Logs # 16 and 40 each yielded one  isolate after 2 months but 5 fungi (log # 40) and 4 fungi (log # 16) were isolated at the initial sampling.  Logs # 1 and 48  yielded no fungi except one isolate cultured from log # 48 at the initial sampling.  Clearly, it can not be assumed that all fungi  were isolated and that the relative frequency of their occurrence represents somewhat a measure of ease of isolation.  Fungi producing red pigmentation on malt agar plates were the most frequent isolates from western hemlock at both sampling times. Similar growth characteristics of these isolates indicated the same  89  TABLE 4: FUNGI1 ISOLATED FROM WESTERN HEMLOCK LOGS SAMPLED AT CHAMISS Bay. FIRST SAMPLING2 #  Fungus  Code  l-l*4  -  1-2  -  1-3*  -  1-4  -  16-1  A5B 5  basidiomycete  16-1  A6B  5  basidiomycete  16-1  A7B  5  basidiomycete  16-2  A2M  SECOND SAMPLING3 Code  -  green/brown mould  16-3  -  16-4  -  Fungus  BY  Yeast -  40-1*  AIM  Penicillium sp.  40-1*  A7S  Phialocephala virens  40-3*  A2S  Ophiostoma piceae  40-3*  A5S  Trichocladium canadense  40-3*  A5aS  Ascocoryne sarcoides  B21S  P. virens  40-2  _  -  40-4  _  42-1  A10S  A. sarcoides  B7S  A. sarcoides  42-2  A11S  A. sarcoides  BIB  A. sarcoides  42-3  A8S  A. sarcoides  42-4  A12S  A. sarcoides  B8S  A. sarcoides  42-4  A9S  A. sarcoides  B9S  A. sarcoides A. sarcoides  -  42-4  -  BIOS  42-4  -  BUS  42-4  -  B20S  A. sarcoides  Leptodentium elatius  BIS  L. elatius  basidiomycete  B2S  L. elatius  44-1*  A1S 6  44-1*  A2B  44-1*  A13S  Phialophora sp.  B3S  L. elatius  44-1*  A6S  Phialophora melinii  B4S  L. elatius  basidiomycete  B13S  L. elatius  44-1*  A4B  6  44-1*  -  B14S  Phialophora sp  44-1*  -  B15S  Phialophora sp  90 44-1*  -  B16S  Leptographium sp.  44-2  A3B  A. sarcoides  B12S  A. sarcoides  44-3*  A4Sa  A. sarcoides  B5S  A. sarcoides  44-3*  A4Sb  black pigmented  44-3*  A14S  T. canadense  B6S  dark pigm.  44-3*  _  B17S  dark pigm.  44-3*  -  B18S  T. canadense  44-3*  _  B19S  A. sarcoides  44-4  A3M  green mould  -  44-4  A3S  P. melinii  _  48-1  -  -  48-2  _  -  48-3 48-4  A1B  basidiomycete -  -  1 = species were tentatively identified by K.A. Seifert, Agriculture, Canada. 2 = logs were sampled latest 3-4 weeks after felling the trees. 3 = logs were sampled following a 2 month storage period (AugustOctober, 93) at the Chamiss bay sorting yard. 4 = sample regions in bold with asterisk refer to regions associated with brownstain 5 = fungi showed a similar growth morphology on malt agar plates. 6 = fungi showed a similar growth morphology on malt agar plates.  91 fungus tentatively identified as Ascocoryne sarcoides  (Jacq. ex.  S.F. Gray) Groves & Wilson by Dr. Keith A. Seifert, Agriculture Canada.  A. sarcoides has been reported to be a common heartwood  inhabitant  of  living  spruce,  true  fir,  pine,  Douglas-fir  (Pseudotsucra menziesii (Mirb.) Franco), hemlock, and larch (Larix sp.) in Canada (L.) Karst)  (Etheridge, 1970) and in Norway spruce  (Roll-Hansen  and  Roll-Hansen,  1979).  (P. abies In western  hemlock this fungus has been demonstrated to be confined to the central heartwood column in the lower one-third of the stem and in the  roots  (Etheridge,  1970).  Etheridge  also  showed that  the  fungus, a primary non-decay invader preceded wood-decay fungi. The fact that A. sarcoides was isolated from disks of log # 42 at both sampling times but wood-decaying fungi were not cultured, supports Etheridge's observations (1970). A connection between A. sarcoides and discolorations has been reported by Whittaker associated it with redheart in lodgepole pine.  (1962b), who  A. sarcoides has  now been cultured from brownstained western hemlock in this study specifically regions 40-3 and 44-3.  Dark-pigmented fungi were isolated from western hemlock both at the initial sampling and after two months storage: Leptodent ium elatius Leptocrraphium tentatively  sp.  Phialophora sp.,  (Mangenot) de Hoog var. elatius de Hoog, and  identified  Trichocladium by Dr. Seifert.  canadense The  Hughes  all  isolates of dark  pigmented fungi were cultured from the disks of log #44 which also showed signs of decay.  Dark pigmented fungi may have infected this  92 tree as secondary invaders following the establishment of decay organisms.  Phialophora sp. and L. elatius have also been reported  in hem-fir lumber by Chung and Smith (1986) and Seifert and Grylls (1991) but Leptocrraphium sp. and T. canadense were not isolated in either previous investigation.  In the current study one 0. piceae  isolate was purified from a freshly felled western hemlock tree. This fungus is not a common invader of standing western hemlock trees yet 0. piceae has been reported to be the most frequent fungus from western hemlock lumber in B.C.  (Seifert and Grylls,  1991).  In the current study dark-pigmented fungi were commonly associated with brownstain regions but a larger sample population would be required to show a definite relationship.  Miller et al. (1983)  reported that Graphium sp. and Leptographium sp. were the most common fungi in brownstained Douglas-fir lumber.  T. canadense was  the fungus most frequently isolated from red heart in white birch (Betula  papyrifera  Marsh.)  (Basham  and  Taylor,  1965) yet  its  capacity to produce red heart in vitro has not been demonstrated (Fritz, 1931; Siegle, 1967).  In the current study strong evidence is provided that the fungi cultured from freshly felled western hemlock trees represent the resident microflora.  Based on available literature it appears  highly unlikely that fungi would have invaded and penetrated the log ends to a depth of approximately 3 0-40 cm from the butt end  93 within 3-4 weeks after felling the trees.  For instance a radial  growth rate of 4.22 mm/day has been reported for Ophiostoma sp. in pine (Breuil et al.,1988) and 4.5 mm/day for 0. piliferum in pine (Gibbs,  1993)  conditions  under  of  anticipated.  forest  laboratory storage  conditions. lower  growth  In  the  rates  cooler  would  be  Fungi isolated in this study did not match these  reported growth rates even on malt agar media at 25°C where growth should be more rapid than in wood.  Although more pigmented fungi  were isolated after 2 months of log storage than at the initial sampling, this was attributed to an improved isolation technique and not to fungal invasion following felling of trees.  Therefore  the evidence indicates that all fungi isolated after 2 months of outdoor storage in this study represented a resident microflora in mature western hemlock trees at the time of felling.  Quantification of bacterial colony-forming units showed no distinct difference between discoloured and stain-free wood following dry-land log storage for two months have  presented  evidence  for  discolorations in standing trees  (Table 5) .  involvement  Several studies of  bacteria  in  (Cosenza et al. , 1970, Shigo,  1971), in freshly cut logs (Zimmermannn, 1974; Bauch et al., 1985) and in discoloured lumber cut from water-stored logs (Hedley and Meder, 1992).  However, wood samples were stored in a refrigerator  for 3-4 weeks prior to undertaking this experiment and this may have decreased viability of the colony-forming units of bacteria. Also, obligate anaerobic bacteria would not be detected using the  94 TABLE 5: MOISTURE CONTENT (MC %) AND COLONY-FORMING UNITS (CFU) IN BROWNSTAINED (BS) AND NON-STAINED (NS) LOGS. LOG #  SAMPLE REGION  MC %  CFU 1 52  1-1  BS  104  1-2  NS  95  4  1-3  BS  88  2  1-4  NS  85  4-5  16-1  NS  55  4  16-2  NS  60  3-4  16-3  NS  137  4  16-4  NS  119  3  40-1  BS  118  3  40-2  NS  144  4-5  40-3  BS  85  3  40-4  NS  85  3  42-1  NS  43  42-2  NS  65  5  42-3  NS  41  n.d.  42-4  NS  61  2  44-1  BS  48  24  44-2  NS  73  l4  44-3  BS  58  34  44-4  NS  54  4  48-1  NS  71  2  48-2  NS  87  3  48-3  NS  81  5  48-4  NS  63  1-2  n.d.3  1 = a dilution 0.1 mL in 1 mL was performed and 0.1 mL was plated. 2 = higher 5number represents more bacteria in sap e.g. 5 = more than 10 CFU/mL. 3 = MC of sample was too low to collect sufficient sap. 4 = a dilution 0.1 mL in 2 mL was performed but 0.1 mL was accidently plated. This could have underestimated CFU counts.  95 procedure employed in this study.  4.3.5  Sap analysis  Generally sap pressed from discoloured regions was distinctively brown, while it was clear or pale yellow when collected from nonstained areas.  It was also observed that sap from brownstained  regions  following  foamed  compounds.  Foaming  was  pressing also  perhaps  noticed  in  brownstained areas of logs after two months. previous  observations  microbial  origin,  (Kreber,  possibly  indicating sap  gaseous  pressed  from  This corresponded to  1993)  suggesting  associated  with  products  of  micro-anaerobic  conditions.  Generally, the pH of sap did not appear to influence brownstain production.  The pH of discoloured sap was slightly lower than that  of non-stained sap (Table 6 ) . However, the difference in pH may have reflected a variation within the disks. sample  1-BS  was  sampled  from  inner  For instance, sap  heartwood  while  collected nearby in another inner heartwood region.  1-NS  was  After two  months of log storage a slightly lower pH in stained as compared to non-stained sap was again observed but pH was higher in some disks. Post-harvest changes, such as the production of acetic acid, have been documented and can occur during log storage as a result of biodeterioration (Packman, 1960) .  96 TABLE 6: MOISTURE CONTENT NON-STAINED SAMPLES. SAMPLE REGION  (MC %) AND pH IN BROWNSTAINED AND  INITIAL MC %  INITIAL pH  pH AFTER 2 MONTHS  1-BS  1  94  5.0  4.9  1-NS  2  92  5.5  5.1  16-NS  104  5.3  5.4  16-NS  114  5.4  5.5  40-BS  118  5.5  5.3  40-NS  116  5.7  5.6  42-NS  48  6.0  5.8  4 2-NS  39  5.7  ND 3  44-BS  78  5.3  5.5  44-NS  76  5.6  5.4  4 8-NS  80  5.2  5.1  4 8-NS  61  5.9  5.3  1 = refers to regions with brownstain 2 = refers to regions without brownstain. 3 = pH was not determined  97 Qualitative HPLC analysis (data not shown) of the gross phenolic composition  showed  discoloured sap.  differences  between  discoloured  and  non-  Discoloured sap contained more low molecular  weight compounds and more oxidized compounds than non-discoloured sap.  This  observation  confirmed  previous  results  seen  when  producing in vitro brownstain in inoculated western hemlock sap (see chapter 3) and may explain the tendency to foaming noted in expressing the discoloured sap.  Quantification of catechin and epicatechin by HPLC showed small amounts (0-10ppm) of these compounds in both discoloured and nonstained wood.  Catechin and epicatechin were not detected in all  the sap samples and there was no clear presence/absence pattern in discoloured and non-discoloured sap relative to hemlock brownstain. This suggests that either other phenols were also involved in the production of brownstain or that catechin and epicatechin can cause colorations  at very  low  concentrations. HPLC  analysis  of  sap  pressed after two months of log storage produced similar results.  Total  soluble  phenol  (TSP)  content  was  determined  after  establishing a standard with gallic acid which produced a linear relationship between 5ppm and 80 ppm  (Figure 16) .  In western  hemlock sap diluted 25 times, the TSP content differed between disks  and  within  stained  and  non-stained  regions  of  disks.  Generally, TSP content was lower in sap collected from brownstained regions of both fresh disks and two month-old disks (Figure 17).  98  Figure 16: Determination of a standard calibration using gallic acid.  99  Figure 17: Total soluble phenols measured in brownstained regions of test logs after 0 (A) and 2 (B) months of storage.  100 The lower mean TSP content associated with brownstained areas was significantly different (95% confidence level) from the higher mean TSP content associated with non-stained regions.  There was no  significant difference at the 95 % level when comparing the mean TSP content of the two non-stained regions associated with control logs (Figure 18) nor when comparing the mean TSP content of the non-stained regions associated with control logs to the non-stained regions  associated  with  the  test  (brownstained)  logs.  Similar  observations were made when determining the total soluble phenols content  in the 4 regions of disks  (e.g. 1-1;  1-2; 1-3;  1-4)  prepared for microbial isolation attempts after 2 months of log storage (Figure 19).  The lower TSP in sap from brownstained regions corresponded with the qualitative HPLC analysis of the gross phenolic composition which also demonstrated less phenolics in these regions. qualitative previously  HPLC  and TSP analysis  (see chapter  3)  supported  that phenolic  observations compounds  Thus, made  are more  oxidized in regions associated with brownstain. It may well be that soluble phenolic brownstain.  