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Black stain in yellow cedar Chamaecyparis nootkatensis (D. Don) Spach Robinson, Robena Claire 1960

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BLACK STAIN IN YELLOW CEDAR CHAMAECYPARIS NOOTKATENSIS (D. DON) SPACH by ROBENA CLAIRE ROBINSON B.A., University of B r i t i s h Columbia, 1957 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in the Department of Biology and Botany We accept t h i s thesis as conforming to the required standard • THE UNIVERSITY OF BRITISH COLUMBIA March, i960 In presenting t h i s thesis i n p a r t i a l f u l f i l -ment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for exten-sive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publ i c a t i o n of t h i s thesis f o r f i n a n c i a l gain shall not be allowed without my written permission. Department of Biology and Botany The University of B r i t i s h Columbia, Vancouver 8, Canada. Date: March, I960 i i ABSTRACT From samples of stained yellow cedar wood, Chamaecyparis nootkatens is (D. Don) Spach, collected at three d i f f e r e n t l o c a l i t i e s on the B r i t i s h Columbia coast, i s o l a t i o n studies consistently yielded two members of the group Fungi Imperfecti. Reinoculation of these . fungi into sound wood produced black s t a i n s i m i l a r to that found in nature. In addition to these Deuteromycetes, three basidiomycetous fungi, Poria w e i r i i Murr., Poria  a s i a t i c a ( P i l a t ) Overholts and Xeromphalina campanella (Batsch. ex Fr.) Kuhner and Maire, were i s o l a t e d . Of these the Poria species are believed to be f i r s t records for the host. Studies on P e t r i plates revealed no marked antagonism between the two Deuteromycetes and P. w e i r i i . The two deuteromycetous fungi gave positive reactions on g a l l i c and tannic acid agars, demonstrating the produc-tion of the enzyme e x t r a c e l l u l a r oxidase. Two series of yellow cedar beams were respec-t i v e l y inoculated with macerated cultures of the Deuteromycetes by means of a special technique described. This technique produced exceptionally rapid and uniform fungal growth in comparison to that obtained under standard c u l t u r a l conditions. i i i Inoculated beams were subjected to weight and strength loss t e s t s , i n comparison to control beams. Weight losses of 0 . 1 7 percent and 8.1j.O percent respec-t i v e l y were noted for the two fungi. No s i g n i f i c a n t difference i n impact modulus of rupture values was detected between test and control for either fungus. Significance of experimental results i s b r i e f l y discussed and recommendations for further study suggested. i v CONTENTS Page Abstract Introduction 1 Literature Review 3 The Host 5 Presentation of Data . . . 6 The Fungi 6 Deuteromycetes . . . . . 6 Cultural Characters of ftmgus "A". 8 Growth Characters 8 Hyphal Characters . . . . . 8 Cultural Characters of Fungus "C". 11 Growth Characters 11 Hyphal Characters 11 Basidiomycetes 1 3 C o l l e c t i o n L o c a l i t i e s 13 Sechelt Peninsula . . . 13 Harrison Lake H 4 . Buckley Bay, Vancouver Island . . . . 1 5 Description of Defects 1 5 Isolation Studies 16 Methods 16 Results of I s o l a t i o n Studies 20 CONTENTS (Cont.) Page Cultural Interactions of Fungi . . . . . . 2 2 H i s t o l o g i c a l Studies 23 Naturally Stained Wood 23 Inoculated Wood 26 Strength and Weight Loss Tests 31 Method of Preparing Beams 31 Method of Preparing Culture Bottles . 3 3 Test Procedure . . . . . . 3 9 Results of Weight Tests Ill Results of Strength Tests . . . . . . I4.3 Conclusions kS Significance of Results k7 Recommendations for Further Study . . . . . . . $0 Literature Cited 51 Appendix I 53 v i LIST OF FIGURES Figure To Follow Page 1 . Fungus "A". Two months old culture on malt agar. X0.9 7 2 . Fungus "C". Two months old culture on malt agar. X0.9 7 3 . Fungus "A". Camera lucida drawings showing various developmental stages of pycnidia . . . . . . 9 l i . Fungus "A". Pycnidia 1 formation. X5"00 1 0 5. Fungus "A". Ruptured pycnidium releasing conidia. X 7 7 5 1 0 6. Fungus "C". Hyphal c h a r a c t e r i s t i c s . X290 1 2 7. Fungus "C". Multiseptate c o n i d i a l structure common i n older cultures. X 7 7 5 1 2 8 . Yellow cedar showing longitudinal streak of black stai n (center) and adjacent i n c i p i e n t decay 17 9. Yellow cedar showing discolouration of almost entire heartwood area 17 1 0 . Conspicuous black stai n i n apparently sound yellow cedar heartwood 18 1 1 . Fungus "A" and Fungus "C" growing together without antagonism i n two months old culture on malt agar. X0.9 . . . 2 l i 1 2 . Tangential section of "stained" yellow cedar showing dark mycelium. X 1 5 0 . . . . 25 1 3 . Radial section of "stained" yellow cedar showing dark mycelium passing through heartwood tracheids. X 6 2 5 . . . . 25 v i i LIST OF FIGURES (Cont.) Figure To Follow Page l k . Fungus MC". Section from inoculated beam with t y p i c a l hyphal penetra-t i o n . X200 28 15. Fungus "C". Section from inoculated beam: hypha penetrating many tracheids in a direct l i n e . Stain hyphae also obvious i n ray c e l l s . X200 28 16. Fungus "C. Section from inoculated beam: penetration of yellow cedar ray tracheids by stain hyphae. X775 . . . . 29 17. Fungus "C". Section from inoculated beam: penetration of tracheid walls by s t a i n hyphae. Note adpressoria. X900 29 18. Fungus "A". Section from inoculated beam: s t a i n hyphae within the tracheids. X250 . 30 19. Fungus "A". Section from inoculated beam: a few st a i n hyphae penetrating tracheids. X 250 30 2 0 . Yellow cedar board. Diagram showing r e l a t i v e placement of test and control beams within s t r i p . Not to scale . . . . . 32 21 . Culture b o t t l e showing glass rods i n place on medium. X 3/5 . . . . . . . i . . 