compounds  oxidize  to quinones when  producing  Attempts to reduce possible quinones with sodium  borohydrate failed because the reducing agent interfered with the Folin-Ciolteau reagent.  101  • REGION-i  REGION-2  70 60 50 -J  40  CD D  30 20 10 0  Figure 18: Total soluble phenols measure in two non-stained regions of control logs after 0 (A) and two (B) months of storage.  102  Figure 19: Total soluble phenols measured in four different regions in test and control logs after 2 months of storage (the 4 regions were sampled as shown in Figure 12).  103 4.4  Conclusions  Generally, brownstain was not observed at the butt cut of the freshly felled trees nor was brownstain noticed at the cross-cuts of the same trees beyond two months of outdoor storage.  Most log endcuts showed brownstain after 9 months of log storage demonstrating  that  storage of  logs beyond  two months promoted  brownstain.  Salt water-stored logs were more discoloured than the land-stored logs. Thus storage conditions affect production of brownstain.  In sawn lumber brownstain disfiguring the wood surface was related to the brownstain regions discolouring the log end-cuts.  Sapwood  and/or the sapwood-heartwood transition zone were more prone to brownstain  than  heartwood.  Brownstain  regions associated with the butt ends.  was  more prevalent  in  Other factors which may  promote the production of brownstain are inhibition of moisture movement, the presence of oxygen and photooxidation.  Fungi isolated from western hemlock logs represented the resident microflora  from  the  living  tree.  A.  sarcoides  was  the most  frequent isolate from both freshly-felled western hemlock trees and hemlock logs following two months of log storage.  104  Dark-pigmented  fungi  were  the  predominant  type  isolated  from  brownstained regions whereas only one dark-pigmented fungus was isolated from a non-discoloured area.  The specially developed method used in this study to quantify aerobic  bacteria  in  wood  produced  consistent  and  repeatable  results.  Finally total soluble phenols were lower in brownstain regions suggesting the presence of more oxidized compounds.  105 5.0  MICROFLORA AND TOTAL SOLUBLE PHENOLS ASSOCIATED WITH BROWNSTAIN IN WESTERN HEMLOCK LUMBER.  5.1  Objective  To understand the relationship between fungi and the  total soluble  phenols in brownstain.  5.2  Materials and Methods  5.2.1  On  Sampling at CIPA sawmill  February  Nanaimo,  22, 94 CIPA's  B.C.  was  visited  (CIPA Lumber to  examine  Co. LTD.) the  hemlock  sawmill  in  brownstain  situation in stored wood awaiting offshore shipment and to sample western hemlock 5 cm x 10 cm material showing hemlock brownstain. Fifty hem-fir boards were selected from one package and trimmed to 1 metre length for laboratory studies. According to CIPA's records the lumber was produced and dip-treated on December 1, 1993 with the fungicide NP-1 (containing didecyldimethylammonium chloride + 3-iodo-2-propynyl-butylcarbamate) staining  fungi.  The 50  to  control  mould  fungi  and  (1 metre long) boards arrived at the  Forintek laboratory on February 25, 1994.  The lumber was stored  inside overnight prior to sorting by wood species (amabilis fir vs western hemlock) microscopically.  106 5.2.2  Sample preparation and isolation of fungi  A 2.5 cm sample was trimmed off the brownstained end of all boards, followed by a 10 cm sample, a 2.5 cm sample, a 20 cm sample and another 2.5 cm sample (Figure 20) . The 2.5 cm samples coded A (end piece), B (centre piece), C (inner piece), were placed in plastic bags and refrigerated for up to 48 hours. wood chips  To isolate fungi small  (10-15 mm long x 2-5 mm wide x 1-3 mm thick) were  prepared from all three positions, A, B and C within a board and also from three different positions across the width of a sample (Figure 20).  Wood chips were then placed on glass rods in petri  dishes overlaying cotton sheet wetted to maintain a high humidity for 2-3 hours.  Five chips were produced from each position, a  total of fifteen from each board.  The wood chips were then surface flame-sterilized and aseptically placed onto MA-T tetracycline).  (2% malt extract and 1.5% agar plus 100 ppm Petri dishes were  sealed  incubated in a chamber at 25°C in the dark.  in plastic  bags and  Incubated wood chips  were frequently checked to enable subculturing of fungi growing out of the wood into the media.  Subcultures were purified on malt agar  prior to transferring them to slant malt agar tubes which were stored at 4°C in the dark.  107  20cm  2.5cm  10cm  X\\\  TOTAL SOLUBLE PHENOLS IN SAP  ISOLATION OF FUNGI  Figure 20: Flowchart showing sampling regime of 5 cm x 10 cm western hemlock lumber (region A represents the end of a board)  108 5.2.3  Determination of TSP content  Following preparation of wood chips the remainder of each 2.5 cm sample used for fungal isolation was individually sealed in a plastic bag and placed in a refrigerator prior to pressing of sap. Sap was collected using a laboratory press as previously described (see and the pressate was immediately placed in a freezer until analysis.  Total soluble phenols was determined using the  Folin-Ciocalteau method as described (see  5.2.4  Assessment of antisapstain treatment  To verify that the western hemlock boards had been treated with NP1 four boards (# 16, 28, 46, 49) were selected at random.  Five  wood chips with a 6.25 cm2 surface area were removed from different positions on these boards. fungicidal  active  The chips were analyzed for the major  ingredient,  didecyldimethylammonium  chloride  (DDAC) according to the HPLC method developed by Daniels (1992) and the surface retention levels were determined.  109 5.3  Results and Discussions  5.3.1  Observations on brownstained lumber  Examination  of  hem-fir  lumber  at  CIPA  demonstrated  that  many  packages contained boards with brown or black discolorations at the cross-cut ends.  The amount and intensity of the discoloration was  variable within and between packages.  The boards sampled were sawn on December 1, 1993 and visibly showed the presence of brownstain.  This observation demonstrated that  hemlock brownstain can develop in the winter season and does not depend  on  summer  conditions.  Brown  discolorations  have  been  observed to develop in spruce and fir lumber both in the cool season  of November  (Rathke,  1991) .  and  the hotter months  In another  study Miller  of  July and  et  al.  August  (1983) have  reported on brown discoloration in Douglas-fir occurring during the winter of 1980/81.  It is also notable that a severe incidence of  hemlock  was  brownstain  reported  in  February  1993  from  Mayo,  Nanaimo, B.C., a sawmill mill close to CIPA (Kreber, 1993).  Upon careful examination of the cross-cut ends green mould fungi, particularly  Penicillium  sp., were  often  seen  in  discoloured  regions. In some cases dark green to black pigmented fungi were also observed.  In contrast, fungi were not visible when the cross-  cut ends were free of brownstain.  This observation, which was  110 consistently  seen  in  different  hem-fir  packages,  indicated  a  relationship between brownstain and fungi on the cross-cut ends.  5.3.2  Isolation of fungi  A total of 570 isolation attempts were performed on 38 western hemlock boards. of  290  boards.  The 12 true fir boards were not sampled.  isolates were  successfully  cultured  from  Two boards, 36 and 47, yielded no fungi.  36  A total  different  Fungi producing  a red-pigmentation on malt agar media were the most  frequently  isolated (total of 104) followed by 70 yeasts and 53 green moulds (Figure 21).  Assuming  it  was  the  same  fungus,  the  red  pigmented  fungus,  tentatively identified as Ascocorvne sarcoides by Dr. K. Seifert, was isolated  from  18 boards and  positions (A, B and C) in 10 boards.  it was present  in all  three  Interestingly, boards 16, 25,  28, which showed no signs of brownstain at the endcuts, yielded A. sarcoides exclusively from all three regions within the board. high isolation  frequency of A. sarcoides  lumber was surprising.  from western  The  hemlock  While this fungus has been reported to be  the most frequent non-decay fungus in living western hemlock trees (Etheridge, 1970) it was not isolated from western hemlock lumber in a recent mill survey in B.C. (Seifert and Grylls, 1991).  Also  Chung and Smith (1986) have only infrequently isolated Coryne sp. from western hemlock lumber, the anamorph genus of the teleomorph  111  Others Dark p1gm. funpi White myceIiu ” 7 A. sarcoides  Green moulds  Yeasts  Figure 21: Frequency of fungi isolated from western hemlock lumber.  112 Ascocoryne sp..  Green moulds, mainly Penicillium sp., were also cultured from 18 boards yet in 3 boards green moulds were the sole fungi isolated from all three positions.  This observation demonstrated  that  moulds deeply penetrated western hemlock lumber and thus agreed with another study  (Spradling, 1936) where Trichoderma liqnorum  (Tode) Harz has been shown to rapidly grow throughout the sapwood of unseasoned southern pine (P. taeda L . ) .  Furthermore, the high  frequency of green moulds in western hemlock lumber observed in this study corresponded with the findings of Seifert and Grylls (1991).  In the current study, only 14 isolates of dark pigmented fungi, possibly  Ophiostoma  sp. , were  found.  investigations of Seifert and Grylls  This  differs  from  the  (1991) and Chung and Smith  (1986) in which 0. piceae was the most  frequent  isolate  from  western hemlock lumber. However, Chung and Smith (1986), surveying for sapstaining fungi on hem-fir lumber in transit, isolated from the discoloured wood surface only.  Also Seifert and Grylls (1991)  did not clarify in their report whether the high incidence of 0. piceae was drawn from the prolific presence of asexual and sexual fruiting structures, which they have observed at the wood surface, or whether this fungus was isolated from the subsurface of western hemlock lumber. dark  pigmented  In the current study, there was no evidence that fungi, for example  0. piceae, predominated  in  113 microflora.  However, it is well possible that some of the whitish  fungi isolated from western hemlock were anamorphs of Ophiostoma sp. , for example Sporothrix  sp. as it has been reported  from  western hemlock (Chung and Smith, 1986).  The DDAC retention levels of the NP-1 fungicide on the boards sampled were at the lower end of the range of the target retention level of 90 jig DDAC/cm2.  Nevertheless it would seem unlikely that  such an abundant fungal invasion had occurred in the cold winter season thereby infecting the boards during the 10 weeks since sawing. Therefore, the majority of fungi isolated from the western hemlock were believed to represent a microflora which were already established at the time of sawing the lumber. confirmed  results  obtained  in  this  study  This conclusion  showing  microflora in freshly felled western hemlock logs 4.4).  Also  in  another  study  (Clark,  1994),  an  endemic  (see section  prolific  fungal  infection was demonstrated in second-growth western hemlock logs after 5 months of forest storage.  The fact that few fungi were  isolated from the freshly cut logs (Clark, 1994) provides further support that fungal infections can occur during log storage.  5.3.3  Sap analysis  TSP content was variable both within and between boards (Figure 22; 23).  In general, boards with brown endstain contained less TSP's  while boards without brown endstain (16, 25, 28, 29) demonstrated  114  140 120 100  I—i—h  •A; 'BIC  •  •  -  •  80  CD ^  60  40 n  20 0  II iillli.illllli lllllll Illi,, I 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 23 24  BOARD #  Figure 22: Total soluble phenols measured in three different regions within a board (board # 16 showed no brownstain).  115  120 •  •  -  A O B Bc  100 --  80 •  (D  -  1  60 --  40  r 20  0  1+1 + [ l l | l l | l l | l l | l l | M M ' i  •III IJI  25 26 28 29 31 32 33 34 36 37 38 40 41 43 46 48 49 50  BOARD #  Figure 23 : Total soluble phenols measured in three different regions within a board (boards # 25, 28 and 2 9 showed no brownstain).  116 a higher TSP content (Figure 22; 23).  The average TSP content of  the boards with brownstain was significantly  (p-value = 0.006)  different from the average TSP content of the non-stained boards when performing a Mann-Whitney test.  In cases (e.g., #23) where  boards  high  with  brown  endstain  produced  amounts  of  TSP  the  discoloration was very light and the discoloured region was small. In another instance a very high TSP content in board #3, which showed brown endstain, was related to bark residue on the sample. Inner bark of western hemlock (Hergert,  1960) .  It  was  is particularly rich in phenols  also  noticed  that  the  TSP  content  decreased from the cross-cut ends to the inner sample region in 21 boards.  This suggested movement of soluble phenols along the grain  to the cross-cut ends, which would be expected in close-piled lumber drying from the cut ends.  Anderson et al.  (196 0) have  demonstrated migration of water-soluble extractives, tannins and polyphenols, from the inner core to the outside of redwood (Sequoia sempervirens  (D. Don)  Endl.)  boards  during  kiln-drying.  authors have also stated that these water-soluble polyphenols  were  largely  responsible  discolorations found in redwood.  for  the  The  tannins and  occurrence  of  117 5.4  Conclusions  Except for two boards all western hemlock boards yielded fungi with A. sarcoides being the most frequently isolated fungus.  The uniformity of fungal isolates from western hemlock lumber which had  received  anti-sapstain  treatment,  suggested  an  endemic  microflora which had infected the wood prior to sawing the lumber.  A spatial relationship between the presence of fungi and brownstain was found on the cross-end cuts of western hemlock lumber.  Total soluble phenol contents were lower in boards with brownstain suggesting that phenol oxidation had occurred.  118 6.0  IN VITRO PRODUCTION OF HEMLOCK BROWNSTAIN  6.1  Immunogold-silver staining of 0. piceae when grown in western hemlock  6.1.1  Objective  To determine whether there is a link between the presence of 0. piceae and the production of brownstain in western hemlock.  6.1.2  Materials and Methods  Sample preparation  A package of 10 cm x 10 cm hem-fir lumber was provided by CIPA, Nanaimo, BC. into  two  Boards with brown endstain were selected and ripped  halves.  The  antisapstain treatments.  faces  were  planed  to  remove  previous  A 30 cm segment was trimmed off the  brownstained end of each board and kept for later reference. A 2.5 cm sample was then cut off the same cross-cut end, followed by a 10 cm, a 2.5 cm and a 12.5 cm piece (Figure 24).  The 2.5 cm pieces  were stored in a freezer until pressate was collected. 3 5 (12.5 and 10 cm) pieces were prepared.  A total of  Fourteen 10 cm pieces  were selected at random and frozen to serve as controls.  The  freezing of control samples was undertaken to control post-mortem changes and growth of resident microflora naturally present in  119  1  2" X 4" WESTERN HEMLOCK  I 30cm I  1 12.5 cm  2. 5  i  10 cm  CEa  J. O.PICEAE#387I INFECTED  W  2.5 cm  - •  T  NON-INFECTED  X  1 ,T  • 2.5 1 1 cm |  '  PRESSATE * TSP * pH * HPLC 1 1 1 2.5 1 |cm|  Jf' 1  i  MICROSCOPY LABELING PRESSATE  Figure 24: Flowchart showing experimental design.  120 wood.  The remaining 10 cm samples and 12.5 cm samples were used as  described below.  Preparation of fungal inoculum  O. piceae isolate # 3871 was cultured on five malt agar plates (2% malt extract, 2% agar) for three weeks. A suspension of spores and mycelial fragments was prepared by slightly scraping the fungus off the malt agar media plates and blending the scrapings in 250 mL of sterile, distilled water.  The inoculum suspension was then added,  while agitating, to 2 litres of sterile, distilled water.  Infection of western hemlock  The 12.5 cm small pieces previously described (see, were individually immersed in the 0. piceae # 3871 suspension for about five seconds.  The inoculated samples were placed in a closed  container on a 2 mm polypropylene mesh underlain with water-wetted filter  paper  to maintain  a  high  humidity  during  incubation.  Between each layer of infected samples a similar 2 mm mesh was placed as spacing.  Each container was sealed with a lid and  incubated at 20°C and RH 68% for six weeks in the dark.  Controls  (10 cm samples) were immersed in distilled water and then incubated in a similar container at 4°C and 88% RH as described above.  121  Visual and microscopic examination of wood samples  After 6 weeks of incubation visual observations were made on both infected and control pieces to determine fungal colonization at the wood surface.  A 2.5 cm sample was then cut from the centre of all  infected 12.5 cm samples and 10 cm control samples (incubated at 4°C and stored in a freezer) and used as detailed below.  Half of  the remaining pieces from all 10 and 12.5 cm samples were ovendried (103°C, 12 hours) to encourage production of brownstain while the other half were stored at room temperature.  The heated samples  were visually examined for brownstain on the cross-cut ends and faces.  Small wood cubes were cut from the centre of the 2.5 cm pieces and 15 jLtm thick, radial sections were prepared for light microscopy. The remaining portion of the 2.5 cm pieces were used to press sap. Both the collected sap and the small wood cubes were frozen until use.  Immunolabeling of 0. piceae # 3 871 in western hemlock  A monoclonal antibody (1F3) raised against O. piceae isolate # 3871 (Banerjee et al, 1994) was provided by Dr. D.L. Brown, University of Ottawa, Canada.  An enzyme-linked immunosorbent assay (ELISA)  was performed in artificial media (data not reported) to determine the reactivity of 1F3 against the 0. piceae #3871 strain used in  122 this study following the procedure described by Banerjee et al. (1994) .  Radial  sections  (15/xm) were  cut  from  infected  and  uninfected wood cubes and stored overnight in distilled water in a refrigerator.  To fix the wood all sections were then incubated for  3 0 minutes at room temperature with gluteraldehyde and Triton X-100 (0.5% gluteraldehyde and 0.2% Triton X-100) buffer  (PB)  in a well  of  a  24-well  in lOOmM phosphate  plate, using  1 mL/well.  Sections were then washed 2x5 minutes in 0.5 mL of PB, followed by a 20 minute incubation with 1 mL/well of Triton X-100  (0.5%).  Sections were washed 3x3 minutes in PB and 1x5 minute in phosphate buffer saline (PBS) before incubation in glycine (50 mM in PBS) for 3 0 minutes using 1 mL/well.  After washing sections in PBS for 2x5  minutes, blocking buffer (washing buffer with 5% goat serum) 0.6 mL/well was applied to the sections for 10 minutes, followed by washing buffer  (0.8% BSA, 0.1% gelatin, 2mM NaN3 in PBS) for 5  minutes (1 mL/well).  The primary antibody (1F3, diluted 500x in  incubation buffer) was applied to sections for 2 hours using 0.5 mL/well, except for the controls which were treated in incubation buffer (washing buffer with 1% goat serum) only. then passed  through washing buffer  The sections were  (3x10 minutes)  followed by  incubation with biotinyl GAM-IgG diluted 200x in 0.35mL/well of incubation buffer for 60 minutes. Sections were then treated (3x15 minutes) in washing buffer prior to incubation with AuroProbe-1streptavidin (0.3 mL/well) diluted 50x overnight at 4°C.  Washing  buffer (3x15 minutes) was then applied to the sections followed by 3x5 minutes treatment with PBS and a 2x5 minutes rinse in filter-  123 sterilized  (0.2 /xm) , distilled water. Silver amplification with  IntenseSE™ M was then performed on wood sections for 8 minutes in the dark using 100 /xL/well while agitating at 200 RPM. minutes)  were  employed  mounting  sections  in  using  filter-sterilized  66% Aquamount.  The  Washes (3x5  water prior  sections  were  to  then  examined with a phase-contrast microscope.  Sap analysis  TSP content was determined in sap collected prior to infection of western hemlock and in sap pressed from either 0. piceae infected, or non-infected, western hemlock following 6 weeks of incubation. The Folin-Ciocalteu method was used as described previously (see section . Qualitative HPLC analysis was also performed to determine the gross phenolic composition according to the procedure described by Daniels (1993b).  124 6.1.3  Results and Discussion  Western hemlock inoculated with 0. piceae showed mycelial growth typical of Ophiostoma sp. and/or their anamorphs on the cross-cut ends after 6 weeks of incubation.  Mycelium and black reproductive  structures were also noticed but were less prolific on the faces of the pieces.  Non-infected  controls stored at 4°C, also showed  similar fungal colonization with reproductive structures typical of Ophiostoma sp. and their anamorphs. Fungal isolation attempts from 3 different controls yielded Ophiostoma-like (unidentified) fungi. It seemed likely that these fungi were resident in the western hemlock lumber used for this study.  Heating infected samples produced dark colorations at the ends whereas non-infected samples showed the chestnut-brown coloration commonly found when brownstain is produced by heating the wood. The dark coloration was either observed across the whole cross section of infected samples or was associated with certain groups of growth rings. infected  samples  Stereo microscopy of the cross-cut ends of showed  hyaline  hyphae  and black  reproductive  structures on the surface thus accounting for the dark coloration on that surface. axially.  A dark-grey coloration penetrated the wood 5 mm  Pigmented hyphae were not found in these discoloured  areas but macroscopically visible brownstain was microscopically associated with non-pigmented hyphae.  125 Observation under a microscope revealed a few brown globules within ray parenchyma cells of western hemlock at the time of starting this experiment.  After incubation a second type of brownstain was  noticed in parenchyma cells and more rarely in tracheids.  This  brownstain pattern showed small rounded particles ranging from yellow to brown in colour which appeared to condense in advanced stages of brownstain to form larger brown deposits within the cells. seen  to  be  In  occupied  (globules)  advanced stages the whole cell lumen was with  brownstained  deposits.  However,  macroscopic brownstain was limited to the edges of the boards but, interestingly, was almost exclusively observed when hyaline fungi were present microscopically.  Similar observations were previously  made in the current study (see Chapter 3.0) and also in another investigation sapstaining  (Smith  and  Spence,  1987).  Why  fungus produced a black pigmentation  0.  piceae,  a  on malt agar  medium, but failed to produce pigment in western hemlock wood is unknown; nonetheless the fungus appeared to promote brownstain.  Even diluted x 104 the monoclonal antibody 1F3 detected 0. piceae in an ELISA, demonstrating high sensitivity of 1F3 to this fungus. However, in western hemlock 1F3 immunolabeling of 0. piceae was less satisfactory.  Effective labelling was still not accomplished  when extending the time for silver amplification from 8 to 20 minutes as this was initially thought to be the factor affecting this outcome.  126 Two factors which may have caused the failure to labell are: the degree of infection was too low (providing insufficient accessible hyphae) or interference may have occurred in the western hemlock samples due to an inherent microflora as observed by Breuil (1994) in non-sterile wood. non-infected  In the current study fungi were isolated from  controls suggesting an inherent microflora  western hemlock used.  in the  Another explanation might be that different  antigens were expressed on the cell wall of the non-pigmented 0. piceae observed in western hemlock, which the monoclonal antibody was unable to recognize.  Interestingly, Banerjee et al. (1994)  labelled 0. piceae with 1F3 in gamma irradiated jack pine (P. banksiana  Lamb.)  sapwood  suggesting  that  the  fungus  expressed  different anigens in the cell wall when growing in jack pine. However,  in  the  current  study  immunolabeling  was  also  not  satisfactory in ultra-thin sections prepared from western hemlock which was thoroughly colonized  with 0. piceae  (Ghariban, 1994).  Thus evidence provided in the current study gave strong indications that the monoclonal antibody, 1F3, lacked specificity against the strain of 0. piceae grown in western hemlock.  However,  an ELISA  might have produced a different outcome on infected ground western hemlock as 1F3 was highly sensitive in the ELISA undertaken to verify its specificity against 0. piceae.  TSP analysis of sap showed differences between and within boards (Table 7) . Variability between boards was expected since phenolics differ qualitatively and quantitatively across the cross-section  127  TABLE 7: TOTAL SOLUBLE PHENOL CONTENT (/ig/mL) MEASURED IN SAP FROM WESTERN HEMLOCK INCUBATED WITH 0. PICEAE FOR 6 WEEKS.  Infected3  A1  B2  1C  1248  1735  1855  1581  2C  513  1099  998  1038  5C  479  370  249  281  3D  324  675  172  540  4D  340  271  768  240  ID  111  181  120  234  5D  68  62  147  448  3B  99  120  247  120  5B  145  110  261  132  IB  1622  1733  1884  1921  4B  1017  678  890  1069  SAMPLE #  C4  1 = total soluble phenol content was measured in 12.5 cm sample prior to infection and incubation for 6 weeks. 