35 22. Culture bottle inoculated with Fungus "A". X 3/5 35 2 3 . Method of dipping yellow cedar beams into macerated fungus suspension 37 2k. Introduction of beams into b o t t l e s . X 1/2 37 25. Test b o t t l e prepared for incubation. X 3/5 38 v i i i LIST OF FIGURES (Cont.) Figure To Follow Page 26. Control bottle prepared f o r incubation. X 3/5 38 27. Tinius-Olsen Universal Testing Machine. Yellow cedar beam i n place p r i o r to t e s t i n g . X l / 5 lj.0 ix LIST OF TABLES Table Page I Fungi iso l a t e d i n a study of black stain i n yellow cedar from three l o c a l i t i e s 21 II Weight loss of yellow cedar heartwood beams exposed to two st a i n fungi for nine weeks ii2 X ACKNOWLEDGEMENT To the Forest Products Laboratories D i v i s i o n , Forestry Branch, Department of Northern A f f a i r s and National Resources, Canada, the author wishes to express her appreciation f o r i n i t i a t i o n of the project and f o r the provision of work f a c i l i t i e s and equipment. Sincere thanks are extended to Dr. J. E. Bier of the Department of Biology and Botany, University of B r i t i s h Columbia, f o r h i s guidance, assistance and encouragement throughout the study and to Mr. R. W. Kennedy of the Faculty of Forestry, University of B r i t i s h Columbia, f o r assistance i n the planning and execution of the strength tests described. The help of a l l others who assisted i n the various phases of t h i s project i s g r a t e f u l l y acknowl-edged. 1 INTRODUCTION The possible significance of black s t a i n i n yellow cedar, Chamaecyparis nootkatensis (D. Don) Spach, was f i r s t recognized as early as 1938 when lumber concerns i n the Vancouver area brought the problem to the atten-t i o n of the Vancouver Laboratory of Forest Products Laboratories D i v i s i o n , Department of Northern A f f a i r s and National Resources. The main reason for concern on the part of commercial interests revolved around the effect of the sta i n on the strength properties of the lumber. Whether to grade stained wood as decayed or sound was a point of debate amongst lumber graders. Unlike many stains, black s t a i n i n yellow cedar i s present i n the heartwood of l i v i n g trees and i s e a s i l y recognizable at the time of cutting. Members of the Vancouver Forest Products Lab-oratory attempted to i s o l a t e the causal organism or organisms but were unable to obtain consistent r e s u l t s . Plantings of samples of stained wood on agar media were large l y unsuccessful. In May 1958 investigation of t h i s problem was reopened, r e s p o n s i b i l i t y being delegated to the writer who, from May u n t i l September 1 9 5 8 , was employed as a Student Assistant at the Vancouver Laboratory. Under 2 the aegis of the University of B r i t i s h Columbia Depart-ment of Botany, and with the continued use of f a c i l i t i e s at the Forest Products Laboratory, further work was car-ri e d out during the winter of 1958-59 with a view to summarizing the experimental results i n thesis form. The objectives of t h i s study were: (1) To determine the cause of black s t a i n i n yellow cedar; (2) To ascertain the effect of t h i s s t a i n on the strength properties of the wood. LITERATURE REVIEW 3 Information on stain-producing organisms i n wood i s not extensive and i n the main i s devoted to sapwood stains. In extensive work on stains i n Sweden, Lagerburg ct al_ (1927 : 2kk) described a blue heartwood s t a i n i n l i v i n g Norway spruce (Picea excelsa Link.) from which the fungus Hormodendrum microsporum Lagerburg and Melin was i s o l a t e d . Christensen and Kaufert (19kl) reported a blue staining fungus inhabiting the heart-wood of certain conifers i n the Lake States. This organism, though unnamed, was compared with the Swedish fungus mentioned above. A heartwood blue s t a i n of balsam f i r (Abies balsamea (L.) M i l l ) and white spruce (Picea glauca (Moench) Voss) i n Quebec was described by Crowell (19k0). In the few samples available the heart-wood was almost e n t i r e l y stained. Though the staining organism was not i d e n t i f i e d i t was stated that " I t i s probably not the same as that causing blue s t a i n of spruce i n Norway". Hubert (1921) i n a discussion of sap s t a i n fungi, noted the occurrence of Ceratostomella spp. on northern white cedar (Thu.ia occidentalis L.). I l l u s -t r a t i o n s accompanying hi s description show the t y p i c a l penetration of c e l l walls by broad, dark-coloured hypbae. The general status of knowledge on heartwood stains was summarized by Hubert (1931). He noted that blue s t a i n organisms had been found i n the heartwood of certain woods. Though tree species were not specifi e d , i t was stated that the woods involved were usually of a type showing l i t t l e difference between heartwood and sapwood and i n which the heartwood possessed a f a i r l y high mois-ture content. More recently, F r i t z ( 1 9 5 D observed that some heartwood discolourations are produced by staining fungi which are not known to cause rot i n t h e i r f i n a l stages and which originate i n the standing tree. In contrast to t h i s type of s t a i n , she mentioned sapwood stains which develop i n f e l l e d timber. With s p e c i f i c reference to yellow cedar, Perry (195>ij-) has said, "Lumber from some areas shows frequent black unsound or loose knots with black stains spreading to adjacent areas." The last mentioned constituted the sole published reference on black s t a i n i n yellow cedar. 