2 = total soluble phenol content was measured in 10 cm sample before incubation for 6 weeks. 3 = total soluble phenols was determined in infected 12.5 cm samples following 6 weeks of incubation. 4 = total soluble phenol content was determined in 10 cm samples following incubation for 6 weeks at 4°C.  128 region  within  western  hemlock  (Barton  and  Gardner,  1966).  Nonetheless the large variations observed in the TSP content within boards were surprising.  However, no distinct patterns emerged when  comparing total soluble phenols in sap from infected samples with sap from non-infected samples and their role in hemlock brownstain was not evident (Table 7 ) .  Qualitative HPLC analysis of sap provided similar variability in the gross phenolic composition both within and between boards.  As  with the TSP analysis a distinct pattern was not noticed when comparing the gross phenolic composition in sap collected prior to infection  with  sap  analyzed  after  infection  and  incubation.  However, hydroxymatairesinol (OHMR) was found in concentrations up to 10 times lower in some infected samples than in non-infected samples.  According  predominately  to  associated  western hemlock sapwood.  Barton with  and  the  Gardner  heartwood  (1966) and  OHMR  less  was  so with  Therefore, brownstain in sapwood may  require phenolics other than OHMR. Furthermore, OHMR produced no coloration when spotted on papergrams (Barton and Gardner, 1966) and the authors explained that the fact that OHMR lacked vicinal hydroxyl groups and a stable ring system precluded oxidation byproducts (Barton, 1973).  This study failed to link changes in TSP and in the phenolic composition with production  of brownstain  in  infected  western  hemlock, differing from the previous observations (see chapter 3.0)  129 which showed oxidation of the gross phenolic composition following infection with 0. piceae. already  present  experiment.  in  the  This may be because some brownstain was western  hemlock  at  the  start  of  the  Subsequent production of brownstain during incubation  with 0. piceae may have been too little to reveal a difference in phenols because soluble phenols already may have been oxidized.  130 6.1.4  Conclusions  O. piceae prolifically colonized the wood surface and produced abundant, black, reproductive structures.  The dark coloration observed in the dried samples was attributed to the presence of the black reproductive structures.  0. piceae produced brownstain mainly in the outer regions of the western hemlock where the fungus showed no hyphal pigmentation.  The monoclonal antibody 1F3 detected 0. piceae in an ELISA assay but 1F3 was unable to detect the fungus when growing in western hemlock.  The total soluble phenols and the gross phenolic composition were not related to the production of brownstain.  In further experiments non-infected and stain-free western hemlock should be used to link production of brownstain produced by 0. piceae to changes in the phenols.  Use of a polyclonal antibody is recommended to link 0. piceae to brownstain as they generally are considered easier to work with.  131 6.2  Production of brownstain in western hemlock sap  6.2.1 Objective  To characterize chemical changes in western hemlock sap incubated with fungi.  6.2.2  Materials and methods  Sample preparation  Hem-fir (10 cm x 10 cm) showing brown cross-cut ends was sampled at CIPA, Nanaimo, B.C. on June 20, 1994.  According to CIPA's record  the lumber had been sawn about a week before sampling.  The boards  were identified microscopically as hemlock and fir and the true firs were discarded.  The ten western hemlock boards were ripped in  half and a 30 cm piece and a 5 cm piece were cut off one end of each boards.  While  the 5 cm samples were oven-dried  (103°C)  overnight to produce brownstain, sap was pressed from each 3 0 cm piece  and  frozen  according  to  the  procedures  described  (see  The remaining western hemlock boards were wrapped and  stored in a freezer until use.  Sap from two (5A; 8A) boards which  showed pronounced brownstain following heating were chosen for the first experiment. Additional sap assays were then undertaken with sap-8A, and also with sap-4A and sap-7D, which similarly originated from boards showing brown endstain after heating.  132  Infection of sap  Sap was thawed mL  of  each  and filtered (4x) through Whatman # 1.  sap was then  filter-sterilized  About 100  (0.2 /xm) using  a  disposable pre-sterilized filter unit (Nalgene Company, Rochester, NY).  Approximately 3-4 mL of the filter-sterilized sap was then  decanted into a pre-sterilized slant agar tube under aseptical conditions.  Sterility of sap samples was verified by placing a few  drops on malt agar plates which were then incubated at 2 5°C in the dark.  The cultures evaluated for their potential to cause browning  of sap (Table 8) , had been previously isolated from western hemlock logs (see chapter 4) and from western hemlock lumber (see chapter 5) . Additional isolates were chosen from the culture collection of Forintek Canada Corp, Vancouver, B.C. (Table 8 ) . A small plug of fungal  inoculum was taken from a slant agar culture  transferred to a culture tube containing sap. were  incubated  at room  temperature  replicate was used for each fungus. no  fungal  inoculum  described above.  were  used  (22 °C)  tube and  All infected tubes for 12 days.  One  In addition controls receiving  for each  sap and  incubated  as  Additional controls were kept in the freezer.  Sap analysis  A pH-meter was used to determine the pH of sap. TSP and qualitative HPLC analyses were undertaken as described previously (see . Data from infected sap, non-infected sap and sap  133  TABLE 8: FUNGI EVALUATED FOR THEIR POTENTIAL TO CAUSE BROWNING IN WESTERN HEMLOCK SAP. ISOLATE  SOURCE  0. piceae 3871  Western hemlock lumber, FCC1  A. sarcoides 12B  Lodgepole pine lumber, FCC1  P. melinii 270B  White birch log, FCC1  A2S  Western hemlock log  Interior2 , Chamiss Bay  A6S  Western hemlock log  Interfor, Chamiss Bay  A7S  Western hemlock log  Interfor, Chamiss Bay  A8S  Western hemlock log  Interfor, Chamiss Bay  A13S  Western hemlock log  Interfor, Chamiss Bay  A14S  Western hemlock log  Interfor, Chamiss Bay  A1B  Western hemlock log  Interfor, Chamiss Bay  A2B  Western hemlock log  Interfor, Chamiss Bay  BIS  Western hemlock log  Interfor, Chamiss Bay  BIBS  Western hemlock log  Interfor, Chamiss Bay  B15S  Western hemlock log  Interfor, Chamiss Bay  B16S  Western hemlock log  Interfor, Chamiss Bay  AIM  Western hemlock log  Interfor, Chamiss Bay  A3M  Western hemlock log  Interfor, Chamiss Bay  B1Y  Western hemlock log  Interfor, Chamiss Bay  C5A1  Western hemlock lum]Der, CIPA3, Nanaimo  C13C1  Western hemlock lum]Der, CIPA, Nanaimo  1 = Forintek Canada Corp., Vancouver, B.C. 2 = International Forest Products Limited, Vancouver, B.C, 3 = CIPA Lumber Co. Ltd., Nanaimo, B.C.  134  kept in the freezer were compared.  135 6.2.3  Results and Discussions  The four sap samples used differed  in their susceptibility to  develop brown colorations. Sap-8A was highly conducive to browning whereas sap-4A and sap-5A demonstrated little colour change upon incubation  with  microorganisms.  No  coloration  developed  in  infected sap-7D.  With regard to the initial trial with sap-8A, it was noticed that different fungi coloured sap at different  intensities and over  different incubation times (Figure 25; Table 9).  For instance, the  first browning was noticed in sap-8A infected with fungus A1B (a basidiomycete)  after  only  two  days  whereas  A13S,  tentatively  identified as Phialophora melinii, needed 4 days to colour sap-8A. In  sap-8A  A6S  produced  brown  precipitates  in  the  solution  demonstrating that the fungus had modified water soluble components in this sap. As a result supernatant from some saps containing the brown precipitate were clearer than the non-infected controls.  The same fungi also caused colour changes in sap-5A, yet at a much lower intensity and over a longer incubation time (Figure 25; Table 10) .  For instance fungus A1B caused the first noticeable colour  changes in sap-5A following 3 days of incubation.  Infected sap-5A  also demonstrated no coloured precipitates indicating that coloured compounds were still water-soluble.  The controls showed light  yellowing following incubation for 12 days at room temperature.  136  •."nil *•«•**••••-  -*•  '"«-IIK.,,/l„„,M|KN ' M l . . , K Ml- IS, U l II !,»,,, | » « • • HI M UN..I VI ,,H "  jm^  Figure 25: Colour changes in western hemlock sap (8A; 5A) incubated with different fungi for 10 days at room temperature.  137  TABLE 9: CHANGES IN WESTERN HEMLOCK SAP (8A) INCUBATED WITH DIFFERENT MICROORGANISMS FOR 12 DAYS AT ROOM TEMPERATURE. TREATMENT  SAP COLOUR  pH  TSP1 /xg/mL  OHMR2 /xg/mL  control/frozen  light yellow  5.1  594  60  control/22°C  yellow  5.2  353  91  0. piceae 3871  light brown  7.2  255  43  A2S  brown  7.3  200  19  A6S  brown  7.5  167  0  A8S  yellow  6.0  399  85  A13S  brown  7.5  172  0  A14S  yellow  4.5  424  86  AIM  light yellow  5.5  355  77  A1B  brown  4.8  0  A2B  light brown  5.3  372  65  BIBS  light brown  6.8  207  10  B1Y  light brown  6.1  198  72  7.5  206  8  B16S  brown  1 = refers to total soluble phenol content 2 = refers to hydroxymatairesinol  0  TABLE 10: CHANGES IN WESTERN HEMLOCK SAP (5A) INCUBATED WITH DIFFERENT MICROORGANISMS FOR 12 DAYS AT ROOM TEMPERATURE. TREATMENT  SAP COLOUR  pH  TSP //g/mL  OHMR /xg/mL  control/frozen  colourless  4.8  85  25  control/22°C  light yellow  4.9  45  34  0. piceae 3871  light yellow  7.5  64  12  A2S  light yellow  7.2  58  10  A6S  light yellow  7.5  0  0  A8S  colourless  6.2  93  28  A13S  light yellow  7.5  0  0  A14S  colourless  5.2  119  35  AIM  light yellow  6.6  54  20  A1B  yellow  4.9  0  0  A2B  colourless  4.9  105  34  BIBS  yellow  6.4  14  0  B1Y  yellow  7.0  71  11  B16S  yellow  7.6  18  0  139 The assays with sap-4A and sap-7D affirmed that saps from different boards differed in their susceptibility to undergo colour changes upon infection with fungi.  Sap-4A developed a yellow coloration  with some fungi, similar to those colour changes produced by the same fungi noted in sap-5A (Table 11). Colorations did not occur in sap-7D upon incubation with fungi (Table 12).  Additional assays with sap-8A, undertaken to verify the initial observations, confirmed browning.  that this sap was very susceptible to  It also affirmed that fungi varied in their potential to  cause sap browning (Table 13). Thus fungi with a high potential, for example A6S, consistently produced brown colorations in sap-8A.  Most fungi shifted the pH to neutral or slightly alkaline when cultured underwent  in  the  four  browning.  different The  fungi  sap  samples  which  but  caused  coloration of sap-8A shifted the pH to alkaline  only  intense  sap-8A brown  (Table 9, 13) .  Interestingly, the fungi which were evaluated for their browning potential, produced similar pH changes in sap-4A, sap-5A and sap-8A but less in sap-7D.  For example, the fungus B16S shifted the pH in  sap-8A from 5.1 to 7.5, in sap-5A from pH 4.8 to 7.6 and in sap-4A from pH 4.7 to 7.6 (Table 9-11).  Two of the three basidiomycetes  tested in sap-8A produced no pH shift yet browning occurred with A1B.  The third  basidiomycete (C5A1) evaluated, decreased the pH  to 3.4 which was accompanied by a colour change to yellow/brown.  140  TABLE 11: CHANGES IN WESTERN HEMLOCK SAP (4A) INCUBATED WITH DIFFERENT MICROORGANISMS FOR 12 DAYS AT ROOM TEMPERATURE. TREATMENT  SAP COLOUR  pH  TSP /xg/mL  OHMR xxg/mL  control/frozen  colourless  4.7  96  6  control/22°C  light yellow  4.9  47  0  0. oiceae #3871  light yellow  7.3  10  0  A2S  light yellow  7.6  0  0  A6S  yellow  7.7  0  0  A13S  yellow  7.8  0  0  B16S  yellow  7.6  0  0  BIBS  yellow  5.1  0  0  colourless  5.8  8  0  colourless  7.6  0  0  colourless  5.0  49  0  yellow  7.6  0  0  A. sarcoides #12B BIS P. melinii # 270B B15S  TABLE 12: CHANGES IN WESTERN HEMLOCK SAP (7D) INCUBATED WITH DIFFERENT MICROORGANISMS AFTER 12 DAYS AT ROOM TEMPERATURE. TREATMENT  SAP COLOUR  PH  control/frozen  colourless  5.3  309  113  control/22°C  colourless  5.2  366  239  0. piceae #3871  colourless  6.7  360  74  A2S  colourless  6.4  340  65  A6S  colourless  7.2  124  0  A13S  colourless  6.7  126  0  B16S  colourless  6.8  267  36  B15S  colourless  6.8  258  36  colourless  6.4  315  45  colourless  6.6  297  36  P. melinii 270B BIS  TSP /xg/mL  OHMR /xg/mL  141  TABLE 13: REPEATED ASSESSMENT OF CHANGES IN WESTERN HEMLOCK SAP (8A) INCUBATED WITH DIFFERENT MICROORGANISMS AFTER 12 DAYS AT ROOM TEMPERATURE. TREATMENT  SAP COLOUR  pH  TSP /xg/mL  OHMR ptg/mL  5.0  537  ND1  yellow  5.0  237  18  A3M  yellow  5.0  253  12  A7S  yellow  6.3  130  9  3.4  5  0  control/frozen control/22°C  C5A1  colourless  ye11ow/brown  C13C1  yellow  6.5  121  11  0. piceae 3871  brown  7.3  69  0  A2S  brown  7.4  17  0  A6S  brown  7.8  0  0  A13S  brown  ND1  0  0  B16S  brown  7.3  31  0  4.7  68  0  yellow  5.0  376  8  brown  7.0  115  0  7.3  38  0  7.7  22  0  BIB A. sarcoides 12B BIS P. melinii 270B l_  B15S  1 = not determined.  yellow/brown  light brown brown  142 Determination of the TSP content showed that sap-5A and sap-4A contained about 6 times less soluble phenols than sap-8A, and sap7D contained about one-half the TSP amount of sap-8A.  Generally,  infected samples demonstrated a pronounced decrease in the TSP content as colorations developed.  For example, fungi A6S and A13S  reduced the TSP content in sap-8A by more than half Both  of  these  isolates  Phialophora sp. (Table 4 ) .  have  been  tentatively  (Table 9) .  identified  as  In another trial with sap-8A the same  fungi modified all the soluble phenols and they were subsequently not  detectable  (Table  13) .  Both  isolates  caused  pronounced  browning in sap-8A. A relationship between coloration and decrease in TSP and pH was thus evident.  Similar relationships between  coloration, pH and TSP content were also seen in infected sap-4A and sap-5A.  For instance A6S and A13S produced an alkaline pH  shift and modified all the TSP present in sap-4A and sap-5A and this caused some yellow coloration (Tables 9, 10). A relationship between the pH and the decrease in TSP was also observed in sap-7D when infected with A6S and A13S,  but colorations were not noticed  (Table 12). However, the other fungi shifted the pH in sap-7D but little changes were recorded in the TSP content.  Interestingly,  an approximate  50% decrease  in TSP content was  observed in the incubated controls (non-infected samples) except for sap-7D  (Table 9-13) .  This suggested that auto-oxidation of  soluble phenols had accompanied a yellowing in sap-8A and a light yellowing in sap-4A and sap-5A.  143 A qualitative difference in the gross phenolic composition was recorded (by HPLC) in the four sap samples.  For example, catechin  was observed exclusively in sap-8A and epicatechin only in sap-5A whereas OHMR was present in each of the four saps.  The sap samples  also varied quantitatively regarding their individual phenols, for example in the OHMR content (Tables 9-13) .  These qualitative and  quantitative differences were expected as the sap originated from different boards.  Barton and Gardner (1966) have shown that the  phenolic composition varied  within and between western hemlock  trees.  Most fungi modified the gross phenolic samples during  composition in the sap  incubation, but the magnitude  between fungi.  of change varied  For example the fungus A6S produced the largest  change in the gross phenolic composition in all four sap samples evaluated and this corresponded with large changes in TSP (Tables 9-13) .  This study has confirmed earlier observations that a non-specific microflora can cause browning of sap (see chapter 3 ) .  However,  this study provided additional information regarding the changes in TSP which decreased as browning occurred.  Similar observations  have been made in discoloured maple (Tattar and Rich, 1973). another study correlated  In  (Siegle, 1967) the decrease in phenolics has been  with  the  increase  in  the  discoloration  rate  incubating hot water extract of birch meal with a fungus.  when  Changes  144 in TSP yielding discolorations have also been reported in cricket bat willow wood infected with a bacterium (Wong and Preece, 1978) and in red maple (Shevenell and Shortle, 1986) .  Several  observations  current study.  correlate  with  the  findings  made  in the  Tattar et al. (1971) have also reported that the  pH shifted from 5.5 in clear maple to 6.4 in discoloured, infected maple. An increase in pH has also been demonstrated in discoloured maple sawdust, infected with bacteria and fungi (Zimmermann, 1974) and in Ilomba infected with bacteria  (Bauch et al. , 1985) .  In  another study (Schmidt and Mehringer, 1989) sap browning was linked to a pH shift caused by bacteria.  Interestingly, Schmidt (1986) has linked the pH shifts produced in infected beech sap, to the availability of sugar and nitrogenous compounds in the sap.  Browning in infected sap has been prevented  by addition of glucose which kept the pH below 7 Mehringer, 1989).  (Schmidt and  In the current study sugar and nitrogen levels  were not determined in the sap specimens.  However, the fact that  some fungi always caused a pH shift while others, for example, mould fungi, a yeast and the A_;_ sarcoides isolates, produced little or no pH changes suggested that the fungi differed metabolically. Clearly,  the  fungi  grew  prolifically  in  the  sap  samples  irrespective of their potential to shift pH, indicating that the sap contained adequate nutrition.  145 The current study also demonstrated that dark pigmented fungi, for instance  0. piceae and  Phialophora sp. shifted the pH to neutral  or alkaline which promoted an intense browning in sap-8A.  This  indicated a relationship between the dark pigmented fungi and the browning in sap, possibly due to the secretion of either enzymes or phenols.  For example, secretion of fungal phenoloxidases into the  environment  may have  present in the sap.  catalyzed  browning  of phenolic  compounds  Rosch et al. (1969) have shown the presence of  laccase, an enzyme involved in the production of fungal melanin in some sapstaining fungi grown in liquid culture.  On the other hand  secretion of fungal phenols into the culture medium has also been reported triggered However, activity  to by  form heterogenous melanin an  alkaline  the current (other  than  pH  shift  (Bell  study was unable shifting  the  (dark-brown pigments),  pH)  and  Wheeler,  to determine was  1986).  if  required  fungal  for  sap  browning. While alkaline conditions alone have been shown to cause browning  (Hathway and Seakins, 1957) slightly acidic conditions  also produced browning in the current study, for example, in sap-8A upon infection with the basidiomycete AlB and C5A1 (Tables 9 and 13, respectively).  This observation suggested involvement of a  phenol oxidizing enzyme.  However, certain compounds seemed to be required in the sap to develop browning.  This was underscored as browning occurred in  sap-8A but not in sap-4A and sap-5A despite similar pH shifts and changes in TSP and in the gross composition of phenols.  Further  146 support was given to the observation that the phenolic composition of the sap is critical to browning when sap-7D, which had a TSP content three times larger than the TSP of both sap-4A and sap-5A, developed no coloration upon inoculation with A6S and A13S although the TSP decreased. other  sap  Catechin was detected in sap-8A and not in the  samples but  it was present  However, catechin was not detectable  in very  (HPLC) after incubation of  sap-8A suggesting its involvement in brownstain. studies  small amounts.  Two recent HPLC  (Hrutfiord et al. , 1985; Daniels, 1994) have also shown  that the concentration of catechin declined rapidly and was related to browning of western hemlock chips and western hemlock sap, respectively.  In the current study, the production of brown precipitates in infected  sap-8A  which  contained  catechin,  was  of  particular  interest, because the development of hemlock brownstain is believed to involve  formation of  insoluble brown compounds  soluble compounds.  Hathway and Seakins  that  very  catechin  was  reactive  condition producing brown polymers.  under  from water-  (1955; 1957) have shown neutral  and  alkaline  Song (1987) also recorded the  production of brown precipitates when investigating brownstain in Douglas-fir.  In the current study catechin was histochemically  demonstrated in brownstained western hemlock  (see chapter 3) and  HPLC analysis provided additional evidence for the involvement of catechin in sap browning in this study.  147 6.2.4  Conclusions  Fungi isolated from western hemlock can promote colour changes in western hemlock sap to produce water-insoluble brown precipitates.  An upward shift in pH in infected western hemlock sap accompanied colour changes.  The phenolic composition of the sap is critical to browning. Chemical extractives, such as catechin, can be involved in the production of hemlock brownstain.  148 6.3  Infection of wood sections with 0. piceae on a glass-slide  6.3.1 Objective  To determine whether 0. piceae can produce brownstain in thin sections of western hemlock.  6.3.2  Materials and Methods  Slide preparation  Glass slides were sterilized in an autoclave prior to coating with water agar (1.5% bacto agar) under aseptic conditions.  One coated  slide was then placed on a bent 2 mm glass rod in a pre-sterilized petri dish with filter paper and distilled, sterile water on the bottom.  Wood sections used  Two wood cubes were prepared from one freshly sawn western hemlock board (4A) containing sapwood, one from the outer region and one from the centre of the board. produced section  aseptically  from  was immediately  Five 15 /xm sections were then  each cube using  placed  a microtome.  on a agar-coated  glass  Each slide,  covered with a sterile cover slip and placed in a Petri dish.  149  Infection of wood sections  Wood sections were infected by placing a small plug of 0. piceae isolate #3871 at the edge of the cover slip. Four sections from each wood cube were infected while a fifth section received no inoculum and served as control.  All plates were incubated in the  dark at 25°C for 4 weeks.  Microscopic examination of sections  Wood sections were removed from the coated glass slide and mounted in lactophenol for microscopic examination.  After the initial  examination sections were removed from the glass slides and washed in distilled water to extract all hyphae attached to the wood surface.  The degree of hyphal colonization within the section was  microscopically examined. optics microscope.  Photographs were taken using a Zeiss  150 6.3.3  Results and Discussions  O. piceae showed prolific growth on the coated glass-slides after 4 weeks of incubation. the wood sections.  However, fungi grew less prolifically in  Interestingly, the hyphae were non-pigmented  when associated with the thin sections. A few pigmented structures were noticed on the coated slides and in instances where the fungus grew on the filter paper at the bottom of a petri dish.  Brownstain developed in numerous ray parenchyma cells which had been brownstain-free and without hyphae prior to infection (Figure 26) .  The  degree  of  brownstaining  varied  within  and  between  infected sections. Brownstain was more pronounced at the side of the sections where the inoculum was placed.  Non-infected samples  developed some brownstain following incubation yet to a much lesser extent than the infected samples.  The degree of staining observed in sections as thin as used in this study was rather surprising considering that the quantity of colour precursors present in the ray cells of the 15 jum sections must be extremely small.  In a previous study, Smith and Spence  (1987)  produced brownstain in 5 cm x 10 cm western hemlock when incubated with 0. piceae and brownstain was also produced in 0.6 cm thick western hemlock wafers following infection with the same fungus in the current study (see chapter 3 ) .  151  " >t  Figure 26: Tsuga heterophylla. Radial section (63x). Brown deposits associated with hyphae of 0. piceae in a 15 jxm section.  152 Brownstain did not develop upon heating of a wood sample from the same board were  (4A) suggesting that both the fungus and the phenols  essential  for production  of  brownstain  in the  sections.  Browning also was not observed when sap from the same board (4A) was inoculated with 0. piceae 3871, or with any of the other fungi previously determined to promote browning of sap. This observation strongly suggested that the phenols responsible for brownstain were not released from the wood (4A) when pressing sap because they were linked to wood components. (Stafford,  1988)  demonstrated  and  they  Phenols can be bound to proteins can  in western hemlock  Barton, 1962) .  also  occur  as  glucosides  as  (Goldschmid and Hergert, 1961;  For example, catechin, which is thought to play an  important role in hemlock brownstain and which was not detected in sap-4A  (see  section  6.2) , may have  sections as glucosides.  been present  in the  thin  However, the exact nature of the browning  induced by 0. piceae is not known yet.  