5 THE HOST Yellow cedar (Chamaecyparis nootkatensis (D. Don) Spach) i n Canada i s confined to the west slope of the Coast Range of mountains i n B r i t i s h Columbia and to the offshore islands from Alaska southward. In the south i t i s found at elevations of 2,000 to 5,000 feet. To the north i t descends gradually u n t i l i t reaches sea lev e l at Knight I n l e t . Usually i t i s associated with amabilis f i r (Abies amabilis (Dougl.) Forb.) and western hemlock (Tsuga heterophylla (Raf.) Sarg.) occur-ring as scattered stems and patches. Only r a r e l y i s i t found i n pure stands (Canada, Department of Mines and Resources, 1951)• Yellow cedar wood has exceptionally desirable working q u a l i t i e s , being fine grained, f a i r l y hard and strong, and possessing a very low shrinkage f a c t o r . It exhibits high d u r a b i l i t y i n situations favouring decay and i s considered valuable for use In conditions of exposure, notably for window frames, exterior doors and i n boat construction (Jenkins et a l . 1951)• The increased use of t h i s tree species as sawn timber i n the coastal region of B r i t i s h Columbia i s e v i -denced by the fact that the t o t a l annual cut for that area has doubled i n the period from 1951 to 1956 (Province of B r i t i s h Columbia, 1952, 1957). PRESENTATION OF DATA 6 The Fungi Deuteromycetes In the course of i s o l a t i o n work, two fungi belonging to the class Fungi Imperfecti were consist-ently i s o l a t e d . I t was observed that these fungi grew d i r e c t l y out of plantings of stained wood. Cultures have been submitted to a s p e c i a l i s t i n the taxonomy of Fungi imperfect!, but to date neither has been i d e n t i -f i e d . For t h i s reason, they have been a r b i t r a r i l y designated as Fungus MA" (Figure l ) and Fungus "C" (Figure 2) and henceforth i n t h i s paper w i l l be so c a l l e d . The two organisms demonstrate somewhat d i f -ferent c u l t u r a l c h a r a c t e r i s t i c s , permitting them to be e a s i l y d i f f e r e n t i a t e d . Both fungi appear very black i n old cultures, while Fungus "C" i s a slate grey colour i n young cultures. Fungus "A" i s j e t black i n colour throughout, even i n young cultures. PLATE I Figure 1. Fungus "A" two months old culture on malt agar. X0.9 Figure 2. Fungus "C" two months old culture on malt agar. X0.9 Figure 1 Figure 2 8 Cultural Characters of Fungus "A" Growth characters Mat j e t black, opaque; a e r i a l mycelium raised almost to the l i m i t of growth, fi n e woolly, uniform. Submerged mycelium growing s l i g h t l y beyond surface of colony. Reverse unchanged. Colonies on malt agar plates att a i n i n g a diameter of k,.$ centimeters i n six weeks. On g a l l i c acid agar,* reaction strong; trace of growth i n one week. On tannic acid agar, reaction moderately strong with a trace of growth over the inoculum i n one week. Hyphal characters Hyphae amber-brown coloured, simple septate, frequently branched at and between septa. Some sugges-t i o n of swellings or expansions on hyphae of advancing zone with some hyphal t i p s having a s l i g h t l y clavate appearance. Hyphal width 2.k. to 3.2 microns. A e r i a l mycelium of older growth (three months old cultures) producing abundant pycnidia (Figures 3» h a n ° ! 5) con-taining conidia (3.II4. by 1.57 microns) which are c y l i n d r i c a l and hyaline. 1 G a l l i c and Tannic Acid agars prepared by adding 0.5 percent of these acids, respectively, to malt agar i n f l a s k s immediately p r i o r to pouring into P e t r i plates. PLATE II Fungus "A": camera lucida drawings showing various developmental stages of pycnidia. Figure 3 10 PLATE III Figure k. Fungus "A". Pycnidial formation. X500. Figure 5>. Fungus "A"'. Ruptured pycnidium releasing conidia. X773>. PLATE III Figure £ 11 On the basis of i t s pycnidia, t h i s fungus can be placed i n the Form family Sphaeropsidaceae, Form order Sphaeropsidales, Form class Deuteromycetes. Cultural Characters of Fungus "C" Growth characters Mat at f i r s t slate grey, opaque, becoming black i n older cultures. Advancing zone even, a e r i a l mycelium raised to the l i m i t of growth; woolly to fin e cottony, uniform. Reverse unchanged. Colonies on malt agar plates attaining a diameter of k.O cm. i n six weeks. On g a l l i c acid agar, reaction strong; no growth i n one week. On tannic acid agar, reaction strong with a trace of growth over the inoculum i n one week. Hyphal characters Hyphae amber-brown coloured, simple septate, infrequently branched (Figure 6). Hyphae 3.2 to 3.9 microns i n width. Many dark, non staining, terminal, multiseptate co n i d i a l structures (Figure 7) up to 15".7 microns i n diameter occurring singly, and occasional swollen terminal c e l l s (up to 18.8 microns i n diameter), the contents of which s t a i n i n phloxine, were observed i n older cultures. 12 PLATE IV Figure 6 . Fungus "C". Hyphal c h a r a c t e r i s t i c s . X 2 9 0 . Figure 7. Fungus "C". Multiseptate c o n i d i a l structure common i n older cultures. X775-PLATE IV Basidiomycetes Three basidiomycetous fungi were isolated in the course of this study: Foria w e i r i i Murr.. Poria  a s i a t i c a ( P i l a t ) Overholts and Xeromphalina campanella (Batsch. ex Fr.) Kuhner and Ma i r e . P. w e i r i i and P. a s i a t i c a are believed to be new records for th i s host, campanella was reported previously (Shaw, 1958 : 3 1 ) . C o l l e c t i o n L o c a l i t i e s The material was collected from three areas on the B r i t i s h Columbia coast: Sechelt: Peninsula; Harrison Lake; Buckley Bay, Vancouver Island. Sechelt Peninsula The stand where material was collected was a mixture of Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco), western hemlock, western red cedar (Thuja  p l i c a t a Donn) and yellow cedar growing in a swampy pocket on a h i l l s i d e at about 3 ,500 feet above sea l e v e l . The sample trees were from 275 to 350 years of age and from 20 to 32 inches i n diameter at breast height 2 Nobles (191+8) was the basis of the writer's i d e n t i -f i c a t i o n of these-fungi. Identity of the Poria species was confirmed by Dr. M. K..Nobles, Plant Research I n s t i -tute, Department of Agr i c u l t u r e , Canada. 111. (k..5 feet above ground l e v e l ) . Most of them exhibited "dryside" about 8 to 10 feet above the ground, possibly as a re s u l t of f i r e scars. Several types of rot were evident i n stumps; but black s t a i n was only occasionally noted, either i n association with advanced decay or i n the outer heartwood of stumps i n apparently sound wood. Stain was usually prominent i n the top of the f i r s t log or beyond, from h.0 to 60 feet above the ground. Harrison Lake The samples from t h i s area were taken from timber that had been f e l l e d three weeks previous to c o l l e c t i o n as part of a logging operation of Canadian Forest Products Limited. The s i t e was well i r r i g a t e d and was located on a very steep east-north-east facing slope at about 3,500 feet above sea l e v e l . The stand was predominantly yellow cedar, amabilis f i r and mountain hemlock (Tsuga mertensiana (Bong.) Carr.). Yellow cedar attained good growth on t h i s s i t e , some stumps being at least Ij. feet i n diameter. Butt rot was not prevalent, but there was a conspicuous yellow ring rot i n the trunks of many of these trees. Black s t a i n appeared to accompany t h i s ring r o t , but i n many cases s t a i n was observed i n wood that showed no signs of decay. 15" Buckley Bay, Vancouver Island This group of samples was brought into the laboratory by the B r i t i s h Columbia Forest Service. This s i t e i s si m i l a r to that of Sechelt Peninsula, the stand composition being mainly yellow cedar, western hemlock, western red cedar, mountain hemlock, western white pine (Pinus monticola Dougl.) and some Douglas-f i r . Sites i n t h i s area are often f l a t and located i n excess of 2,000 feet above sea l e v e l . Samples from seven trees were submitted, one of these being a plank that had been sawn from a butt log. This sample contained a brown cubical rot i n addition to black heartwood s t a i n . The log from which t h i s plank had been recently sawn was from a tree f e l l e d approximately si x months previously. Description of Defects Black s t a i n appeared very frequently i n the heartwood of the trees examined. Several forms were observed, the most common one being prominent lo n g i -tudinal black streaks made up of short black l i n e s , running i n a r a d i a l d i r e c t i o n i n the wood (Figure 8). The location of the s t a i n r e l a t i v e to the center of the tree was variable. Occasionally most of the heartwood 16 was stained a dull grey-black (Figure 9). Knots were frequently blackened, but no consistent relation-ship between knots and stained heartwood was apparent. In some instances, the stain was accompanied by obviously decayed wood; but in others i t occurred in apparently sound wood (Figure 10). Several types of rot were noted in the sample trees. These included the following. 1. Yellow laminated heartrot: associated with P. weirii 2. Brown cubical butt rot: associated with P. asiatica 3. Firm red-brown heartwood discolouration: associated with X. campanella Isolation Studies Methods Fresh wood samples were used for the isola-tion work. Inoculations on three types of media were made from a l l of the samples In an effort to provide for a range of nutritional requirements. The media used were; standard Bacto-difco malt agar, standard Bacto-difco potato dextrose agar and acidified malt agar. 17 PLATE V Figure 8. Yellow cedar showing longitudinal streak of black stain (center) and adjacent incipient decay. Figure 9. Yellow cedar showing discolouration of almost entire heartwood area. PLATE V F i g u r e 9 1 8 PLATE VI Figure 10. Conspicuous black stain in apparently sound yellow cedar heartwood. PLATE VI Figure 10 19 Because d i f f i c u l t y had been encountered in previous isolation work with yellow cedar, i t was decided to explore the usefulness of malt agar acidified with lactic acid to pH of li .5» in addition to the two previously noted media. A l l of these media were harden-ed at 2 percent agar concentration. The malt and potato dextrose agars were poured into culture tubes and slanted, and the acidified malt agar was poured into Petri dishes. Inoculations were made in the conventional manner by aseptically placing small pieces of wood on the media. Plantings were taken from the different stages of stain and decay on the samples in an attempt to explore a l l the pos s i b i l i t i e s of fungal infection. The plantings were selected after splitting the bolts of wood to expose areas of stain and/or decay that would be free from surface contamination. Several plantings were made from each situation so exposed. Two hundred sixty four areas of stained and/or decayed wood were explored in this manner on the 2k, samples, giving a total of 600 culture tubes and l£0 Petri plates (three inocula per plate) of attempted cultures. Results of Is o l a t i o n Studies The r e s u l t s of these i s o l a t i o n studies are summarized i n Table I. The three types of media used appeared equally suitable for i s o l a t i o n work, fungal growth being obtained i n roughly the same proportion and v a r i e t y on each. Of the stained and/or decayed areas from which cultures were obtained, k2 percent yielded Fungus "C", 28 percent Fungus "A", 5 percent P. w e i r i i . about 1.5 percent P. a s i a t i c a and about 1.5 percent X. campanella. About 3 percent of the stained and/or decayed areas yielded a mixture of Fungus "A" and another imperfect fungus. The remainder of growth was made up of a heterogeneous group of unidentified Fungi Imperfecti. 21 TABLE I FUNGI ISOLATED IN IN YELLOW CEDAR A STUDY OF FROM THREE BLACK STAIN LOCALITIES Sechelt Peninsula Harrison Lake Buckley Bav. V.I. Number of trees sampled-*?-. . 7 8 8 Situations from which cultures were attempted . . . 63 lk7 5k Situations producing . 56 10k k2 Fungus "A" 31 29 11 25 Funaus "C" . 