It is possible that the  fungus, as it colonized western hemlock, released phenols from their sugar moieties, which then accompanied by a pH shift produced brownstain  similar  to  the  pH  promoted  browning  observed  in  inoculated sap (see chapter 6.2).  On the other hand 0. piceae may also have secreted substances into the wood  cells which  extracellular melanin.  then caused  the  coloration,  Bell and Wheeler  for example  (1986) reported  that  production of extracellular fungal melanin can occur either by releasing phenol oxidase into the environment to oxidize phenolic  153 compounds or by releasing phenols into the environment, where they are auto-oxidized.  Bell and Wheeler (1986) also showed that fungi  devoid of wall-bound melanin could still produce brown pigments. wall-bound  extracellular  In the current light microscopy study a distinct  melanization  was  not  observed  in  0.  piceae  when  associated with brownstain. However, further experimentation would be  required  to  determine  whether  this  fungus  can produce  an  extracellular brown pigment.  In the current study, dark pigmented hyphae grew out of the thin sections  when  placed  on  malt  agar  media.  This  observation  demonstrated that this fungus had the enzymes, either induced or constitutive, required for melanin production. also  suggested  that  certain  chemical  This observation  constituents  in  western  hemlock greatly reduced pigmentation of 0. piceae when growing in wood as it was not detectable by light microscopy.  154 6.3.4  A  Conclusions  technique  has  been  developed  which  demonstrated  fungal  participation in the brownstaining process in wood.  Brownstain  formation  can occur  in the presence  of very  small  amounts of chemical precursors.  Cell wall pigmentation of 0. piceae is greatly reduced or not apparent by light microscopy when the fungus grows within the cells of western hemlock.  155 6.4  Infection of western hemlock and lodgepole pine with 0. piceae  6.4.1 Objective  To determine whether 0. piceae  produces pigmented hyphae when  growing in western hemlock.  6.4.2  Materials and Methods  Wood used  A freshly sawn western hemlock board (2.5 cm x 5 cm) with a high proportion of sapwood, which had been stored in the freezer for two weeks, was used.  Two 3 0 cm pieces and two 25 cm pieces were  produced after thawing.  In addition a lodgepole pine bolt which  had been stored in a freezer since 1990 was sawn to 30 cm length and four 5 cm x 5 cm pieces and four 2.5 cm x 10 cm pieces were produced from the sapwood and heartwood.  The wood samples were  planed on all faces prior to infection.  Preparation of inoculum and infection of wood  0. piceae # 3871 was cultured on malt agar (2.0% malt extract; 1.5% agar) for two weeks at 25°C in the dark.  A suspension with fungal  fragments and spores was prepared by adding a small amount of sterile, distilled water to a culture plate and lightly scraping  156 the surface mycelium.  This procedure was repeated three times.  The suspension prepared from three plates was then blended (2x15 seconds) in approximately 250 mL of sterile, distilled water using a  Waring  blender.  The  blended  suspension  was  added  to  approximately 2500 mL of sterile, distilled water.  The wood samples were dipped in the fungal propagule suspension for 5-10 seconds and they were then close-piled in a container with moist filter paper and glass rods on the bottom.  Four stacks were  assembled in the container which was covered with a lid and placed at 20°C and 68% RH in the dark.  After four weeks of incubation the  infected samples were visually examined and a 1 cm slice was cut from one end of a lodgepole pine sample and also a western hemlock sample and  15 ptm radial  microscopic examination.  sections were prepared  from them for  The infected samples were then incubated  for another 8 week period.  After a total of 12 weeks the four  faces of each sample were planed  (1 mm) to remove the fungal  surface flora and to disclose the degree of surface coloration.  A  1 cm section was then cut from one end and from the centre of a lodgepole  pine  and  sections.  A Leitz photomicroscope was used for examination of the  15 fJLrci specimens.  a western  hemlock  board  to produce  15 /xm  157 6.4.3  Results and Discussion  Incubated lodgepole pine sapwood samples were covered with a thick mycelial  mat  after  three  weeks.  In  heartwood was free of mycelial growth.  contrast  lodgepole  pine  Fungal growth was also well  established at the wood surface of western hemlock. After 4 weeks of incubation some mold fungi, for instance Penicillium sp., were also growing on the surface of both lodgepole pine and western hemlock pieces.  Visual inspection after 8 and 12 weeks demonstrated an unidentified decay  fungus  Lodgepole  colonizing  pine  heartwood  some  of  the  also  showed  lodgepole  pine  samples.  localized mould  (orange)  infections on the surface but no mycelium of 0. piceae.  The  surface of the western hemlock samples was thoroughly colonized with 0. piceae except for a few localized, green mould fungi.  Inspection of the 1 cm piece trimmed off a lodgepole pine sample demonstrated that sapstain was established after 4 weeks. lodgepole pine sample  showed the classical blue  frequently seen in pines. sapwood was parenchyma  due  axial  stain pattern  The discoloration of lodgepole pine  to abundant, pigmented  cells and  The  tracheids  as  hyphae growing seen  in ray  microscopically.  After 12 weeks of incubation bluestain was seen on the faces of the planed lodgepole pine sapwood.  However, the degree of bluestain  varied within and between lodgepole pine pieces.  For example,  158 bluestain was more pronounced on the 1 cm cross-end samples whereas much less coloration was noticed on the cross-section of the centre samples.  This observation was also confirmed microscopically as  very few pigmented hyphae were found in sections cut from the centre of the lodgepole pines.  In contrast visual inspection of the 1 cm slice trimmed off western hemlock  demonstrated  no sapstain after 4 weeks of  incubation.  However, microscopic examination showed prolific growth of hyaline hyphae within the wood. outer  Pigmented hyphae were only present in the  (1-5) cells close to the wood surface.  Brownstain also  developed in parenchyma cells containing hyphae where there was no brownstain and no visible hyphae existed prior to infection. After 12 weeks of incubation visual examination of the planed western hemlock showed that the board surfaces were stain-free except for a  brown  coloration  Microscopic brownstain  disfiguring  examinations  of  in ray parenchyma  the  these and  edges stained  tracheids  of  the  regions and  surfaces. revealed  abundant  non-  pigmented hyphae. Very few pigmented hyphae and a few coremia were associated with brownstain but they seemed to be closely related to the wood surface. depth of 2-5 mm.  The brown coloration penetrated the wood to a The western hemlock slice cut from the centre was  free of brownstain and hyphae were not observed microscopically.  This experiment reconfirmed that 0. piceae can produce brownstain in western hemlock as observed many times during the course of this  159 study. Macroscopic brownstain was concentrated at the edges of the board surfaces and penetrated about 5 mm.  Smith and Spence (1987)  also demonstrated that 0. piceae can produce brownstain and a deeper penetration of the brownstain was shown by these authors. However,  the presence  of non-pigmented  hyphae  brownstain was consistent in both studies. hyphae were  absent  in the  centre  of  the  associated  with  Both brownstain and sample  suggesting  a  relationship between brownstain and 0. piceae.  The current study has also shown that pigmentation of 0. piceae was promoted in lodgepole pine sapwood whereas it was suppressed in western hemlock sapwood.  0. piceae produced pigmentation on the  surface of western hemlock wood but not when deeply penetrating the wood.  However, at present the factors suppressing pigmentation of  0. piceae moisture  in western hemlock content  may  have  have not been elucidated.  influenced  the  staining  Wood  intensity.  According to Lagerberg et al. (1927) maximum pigmentation occurred in pine and spruce at a wood moisture content of 60-80%.  Sapstain  has been controlled by keeping Scots pine (P. sylvestris L.) above a wood moisture content of 100-120% (Liese and Peek, 1984).  In the  present study both, western hemlock and lodgepole pine, showed a wood moisture content of 120%.  On the other hand wood species has  also been reported to influence the degree of fungal pigmentations (Liese and Schmidt, 1961, Smith, 1994).  It is well possible that  western hemlock extractives inhibited pigmentation of 0. piceae but not its growth.  160  Therefore, evidence from the current study suggested that this fungus was not capable of causing sapstain in western hemlock.  The current study also demonstrated that the association between brownstain and 0. piceae occurring in western hemlock does not occur in lodgepole pine.  161 6.4.4  Conclusions  0. piceae developed pigmented hyphae when grown in lodgepole pine but, except for a small amount of surface growth, the same fungus remained hyaline when colonizing western hemlock.  Brownstain developed macroscopically and microscopically in western hemlock infected with 0. piceae.  A chemical difference must exist between lodgepole pine and western hemlock  to  account  for  the  fact  that  0.  piceae  develops  pigmentation when growing in the former and not in the latter wood species.  162 7.0  ELUCIDATION OF THE MECHANISMS OF SAP BROWNING  7.1  Objective  To determine factors involved in the browning of western hemlock sap.  7.2. Materials and Methods  7.2.1  Effect of pH on sap browning  Filter-sterilized (0.2/xm) western hemlock sap (8A) was inoculated with different microorganisms according to the procedures described (see . Additional control sap-8A samples adjusted to pH 7 with NaOH and unchanged sap-8A (pH 5) were set up but they received no inoculum.  The sap samples were incubated at room temperature  (22°C) for 12 days.  The changes in colour, pH, TSP and qualitative  changes of the gross phenolic composition in inoculated and control sap-8A  were  compared  according  to  the  procedures  outlined  previously (see  7.2.2  Effect of oxygen on sap browning  Four mL of filter-sterilized (0.2 fxm) sap-8A was transferred into a sterilized (121°C, 15 minutes) glass ampoule.  Additional sap-8A  was adjusted to pH 7 with NaOH prior to filter-sterilization  163 (0.2  nm)  and  ampoules.  4 mL  was  then  delivered  into  additional  glass  The ampoules were each sealed with a cotton plug and  stored in a freezer at -2 0°C overnight.  Two ampoules, one with pH  7 adjusted sap and one with unchanged (pH 5) sap were degassed bydrawing a vacuum over the frozen sap to remove the oxygen present in the ampoule.  The sap was then thawed to release the dissolved  oxygen prior to rapid freezing of the sap with liquid nitrogen.  A  vacuum was drawn again and the total procedure (thawing-freezingvacuum) was repeated three times.  Nitrogen was then introduced  into the ampoules prior to sealing them with a Bunsen burner. other  two ampoules, one with pH  unchanged  7 adjusted  gross  sap and one with  (pH 5) sap, were neither degassed  nitrogen and they were then sealed. phenolic  composition  were  The  nor  flushed with  The coloration, TSP and the  determined  after  12  days  of  incubation at room (22°C) temperature as described (see  7.2.3  Effect of heat on sap browning  Sap-8A was autoclaved at 121°C for 15 minutes, poured into presterilized test tubes using 4 mL per tube and then inoculated with various fungi.  Two additional test tubes containing heated sap  without inoculum served as controls.  After 12 days of incubation  the colour, pH and TSP was determined in heated/inoculated sap and in controls  according  to procedures  described  previously  (see  164 7.2.4  Investigation  of  sap  browning  by  heat  and  by  pH  alteration  Sap from boards # 2B, 5B, 5D, 8B, 9A and 9D, which were sampled at CIPA, Nanaimo, B.C.  (see,  minutes in an autoclave.  was heated at 121°C for 15  Additional sap samples from the same 6  boards were adjusted to about pH 7 with NaOH, filter-sterilized and then incubated at room temperature (22°C) for 12 days.  The heated  and the pH adjusted sap specimens were compared to controls (nonheated and non-pH adjusted) regarding coloration, changes in pH and TSP as described (  7.2.5  Amendment of water with phenols  Distilled water was adjusted to pH 7 with NaOH prior to making up solutions  containing  epicatechin,  approximately  hydroxymatairesinol  respectively.  500  |iig/mL  and  of  catechin,  alfa-conidendrin,  In addition a single solution was prepared mixing  together approximately 150 /xg/mL of each compound.  