35 1*9 19 k2 Funqus "A". Mixed Culture — --Poria w e i r i i — Xeromphalina campanella MM » 3 7 Poria a s l a t i c a 3 7 Unidentified Fungi Imperfecti 5 26 25 9 19 •JfrOne bolt per tree. **Percentage of the t o t a l "situation-fungus-growth types." Because of the i s o l a t i o n of more than one fungus from some situations, the t o t a l for Sechelt Peninsula, for example, i s 60 and not 56: i . e . , i f a s i t u a t i o n yielded Fungus "A" and P. w e i r i i . It would be represented i n both instances. Cultural Interactions of Fungi 22 Exploration of the poss i b i l i t i e s of inhibi-tion or stimulation between cultures of Fungus "AM, Fungus "C", Poria weirii and an undesignated imperfect fungus (this fungus was occasionally isolated during culture work) was carried out. Small squares of vigorously growing cultures of these fungi were inocu-lated on Petri dishes of malt agar. The inocula were placed at the periphery of the culture medium as far apart as the combinations permitted. Inoculations were made of the four fungi in a l l possible combinations and singly. The series was replicated four times, making a total of 60 plates. The cultures were grown in the dark at room temperature for six weeks, measurements and descriptions being recorded at weekly intervals. At the end of this period, measurements of the fungi plated singly were compared with those plated in com-bination. No marked synergistic or antagonistic effects were observed, but the growth of P. weirii appeared to be slightly inhibited by both Fungus "C" and the undesignated imperfect fungus. Fungus "A" and Fungus MC" grew together with no effect of inhibition or 23 stimulation. These two fungi were subsequently replated (with inocula i n closer proximity than i n this series) to i l l u s t r a t e t h i s fact (Figure 1 1 ) . H i s t o l o g i c a l Studies Naturally Stained Wood Blocks of wood exhibiting t y p i c a l black s t a i n were prepared f o r microtome sectioning. Unstained sections were mounted i n g l y c e r i n and examined under a microscope. Numerous amber-brown, broad, septate hyphae were observed (Figure 1 2 ) . O i l droplets were frequent i n the hyphal contents, and many of the hyphal walls were somewhat rough i n appearance. In some cases structures resembling adpressoria (Lagerburg et a l . 1928 : 172) were present. These hyphae t y p i c a l l y penetrated the tracheid walls and grew i n a transverse d i r e c t i o n (Figure 13)• Marked c o n s t r i c t i o n was noted when a hypha penetrated a tracheid wall with immediate widening on the other side of the wall. A single hypha could be traced passing through many tracheids i n a straight l i n e . Some sim i l a r hyphae were also found growing i n a longitudinal d i r e c t i o n within individual tracheids. The mycelium was occasionally found within the ray c e l l s PLATE VII Fungus "A" and Fungus MC M growing together without antagonism in two months old culture on malt agar. X0.9 PLATE VII Figure 1 1 PLATE VIII Figure 12. Tangential section of "stained" yellow cedar showing dark mycelium. XI50 Figure 1 3 . Radial section of "stained" yellow cedar showing dark mycelium passing through heartwood tracheids. X625> PLATE VIII Figure 1 3 26 but did not generally appear to follow the rays, i n contrast to most sapwood-staining organisms. Narrow hyaline hyphae were also occasionally observed. These may have been of t y p i c a l decay mycelium or the e a r l i e r stages of s t a i n hyphae. Inoculated Wood Two series of yellow cedar beams were inocula-ted (see page 33) with Fungus "A" and Fungus "C" respectively. These beams were incubated for nine weeks at room temperature (about 22° C) a f t e r which they were removed from the culture bottles and placed i n an "Airegulator" 1^ to bring to a uniform moisture content before t e s t i n g for possible losses of strength and weight. Subsequent to these t e s t s , the beams were oven dried; and microtome sections were made from some of the beams of both series to observe the habit of the fungi when inoculated into wood under laboratory conditions. In the case of the beams inoculated with Fungus ,,C,,, the s t a i n hyphae could be found penetrating to the center of the beams i n the c h a r a c t e r i s t i c manner (Figure l k ) . As i n n a t u r a l l y stained wood, a single 3 Custom made machine fo r maintaining constant condi-tions of temperature and humidity, located at Forest Products Laboratory, Vancouver. hypha could be seen passing through many tracheids i n a direct l i n e (Figure 1 5 ) . Macroscopically, the wood had a greyish color; and microscopically, hyphae were found i n great abundance i n the tracheids (Figures 1 6 , 1 7 ) . Examination of beams from the series inoculated with Fungus "A" did not show as many hyphae i n the tracheids as i n the "C" series (Figure 1 8 ) . Stain hyphae were found penetrating tracheids i n the t y p i c a l manner but usually i n the outer part of the beams and ra r e l y i n the center (Figure 1 9 ) . 2 8 PLATE IX Figure Ik. Fungus "C". Section from inoculated beam with t y p i c a l hyphal penetration. X200 Figure 1 5 « Fungus "C". Section from inoculated beam: hypha penetrating many tracheids i n a di r e c t l i n e . Stain hyphae also obvious i n ray c e l l s . X200 PLATE IX Figure 1 5 PLATE X Figure 16. Fungus "C". Section from inoculated beam: penetration of yellow cedar ray tracheids by sta i n hyphae. X775 Figure 17. Fungus "C". Section from inoculated beam: penetration of tracheid walls by sta i n hyphae. Note adpressoria. X900 PLATE X Figure 17 PLATE XI Figure 18. Fungus "A". Section from inoculated beam: stain hyphae within the tracheids. X250 Figure 19. Fungus "A1". Section fron inoculated beam: a few stain hyphae penetrating tracheids. X25"0 PLATE XI Figure 19 31 Strength and Weight Loss Tests Method of Preparing Beams A flat-sawn, cle a r , yellow cedar board approximately 8 inches by 2,/k, inch by 5 feet was obtained from K i t s i l a n o Lumber Company, Vancouver. This board was s p l i t into two pieces, each approxi-mately 0.30 inches i n thickness. One side of the board was used f o r each of the two series of inocu-lat i o n s and matching controls. For each series the halfboard was ripped into approximately 0.25-inch wide s t r i p s which were subsequently planed to a nearly uniform thickness of 0.20 inches. The s t r i p s were cut into four-inch beams and these were immediately numbered. Alternate beams within a single s t r i p were chosen for " t e s t " and "control" pieces (Figure 20) so that they would be end matched and a comparison could be made between beams from the same annual growth rings, thus avoiding some of the natural v a r i a t i o n of wood properties within a single tree. Light sanding of the ends of the samples was done with a fi n e sandpaper to remove any loose s p l i n t e r s . Preparatory to weighing, the beams were placed i n the "Airegulator" to bring them to a uniform moisture content. After two days and d a i l y thereafter, dummy beams were taken out, 3 2 P L A T E X I I Figure 2 0 . Yellow cedar board. Diagram showing relative placement of test and control beams within strip. Not to scale. PLATE X I I i < u OQ U. o z o Iii i nr STRIP NUMBER F i g u r e 2 0 weighed, oven dried and reweighed to check mois-ture content. After one week, during which time the moisture content of the beams was found to have reached equilibrium, the beams were removed i n small groups to polyethylene bags and quickly weighed. The test pieces were weighed i n d i v i d u a l l y , and the controls were weighed i n the two groups i n which they were to be used. Before placing i n the inoculation chambers, a l l of the beams were placed i n an autoclave under flowing steam (without pressure) for 2 5 minutes for the purpose of eliminating surface contamination. Method of Preparing Culture Bottles Pyrex "milk d i l u t i o n b o t tles" of outside dimensions 5 « 5 by 1 . 7 5 by 1 . 7 5 inches were used as inoculation chambers for the beams. Thirty cubic centimeters of malt agar were placed i n each bottle which was then closed with a cotton plug to allow aeration. The bottles were s t e r i l i z e d i n an auto-clave for 2 0 minutes at 1 5 pounds pressure per square inch. They were then placed i n a horizontal p o s i t i o n to provide the maximum culture medium sur-face upon cooling. Because the two sta i n fungi have very slow growth rates, a special method of inoculation of the culture medium was devised that produced good growth over the entire agar surface i n a week. Two-month-old P e t r i plate cultures of Fungus "A" and Fungus "C", growing on malt agar, respectively, were macerated i n a s t e r i l e Waring blendor with s t e r i l e d i s t i l l e d water. The mycelial colonies were scraped from the agar surface with a s t e r i l e s c a lpel, and care was taken to include as l i t t l e of the substrate as possible. In each case, the blendor cylinder was approximately one-third to one-half f i l l e d ; and blending was done for 1 .5 minutes. Two bent glass rods were placed a s e p t i c a l l y on the agar i n each bottle (Figure 2 1 ) . The rods were heated s l i g h t l y so that they would adhere to the surface of the medium. These were used to support the beams i n contact with the mycelial mat, but not touching the agar surface. Using a s t e r i l e pipette, 5 cubic centimeters of the appropriate macerated fungus suspension was placed i n each upright b o t t l e . The bottles were then tipped so that the suspension spread evenly over the agar surface. Vigorous uniform growth was obtained i n a l l the bottles i n about one week (Figure 2 2 ) , i l l u s t r a t i n g the usefulness of t h i s method for obtaining uniform growth of slow-growing fungi on an agar surface. Care In observing aseptic technique throughout the experiment completely obviated the PLATE XIII Figure 21. Culture bottle showing glass rods In place on medium. X 3/5 Figure 22. Culture bottle inoculated with Fungus "A". X 3/5 P L A T E X I I I F i g u r e 22 3 6 common problem of air-borne contaminants. Before being introduced into i t s culture b o t t l e , each beam was dipped i n a macerated suspension of the appropriate fungus (Figure 23). This was done i n an attempt to accelerate i n f e c t i o n of the beams on a l l sides. Three beams were placed i n each bottle (Figures 2k., 25). The control beams for each series were treated i n exactly the same manner as the test beams, except that the agar i n the control bottles was not subjected to any fungus inoculation (Figure 26). Both series (Fungus "A" and Fungus "C") and t h e i r controls were incubated at room temperature i n humidity chambers for nine weeks. PLATE XIV Figure 2 3 . Method of dipping yellow cedar beams into macerated fungus suspension. Figure 2 k . Introduction of beams into bottles. Xi PLATE XIV Figure 23 Figure 2k PLATE XV Figure 25* Test bottle prepared for incuba-tion. X 3/5 Figure 26. Control bottle prepared for incubation. X 3/5 PLATE XV Figure 25 Figure 26 39 Test Procedure At the end of the nine-week period, a l l of the beams were removed from the bottles; and the sur-face mycelium was gently removed from the test beams with a nylon brush. Both test series and controls were then placed i n the "Airegulator" to bring them to a uniform moisture content (approximately 13 percent). When t h i s was established, the beams were removed to polyethylene bags i n preparation for im-mediate t e s t i n g . The width and the depth of each beam was i n d i v i d u a l l y measured by a micrometer to the nearest 0.001 inch. They were then subjected to a semi-impact bending test using a Tinius-Olsen Universal Testing Machine (Figure 27). This test was chosen because i t i s considered to be one of the most sensi-t i v e of accepted methods to changes brought about by decay-causing organisms. The machine had a capacity of 200 pounds and a load s e n s i t i v i t y of 0.2 pounds. Samples were center-loaded over a three-inch span and the load was applied at a head speed of 9.5 inches per minute. The time required f o r f a i l u r e of the beams was approximately three seconds. At the completion of the tes t , a l l beams were oven dried and weighed for the purpose of computing any weight losses. ko PLATE XVI Figure 27 . Tinius-Olsen Universal Testing Machine. Yellow cedar beam i n place p r i o r to t e s t i n g . X 1/5 PLATE XVI Figure 27 Results of Weight Tests The weight test data are summarized i n Table I I . Each of the four groups (A t e s t , A control, C t e s t , C control) of beams was weighed, and the resultant value was compared with the computed o r i g i -nal oven-dried weight of the groups. Weights of both sets of control beams remained r e l a t i v e l y constant while the test beams suffered weight losses of 0 . 