The solutions  were placed in an ultrasonic bath for 1 hour to promote a thorough dissolving of the compounds. different  solutions, were  temperature TSP.  Test tubes, each with 4 mL of the  then  incubated  for  12 days  at room  (22°C) prior to assessing the changes in colour and  165 7.2.6  Amendments of sap with known phenols  Sap-5a, which had develop no browning upon infection with fungi in this study, was filtered (3 x) through Whatman # 1 and adjusted to pH 7 with NaOH. Aig/mL  of  The sap-5A was then amended with approximately 500  either  catechin,  hydroxymatairesinol.  epicatechin,  alf a-conidendrin  or  Additional sap-5A was amended with a mixture  of approximately 150 Aig/mL of each of the four individual compounds or with about 50 Aig/mL of catechin.  Furthermore,  sap-5A adjusted  to pH 7 and unchanged sap-5A were included without amendments. The sap solutions were placed  in an ultra-sonic  Approximately 4 mL of each filtered-sterilized  bath  for 1 hour.  (0.45 Aim) sap was  placed in individual test tubes and incubated for 12 days at room temperature  (22°C) prior to evaluating  the colour and the TSP  changes as described previously (see  7.2.7  Effect of buffer on sap browning  Sap-9A was thawed and filtered through Whatman #1 filter paper.  A  biological buffer, containing 4.8 g of MES (Sigma, St Louis, MO) and 0.1 g NaOH, was then added to 50 mL of sap prior to filtersterilization  (0.2 Aim).  Additional sap which received no buffer  was also filter-sterilized.  About 3-4 mL of the sterile sap was  then decanted into pre-sterilized test tubes and inoculated with a fungus.  One replicate was established for each fungus evaluated  and also for the controls (with buffer and without buffer).  The  166 test tubes were incubated at room temperature (22°C) for 12 days. Changes in coloration, pH and TSP were compared to controls kept at room temperature or in a freezer using the procedures outlined (see  167 7.3  Results and Discussions  7.3.1 Effect of pH on browning  Brown  colorations  developed  in  sap-8A  upon  infection  with  microorganisms. The browning process was accompanied by a pH shift from slightly acidic to slightly alkaline and a decrease in TSP and OHMR (Table 14).  However, sap-8A adjusted to pH 7 and incubated  without fungal inoculum also underwent browning (Table 14) . A very slight colour change was already noticed at the time the sap was pH altered.  The brown colour and the decrease in TSP and OHMR were  similar in the pH 7 adjusted sap-8A and in the infected sap-8A. This observation demonstrated that the phenols were highly reactive in sap-8A at pH 7.  However, the pH decreased in the adjusted sap-  8A from 7 to 6.1 over incubation time,  contrasting with the pH  increase recorded in the inoculated samples.  This observation  indicated that carbon dioxide was absorbed from the atmosphere into the pH 7 adjusted sap and possibly reacted with protons released from phenols as oxidation and condensation occurred.  Interestingly, brown precipitates formed in the pH altered sap-8A which looked similar to that observed in inoculated sap-8A. observation  demonstrated  that neutral pH  conditions  alone  produce brown reaction products in western hemlock sap. and Seakins  This can  Hathway  (1955; 1957) have demonstrated that catechin formed  quinones followed by oxidative condensation at a neutral and a  TABLE 14: CHANGES IN pH 7 ADJUSTED AND NON-ADJUSTED SAP (8A) FOLLOWING INCUBATION FOR 12 DAYS AT ROOM TEMPERATURE. TREATMENT  SAP COLOUR  pH  TSP /xg/mL  control/frozen  colourless  5.1  537  16  control/22°C  yellow  5.1  237  13  Sap-8A/pH 7/22°C  brown  6.1  105  0  0. piceae #3871  brown  7.6  122  0  A2S  brown  7.8  114  0  A6S  brown  7.9  70  0  A13S  brown  7.9  86  0  A16S  brown  7.6  119  0  BIB  yellow  5.5  135  9  BIS  yellow  7.4  112  0  B15S  brown  8.0  78  0  OHMR /xg/mL  169 slightly alkaline pH.  In the current study catechin might have  condensed to brown polymers as it was detected before, but not after, incubation.  in the sap-8A  In addition, the yellowing  observed in the controls of sap-8A perhaps suggested formation of quinones  from  catechin  as  its  catechin  content  decreased  considerably over incubation time.  7.3.2 Effect of oxygen on browning  A brown coloration and a brown precipitate developed in sap-8A, adjusted to pH 7, under atmospheric coloration formed under nitrogen slight  colour  Unchanged  (pH  change 5)  conditions whereas little  (Figure 27) .  Interestingly, a  occurred when adjusting  sap-8A to pH 7.  sap-8A  developed  incubated under oxygen but  remained  a  yellow  colourless  coloration  when  under nitrogen  (Table 15) . As previously noticed the changes in colorations were accompanied with TSP changes (Table 15). Thus neutral conditions and oxygen were required to produce browning in sap-8A containing catechin while colorations were absent in the unchanged (pH 5) sap8A when kept under nitrogen.  Hathway and Seakins (1955) have also  shown that absence of oxygen arrested colorations of catechin at neutral pH.  However, the findings made in the current study  disagreed with an investigation (Miller et al. , 1983) reporting on browning in Douglas-fir sap incubated with nitrogen after 47 days. In another study (Arvey, 1993) colorations were also observed with phenols extracted from Douglas-fir and incubated in a helium  170  l ' l l , l I l K - A I IMV.1 I . I / . V . l l  V»lii>IE-t*J>  HEMLOCK SAP AFTER 4 DAY OF INCUBATION (October 10, 94)  OXYGEN  Figure 27: Production of sap coloration under oxygen.  171  TABLE 15: EFFECT OF OXYGEN ON COLOUR AND TSP (jug/mL) CHANGES IN WESTERN HEMLOCK SAP. TREATMENT  SAP COLOUR  PH  TSP /xg/mL  Nitrogen  light brown  7  225  Nitrogen  colourless  5  425  Oxygen  brown  7  125  Oxygen  yellow  5  250  172 atmosphere after 32 weeks (Arvey, 1993) . It is possible that Arvey (1993) and Miller et al. (1993) did not completely remove oxygen from their systems as other studies have also reported on oxygen requirements for colorations in redwood (Anderson et al. , 1960), in rosewood (Millettia sp) (Kondo et al. , 1986) and in oak (Wassipaul et al. , 1987) .  Also vacuum kiln-drying  has  controlled  brown  colorations in oak (Charrier et al., 1992).  7.3.3 Effect of heat on browning  Heating  of  sap-8A  (121°C,  15  minutes)  produced  a  brownish  coloration without changes in pH and in TSP (Table 16). However, TSP decreased in the heated sap-8A  (controls) during incubation  even though no changes in colour or pH occurred  (Table 16) .  In  contrast, a shift in pH and a drastic drop in the TSP were recorded in the heated sap-8A following incubation with fungi and the colour of the sap further darkened  (Table 16) . The fungi which produced  a dark brown coloration also caused the most changes in the TSP and in pH. These observations suggested that resident sap enzymes were not involved in the browning which developed upon infection with fungi.  Barton and Gardner  (1966) have suggested that enzymes  inherent in western hemlock sapwood, were involved in brownstain. In another study (Azim-Musbah, 1993) phenol oxidases were isolated from Douglas-fir sapwood and they were implicated in brownstains in this wood species. Residual tree enzymes or bacterial enzymes have also been suggested as causing brownstain in sugar pine and in white  173  TABLE 16: CHANGES IN HEATED 1 SAP-8A AFTER INCUBATION WITH DIFFERENT MICROORGANISMS FOR 12 DAYS AT ROOM TEMPERATURE. TREATMENT  SAP COLOUR  PH  control-frozen  colourless  5.0  494  control-heated-frozen  light brown  5.0  465  control-heated-22°C  light brown  5.0  306  A3M  light brown  5.0  305  A2S  brown  6.4  205  A6S  dark brown  6.8  32  B15S  dark brown  6.7  122  dark brown  6.8  63  P. melinii 270B  TSP /xg/mL  1 = refers to heating of sap in an autoclave at 121°C for 15 minutes.  174 pines  (Stutz, 1959) .  However, it was reasonable to assume that  heating denatured proteins in sap-8A in the current study and thus subsequent sap colorations were induced by the fungi.  7.3.4 Production of sap browning by heat and by pH alteration  Heating  and  pH-modification  of  six  different  sap  specimens  produced brown precipitates in four of the sap samples while both heat and pH caused little coloration in the other two saps (Table 17).  Thus heating and pH modification were able to identify sap  specimens which were susceptible to browning.  Evans and Halvorson  (1962) have also shown that heating of western hemlock sap produced browning when collected from boards with brown endstain.  Heating itself produced no changes in the pH of the sap samples corresponding with results obtained when heating water extracts of redwood  (Anderson et al. , 1960) .  These authors have also shown  that heating of aqueous redwood extract, adjusted to pH 7, produced greatly intensified colorations compared to heating at pH 3 and pH 5.  As expected, incubation of the pH-altered sap samples produced browning  and  changes  specimens (Table 17). pH-altered  and  heated  in  the  TSP  content  in  susceptible  sap  Similar TSP changes were produced in both samples  of  sap-2B  and  sap-9D.  This  observation clearly suggested heat-modification of sap phenols.  175  TABLE 17 : CHANGES IN HEATED SAP AND IN pH MODIFIED SAP. pH-ALTERED1  HEATED SAMPLE #  Sap Colour  TSP /xg/mL  Sap Colour  TSP /xg/mL  2B  brown  225 (350)2  brown  200 (350)  5B  light yellow  450 (475)  yellow  350 (425)  5D  light yellow  150 (125)  yellow  50 (100)  8B  brown  775 (725)  brown  300 (500)  9A  brown  300 (300)  brown  100 (325)  9D  brown  125 (400)  brown  200 (475)  1 = refers sap adjusted to pH7 and incubated for 12 days at room temperature. 2 = numbers in brackets refer to TSP in sap before treatment.  176 Polcin and Rapson (1971), when studying the heat stability of some western hemlock extractives, have demonstrated that heat treatment produced  brown  colorations  with  flavan-3ols  (e.g.,  catechin)  whereas lignans were quite stable.  However, browning developed in the heated sap-8B and sap-9A without TSP decrease (Table 17) suggesting that sap compounds other than phenols also formed colorations upon heating.  Millett (1952) has  implicated sugars in brownstaining of kiln-dried sugar pine (P. lambertiana Doul.) and reported that sugars produced an even more intense coloration in the presence of amino acids.  In the current  study, involvement of sugars in the browning of heated sap was not shown because similar infrared spectra were recorded for brown precipitates formed in heated sap-8B and in pH adjusted sap-8B (Weigel, 1994).  Both infrared spectra indicated free phenolic,  carbonyl and ether groups.  The presence of ether groups was of  particular interest because condensed tannins built from catechin monomers, have shown this linkage (Hemingway, 1989; Goldschmidt and Hergert, producing  1960) .  Catechin  browning  upon  pH  was  recorded  alteration  decreased by 50 % during incubation. suggested process.  that  catechin  played  only  and  in  its  the  samples  concentrations  This observation strongly  a major  role  in  the  browning  177 7.3.5 Amendment of water and sap with known phenols  A yellow coloration developed in water amended with catechin and epicatechin,  whereas  hydroxymatairesinol incubation.  the  solutions  remained  with  colourless  alfa-conidendrin after  12  and  days  of  Less coloration developed in water supplemented with  a mixture of the four phenols than in the samples with catechin and epicatechin.  This demonstrated that flavan-3ols were unstable and  were auto-oxidized in water. However, brown precipitates were not formed with catechin or epicatechin, indicating that any oxidation products were still water soluble.  It also suggested that other,  unknown chromophores contribute to sap browning.  Sap-5A, which had developed no brown coloration upon inoculation with  microorganisms,  nor  following  heating,  produced  brown  colorations under alkaline conditions when amended with catechin, epicatechin and a mixture containing the four phenols (Table 18). In contrast, neither  alfa-conidendrin  nor OHMR  alone  produced  browning in amended sap-5A, substantiating the observation made with water.  TSP analysis also demonstrated that OHMR changes did  not occur in sap-5A even when incubating under neutral conditions. This confirmed Barton's (1968) observation that OHMR's were stable and probably not involved in brownstain. However, alfa-conidendrin seemed more reactive under neutral conditions, as indicated by the large  decrease  in  TSP;  the  poor  water  conidendrin may have cause this outcome.  