1 7 percent i n the case of Fungus "A" and 8.U.0 percent i n the case of Fungus "C". k2 TABLE II WEIGHT LOSS OF YELLOW CEDAR HEARTWOOD BEAMS EXPOSED TO TWO STAIN FUNGI FOR NINE WEEKS Condition- Computed Oven-dried Weight Weight ed weight oven-dried weight sub-- loss loss as of beams weight of sequent to percent prior to beams prior testing age of inocula- to inocu- column 3 Series tion lation* grams grams grams grams percent A (test) 121.190? 106.6819 106.k992 0.1827 0.17 A (control) 131.k$68 115.6579 115.6570 0.0009 0.0007 C (test) 128.3239 113.2602 103.7k70 9.5132 8.I4.O C (control) 123.8695 109.3287 109.3170 0.0117 0.01 #Oven-dried weight prior to inoculation was computed using the following formula: O.d. wt. = wt. (as group) 1 + moisture content Moisture content was computed using the following formula: ra.c. = conditioned wt. - oven-dried wt. oven-dried weight (Brown et a l , 19k9: 60). k3 Results of Strength Tests After conducting strength tests on the beams, the modulus of rupture (R) was calculated for each beam using the formula: (Brown ct § _ 1 , 1 9 5 2 - Wakefield, 1 9 5 7 ) R = 1 . 5 x p x 1 b x h 2 where 1 = length of span p = maximum load (lbs) required for f a i l u r e of the beam b = width of beam h = depth of beam The experimental design was that of Random-ized Complete Block (Cochran and Cox, 1 9 5 7 ) . Test values of R were compared with control values of R i n an analysis of variance. The s t r i p s from which test and control beams were cut represented r e p l i c a t e s , 1 1 s t r i p s i n the case of "A" and 1 2 s t r i p s i n the case of "C". "Test" and "'control*" constituted two tr e a t -ments, of 5 5 and 60 beams each for "A" and "C" respectively. Calculated F values were compared with tabled F values. No s i g n i f i c a n t differences (at the 1 and 5 percent levels of confidence) were found between modulus of rupture values of test and control beams in either series. A comparison was also made between the strips (blocks or replicates). A significant difference at the 5 percent level was found between the strips used in series "Cw. No difference was noted for series "A". 1 £ CONCLUSIONS Two members of the group Fungi Imperfecti were consistently isolated from stained wood collected at three different l o c a l i t i e s . Three basidiomycetous fungi were also isolated during the course of the study. These were Poria  weiri i. Poria asiatica and Xeromphalina campanella of which the two f i r s t named are believed to be new records for the host. The results of a series of plate cultures indicated no marked antagonism between the two Deuteromycetes and Poria weirii« Similarly, no synergistic action was observed between any of these fungi. Positive reactions of the two Deuteromycetes on g a l l i c and tannic acid agars indicate the produc-tion of extracellular oxidase. This enzyme is generally considered to be a product of white-rotting fungi and to some extent i s used as a taxonomic tool for separating the latter group from brown-rotting fungi (Nobles, 1 9 5 8 ) . Two series of yellow cedar beams were respectively inoculated with macerated cultures of the two Deuteromycetes employing a special technique devised for this purpose. This technique was successful i n obtaining rapid and uniform growth of those fungi which had demonstrated a slow rate of growth under standard c u l t u r a l conditions. As a result of the inoculations, stains s i m i l a r to those observed i n nature were produced i n the beams. Following inoculation, these beams were subjected to weight and strength loss t e s t s , i n comparison to uninoculated controls. Results of these tests showed weight losses of 0.17 percent and 8.k.0 percent respectively for series "A" and series "CM while analysis of variance detected no si g n i f i c a n t differences between modulus of rupture values for test and control samples of either series. SIGNIFICANCE OF RESULTS Two Fungi Imperfect! were consistently iso l a t e d from stained wood. These fungi produced t y p i c a l s t a i n i n yellow cedar beams when inoculated under experimental conditions. Though i t cannot be said that these fungi are the sole cause of black st a i n i n yellow cedar, i t would appear that they both may be of major importance. When grown together on a r t i f i c i a l media, these fungi exhibit no antag-o n i s t i c nor stimulatory e f f e c t s . They can grow together i n nature, both having been isolated from the same piece of stained wood i n several instances. That these fungi inhabit the heartwood of l i v i n g trees and are capable of producing extra-c e l l u l a r oxidase (under experimental conditions) indicate marked differences from the fungi generally associated with sapwood stains and ce r t a i n p a r a l l e l s with wood-rotting fungi. Wood-rotting fungi are well represented as heartwood inhabiters whereas the fungi causing "blue s t a i n " are usually r e s t r i c t e d to sapwood. It i s not known just how general the produc-t i o n of e x t r a c e l l u l a r oxidase by wood-inhabiting fungi i s , but t h i s c h a r a c t e r i s t i c Is considered to be a feature of white-rotting fungi and a means of separating t h i s group from the brown-rotting fungi. Penetration of tracheid walls of yellow cedar heartwood by the hyphae of black s t a i n fungi i s s i m i l a r i n appearance to the habit of decay fungi. Under the conditions of t h i s experiment, weight losses of 0.17 percent and 8.L0 percent respectively were produced as a r e s u l t of inoculation of test beams with s t a i n fungi. Modulus of rupture values i n impact were not s i g n i f i c a n t l y d i f f e r e n t in comparison with values for controls. Weight loss i s generally considered to be a less sensitive test of decay p r o c l i v i t y of fungi than various strength t e s t s including modulus of rupture (Kennedy, 1958). This appears to be p a r t i c u l a r l y so i n the case of brown-rot fungi, white-rot fungi having been found, i n some cases, to produce s i g n i f i c a n t weight losses accompanied by a r e l a t i v e l y low effect on modulus of rupture values (Cartwright, Campbell and Armstrong, 1936). Other than the preceding comparison, no attempt i s made here to interpret the effect of t h i s s t a i n i n terms of decay. Neither can i t be i n t e r -preted i n terms of sapstain though tests have s i m i l a r l y shown that the strength of sapwood i n some species i s not appreciably lessened by staining (Cartwright and Findlay, 1958). More work should be carried out to add to the evidence provided by t h i s experiment that black s t a i n does not reduce the strength of yellow cedar heartwood. 50 RECOMMENDATIONS FOR FURTHER STUDY Certain p o s s i b i l i t i e s for further study of t h i s problem become apparent: 1. Determination of mechanical properties of wood subjected to similar t e s t s over a longer period of time. 2. Determination of the effect of stain fungi on the chemical wood components, notably l i g n i n . 3. Comparison of decay resistance i n stained and unstained wood. k. Study of enzyme production of these fungi. 5. F i e l d study of mode of i n f e c t i o n of the fungi causing black stai n of yellow cedar. 6. I d e n t i f i c a t i o n and/or description as new species of the fungi herein designated as Fungus "A" and Fungus "C~". 51 LITERATURE CITED Anonymous. Anonymous. Canada, Department of Mines and Resources. 1951* Native Trees of Canada. Fourth Edition. Ottawa. Province of British Columbia. 1952. Report of the Forest Service for the year ending December 31st, 1951• Victoria. . Province of British Columbia. 1957' Report of the Forest Service for the year ending December 31st, 1956. Victoria. Brown, H. P., A. J. Panshin and C. C. Forsaith. 19k9. Textbook of Wood Technology. Vol. I. McGraw-Hill Book Company, Inc. . 1952. Textbook of Wood Technology. Vol. II. McGraw-Hill Book Company, Inc. Cartwright, K. St. G., W. G. Campbell and F. H. Armstrong. 1936. "The effect of progres-sive decay by Polyporus hispidus Fr. on the strength of English ash, (Fraxinus  excelsa L.)" Ser. B., Proc. Roy. Soc. London 120 : 76-95. Cartwright, K. St. G. and W. P. K. Findlay. 1958. Decay of Timber and Its Prevention. Second Edition. H.M.S.O. London. Christensen, C. N. and F. H. Kaufert. 19kl. "A blue-staining fungus inhabiting the heartwood of certain species of conifers." Phytopathology 31, 8 : 735-736. Cochran, W. G. and Gertrude mental Designs. Second Edition. 4. Cox. 1957. Experi-John Wiley and Sons, Inc. Crowell, Ivan H. 191+0. "Heart blue-stain of white spruce and balsam f i r . " Pulp and Paper  Magazine of Canada. XLI, 7 : k51-k52. Hubert, E. E. 1921. "Notes on sap stain fungi." Phytopathology. Vol. II : 21k-22k. . 1931. Outline of Forest Pathology. John Wiley and Sons, Inc. 52 Jenkins, J . H., Clara F r i t z et a l , 195l« Canadian woods their properties and uses. Canada, Depart-ment of Resources and Development, Forestry-Branch, Forest Products Laboratories, Ottawa. Kennedy, R. W. 1958. "Strength retention in wood decayed to small weight losses." Forest  Products Journal, V o l . V I I I , No. 10 : 308-31k. Lagerburg, T., G. Lundberg and E. Mel i n . 1927. " B i o l o g i c a l and p r a c t i c a l researches into blueing in pine and spruce." Svenska  Skogsvardsforeningens T i d s k r i f t , II : 114 .5-272. : . Nobles, Mildred K. -19k8. Studies in Forest Pathology: VI. " I d e n t i f i c a t i o n of cultures of wood-ro t t i n g fungi." Canadian Journal of  Research ' c . 26.: 2 o l - k 3 1 . . . 1958. "Cultural characters as a guide to the taxonomy and phylogeny of the Polyporaceae." Canadian Journal of  Botany, 3 6 : 883-996. Perry, R. S. 195k. Yellow cedar, i t s c h a r a c t e r i s t i c s , properties and uses. Canada, Department of Northern A f f a i r s and National Resources, Forestry Branch, B u l l e t i n no. I l k * Shaw, C. G. 1958. Host fungus index for the P a c i f i c Northwest, 1, hosts. Stations C i r c u l a r no. 335 . Washington A g r i c u l t u r a l Experi-ment Stations. State College of Washington, Wakefield, W. E. 1957* Determination of the strength properties and physical c h a r a c t e r i s t i c s of Canadian woods. Forest Products Labora-tor i e s of Canada, Forestry Branch, Department of Northern A f f a i r s and National Resources. B u l l e t i n no. 119. 53 APPENDIX I - SUMMARY TABLE OF ANALYSES OF VARIANCE FUNGUS "A" Source of Variati o n Sum of d.f. Squares Mean Calc. Tabled F at Square F p = 0.05 Treatments Blocks T x B Error 1 2,109,k90 10 12,975,679 10 12,9k9,96l 88 7k.176.219 2,109,k90 2.50 1,297,568 1.5k 1,295,996 1.5k 8k2.912 3.95 1.93 1.93 Total 109 102.211.3k9 Mean Standard Deviation Control Test 11,155 ll.ii.32 ± 976 + 990 FUNGUS "C" Source of Var i a t i o n Sum of d.f. Squares • Mean Calc. ' Square F rabled F at p = 0.05 Treatments Blocks T x B Error I k,600 k,600.000 0.00k3 3-9k 11 23,7k6,808 2,l58,982.5k5 2.0052* 1.89 11 l5,6kk,322 l,k22,211.090 1.3209 I.89 96 103.362.080 1.076.688.330 Total 119 lk2.759.810 Mean Standard Deviation Control Test 12,050 12,038 + 1095 + 1099 -«-Significant at the 5 percent lev e l of confidence. 1 

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