solubility  of  alfa-  178  TABLE 18 : COLOUR AND TSP IN pH ADJUSTED SAP-5A AMENDED WITH PHENOLS AFTER 12 DAYS OF INCUBATION AT ROOM TEMPERATURE. AMENDMENT  SAP COLOUR  TSP /xg/mL  Control-5A/22°C  light yellow  98  (195)1  Control-5A/pH7/22 °C  yellow  75  (138)  Catechin  brown  283  (703)  Epicatechin  brown  253  (705)  alfa-conidendrin  yellow  110  (320)  hydroxymatairesinol  yellow  445  (525)  Mixture2  yellow  373  (753)  Catechin (50 /xg/mL)  brown  not determined  1 = numbers in brackets refer to TSP of sample kept in a freezer 2 = refers to sap-5A containing a mixture of approximately 150 /xg/mL of each individual compound.  179 Browning also occurred in the pH 7 altered sap-5A when amended with 50 /xg/mL of catechin; only yellowing developed in additional sap-5A with 50 /xg/mL of catechin at pH 5.  Interestingly, catechin was  rapidly oxidized under neutral conditions to the point that it was not detected  (HPLC) after 24 hours of incubation.  Once again  observations underscored that catechin was extremely reactive at a neutral pH and demonstrated that very small amounts of catechin can produce pronounced colour changes in sap. However, other compounds may contribute to the browning process as only a yellow coloration was  produced  in water  Anderson et al.  amended  with  catechin  and  epicatechin.  (1960) showed that a mixture of redwood water  extractives had a darker colorations than any of the individuals extracts  alone.  However,  analytical  extractive  chemistry  is  required to determine which compounds can contribute to browning in western hemlock sap.  One striking observation was the fact that naturally occurring epicatechin, which was detected in controls produced no browning  (unamended sap-5A),  under neutral conditions.  However, browning  occurred when sap-5A was amended with additional epicatechin, which resembled  the coloration observed  catechin (Table 18).  in sap-5A when amended with  It is possible that the natural epicatechin  content of about 10-15 /xg/mL was too little to support browning in sap-5A even under highly reactive (neutral) conditions.  180 7.3.5  Buffer experiment  Browning occurred in the non-buffered sap-9A upon inoculation with different fungi (Table 19). As expected, browning was accompanied by a pH shift towards neutral and a decrease in TSP. the  same  fungi were  unable  to produce  colour  In contrast,  changes  in the  buffered sap-9A and both pH and TSP remained unchanged (Table 19). Because sap-9A contained catechin, this demonstrated that catechin was much more reactive at a near neutral pH than at a slightly acidic pH.  Fungi grew well in both the buffered sap and non-buffered sap-9A. Since dark color accompanied the rise in pH, stabilization of the pH of sap-9A effectively controlled browning.  Bauch  (1986) has  reported that pH stabilization of the surface of Ilomba inhibited browning, related to a pH shift by bacteria (Starck et al. , 1984) . Oldham and Wilcox  (1981) also controlled surface brownstain in  solid-piled sugar pine lumber when keeping the pH of the wood surface low with phosphoric acid. browning  in  western  hemlock  Springer (1983) has prevented  chips  with  sodium  recorded a decrease in pH of sap from 5.4 to 2.6.  bisulfite  and  Hathway and  Seakins (1955) have also arrested browning of catechin with sodium hydrogen sulfite.  In the current  study, pH seemed critical  to promotion of sap  browning: for example, the fungus B15S produced browning at pH 6.6  181  TABLE 19: CHANGES IN BUFFERED AND NON-BUFFERED WESTERN HEMLOCK SAP (9A) INCUBATED WITH DIFFERENT FUNGI FOR 12 DAYS AT ROOM TEMPERATURE. BUFFERED1 Fungus  1 2 3 4  Colour 3  pH  TSP /xg/mL  NON-BUFFERED Colour  Control  2  yellow  5.2  300  yellow  Control  4  yellow  5.1  278  Op 3871  yellow  5.2  A2S  yellow  A6S  3  pH  TSP  5.2  300  yellow  5.1  203  303  yellow  6.3  300  5.2  325  yellow  6.3  305  yellow  5.2  300  brown  6.9  140  A13S  yellow  5.2  295  brown  6.9  135  B15S  yellow  5.2  318  brown  6.6  157  = = = =  iiq/mL  50 mL of sap was buffered with 4.8 g of MES and 0.1 g NaOH. sample kept in the freezer during incubation. coloration was light yellow. sap incubated at room temperature.  182 but not at pH 6.3.  However, 0. piceae and A2S also shifted pH to  6.3 but caused no browning.  A critical pH has been demonstrated  for browning in beech sap (Schmidt and Mehringer, 1989).  In this  study the composition of the sap and the metabolism of the fungi were also suggested to play a role in the pH changes of the nonbuffered sap.  Clearly, the fungi differed in their potential to  induce pH shifts accompanied by colorations.  183 7.4  Conclusions  Alkaline conditions alone can promote browning in western hemlock sap.  Oxygen is essential for browning of sap.  Browning  of  inoculated  sap  following  heat  treatment  was  not  produced by an enzyme resident to western hemlock sap.  Heat and a neutral pH produced browning in sap predisposed to colorations.  Catechin played a major role in browning but other sap constituents contributed to the coloration.  Stabilization  of  the  sap  pH  in  the  acidic  controlled sap browning produced by fungi.  range  effectively  184 8.0  Summary and Recommendations  In this study, conclusive evidence was presented that fungi can produce brown colorations in western hemlock during seasoning.  Brown discolourations can disfigure both amabilis-fir and western hemlock, but the research concentrated on the latter species which is economically more important.  These discolourations, clearly  different from sapstain, can occur in several types and intensities and are a serious problem in high-value markets. Because little is known  about  their  unavailable.  causes, means  for  their  control  are  still  Therefore, fundamental research was initiated to  elucidate the biology and chemistry of hemlock brownstain and to suggest control measures.  As  a  first  approach  into  the  cause  of  hemlock  brownstain  microscopic examinations of hem-fir samples were performed.  While  samples exhibited different macroscopic types of colorations, a similar  microscopic  demonstrated. evidence Because  Subsequent  that fungi  brownstain,  distribution  as  the and  brown  the  histochemical colouring  bacteria  seen  of  were  colouring  matter  studies provided  matter  contained  frequently  microscopically,  in  catechin.  clearly  demonstrated  that  a  broad  with  brownstain  experiments were performed on western hemlock sap and wood. experiments  first  associated vitro  was  These  microflora  can  185 produce brownstain, which led to the hypothesis of this study that microorganisms are involved in hemlock brownstain.  To determine the role of microorganisms in hemlock brownstain, two field studies were performed with emphasis on the brown colorations disfiguring western hemlock logs and lumber during seasoning, the most troublesome discoloration to industry.  In  the  first  field  study  dark-pigmented  fungi  were  isolated  predominatly from western hemlock logs showing brownstain and it was shown that the brownstained regions contained a lower quantity of soluble phenols than non-stained areas.  While this observation  suggested a link between brownstain and the presence of fungi in western  hemlock  logs,  additional  factors  promoting  brownstain  became evident when monitoring the western hemlock logs and sawn lumber produced from the logs over time. time promoted discoloration.  Prolonged log storage  Salt water storage caused severe  colorations in logs and lumber much more than in the logs stored on land.  However,  several  limitations  of  this  field  trial were  recognized; for example the small sample size with logs from one growth site, one age class and harvested in one season of the year. Future research in this area might address the following questions: *  Do felling season, log age and growth site influence the extractive compositions with respect to brownstain?  *  Does death of parenchyma cells influence hemlock brownstain?  186 *  Is brownstain formed in lumber even when sawn from freshly, felled logs?  *  Are metal ions involved in the dark-brown colorations observed in water-stored logs?  In a second trial, fungi were clearly associated with brownstain on the endcuts of sawn lumber.  The fungi isolated were most likely  resident in the wood prior to the sawing of lumber.  Longitudinal  movement of water-soluble phenols was shown towards the cross-end cut.  However, shortcomings of the study, for example that this  survey dealt with one sawmill only, at one time of the season and with no information on log source and log storage conditions, could be addressed in a future study  To demonstrate microbial involvement in the phenomenon of hemlock brownstain, an immunolabeling technique was applied on infected western  hemlock.  While  in situ production  of brownstain was  associated with hyphae, the antibody was unable to detect the fungus in wood, possibly due to a lack of specificity.  In future  research in this area a polyclonal antibody, which is much easier to work with, should be used instead of a monoclonal antibody providing  that  interference problems, which can arise  from an  inherent wood microflora, can be eliminated.  Fungal involvement in brownstain was then conclusively demonstrated in sap assays.  Fungi shifted the sap pH from slightly acidic to  187 near neutral, or above neutral, which ionized and oxidized phenols causing browning  of  sap.  Oxygen and a near neutral pH were  essential to produce colorations.  However, fungi did not produce  colorations  it was not possible  in buffered  sap and  to study  production of browns tain in the absence of oxygen, due to the aerobic nature of these organisms.  Furthermore, 0. piceae infection of 15 /xm wood sections, which must have contained very small amounts of brownstain precursors, and also of infection of wood blocks, confirmed that the fungus can produce brownstain in solid western hemlock.  Interestingly, the  hyphae of 0. piceae remained hyaline in western hemlock, whereas pigmentation was observed when it grew in lodgepole pine and on a nutrient medium.  Thus, physiological factors appeared to trigger  production of brownstain in western hemlock when infected with 0. piceae as well as suppress the development of sapstain.  Catechin was demonstrated to play a major role in the browning of western hemlock.  However, other yet unknown sap  probably were also involved in brownstaining. this  area  chemistry.  would  involve  thorough  constituents  Future research in  extractive  and  For example, western hemlock extractives  analytical could be  fractionated based on their ability to produce browning followed by isolation, purification and identification of individual compounds. This approach may  clarify which of  the compounds, other  catechin, are involved in hemlock brownstain.  than  However, based on  188 information gained from the current study, the following simplified mechanism  is  proposed  for  the  involvement  of  fungi  in  the  brownstain developing in western hemlock during seasoning.  In  addition to any inherent tree microflora, which could be extensive especially in older trees, western hemlock is likely colonized within hours after the felling of the trees.  Nutrients and water-  soluble wood extractives migrate to the wood surface during storage of logs and lumber which leads to an accumulation of phenols.  As  wood moisture content decreases, fungal colonization can progress axially and can alter wood extractives within the wood, for example by hydrolysing glycosides.  Oxidation, for example of flavan-3ols,  can then readily occur upon exposure to air, forming coloured condensation  and  polymerization  accompanied by an increase in pH. production  of  brownstain  is  products  especially  when  However, it is emphasized that  likely  more  complex  and  other  the  factors  involved  mechanisms can not be excluded.  Based  on  the  elucidation  of  some of  in  brownstaining the following means for control are suggested:  *  Because fungi are implicated, biocides should be reassessed but might need a supplement for example a chelating agent or a reducing agent.  *  Because  phenols  are  highly  reactive  at  higher  pH,  stabilization of the pH of the wood substrate with a strong  189 buffer or acidification of the wood substrate should be investigated.  *  Because  oxygen  is essential  for browning,  kiln-drying  of  lumber in the absence of oxygen should be investigated, for instance the use of a super-heated steam vacuum dryer.  *  Because high temperatures promote coloration, drying schedules using lower temperatures should be investigated for high-value western hemlock.  *  Because precursors to brownstain migrate to the surface, pre-steaming western hemlock before kiln-drying  should be  investigated to remove water-soluble wood extractives.  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