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An investigation of the mycorrhiza of Douglas-fir seedlings. Pentland, Gertrude Draper 1959

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AN INVESTIGATION OF THE MYCORRHIZA OF DOUGLAS-FIR SEEDLINGS by GERTRUDE D. PENTLAND B.A., U n i v e r s i t y of B r i t i s h Columbia, 1956 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of B i o l o g y and Botany We accept t h i s t h e s i s as conforming t o the re^tSred. st«xndai>>d/ THE UNIVERSITY OF BRITISH COLUMBIA January, 1959 i i ABSTRACT A study was made to determine i f there i s a growth response i n D o u g l a s - f i r s e e d l i n g s to the presence of m y c o r r h i z a l a s s o c i a t i o n s . Nursery, p o t t e d and pure c u l t u r e experiments were c a r r i e d out to induce the f o r m a t i o n of mycorrhiza on Douglas-f i r s e e d l i n g s . S e e d l i n g s from n a t u r a l areas were examined to give an i n d i c a t i o n of the types of mycorrhiza which occur and the age at which they are formed. V a r i a t i o n i n the number and type of mycorrhiza as determined by s i t e f a c t o r s was s t u d i e d . Well-developed mycorrhiza were found i n n a t u r a l s e e d l i n g s as young as three months o l d . Most of the s e e d l i n g s examined showed well-developed mycorrhiza by the end of the f i r s t growing season. Only t y p i c a l e c t o t r o p h i c mycorrhiza were found i n D o u g l a s - f i r s e e d l i n g s . They occurred i n a range of s i t e s , but the occurrence could not be c o r r e l a t e d w i t h v a r i a t i o n i n s i t e f a c t o r s . In the nursery and p o t t e d experiments there were no s i g n i f i c a n t d i f f e r e n c e s between the treatments as i n d i c a t e d by the h e i g h t , r o o t volume or number of mycorrhiza of the s e e d l i n g s . A l l attempts to induce the f o r m a t i o n of mycorrhiza on Douglas-f i r s e e d l i n g s i n pure c u l t u r e were u n s u c c e s s f u l . I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Columbia^ Vancouver 3, Canada. Department o f i i i CONTENTS Page Abstract Introduction 1 Literature Review 2 Mycorrhiza in General 2 Mycorrhiza of Douglas-Fir 4 Experimental Studies 8 Nursery and Pot Experiments 8 Introduction 8 Methods 9 Design of Experiment . . 9 Preparation of Treatments . . . . . . 10 Growth Measurements 11 Pot Experiment 12 Results of Nursery and Pot Experiments . . 14 Main Effects 14 Secondary Effects of Treatments . . . 16 Pure Culture Techniques to Induce Mycorrhiza Formation 18 Introduction 18 Methods 18 Discussion 22 i v Contents (Cont.) Page D e s c r i p t i v e S t u d i e s 24 Morphology of S e e d l i n g Mycorrhiza 24 Types of Branching 24 Mantle Formation 25 Renewed Growth 27 Occurrence of My c o r r h i z a on D o u g l a s - F i r S e e d l i n g s Under N a t u r a l C o n d i t i o n s . . . . 27 S e e d l i n g s i n the Nanaimo R i v e r V a l l e y . . . 27 I n t r o d u c t i o n 27 Methods of Examination . . 28 R e s u l t s 29 a) M y c o r r h i z a f o r m a t i o n 29 b) Height growth 29 c) Root volume 30 d) E f f e c t s of s i t e 30 e) E f f e c t s of burning 31 f ) E f f e c t s of l i g h t 33 S e e d l i n g s i n Other C o a s t a l Areas 33 U n i v e r s i t y F o r e s t Area 34 Powell R i v e r Area 34 G u l f I s l a n d s Area 35 C o n c l u s i o n s , 36 Summary 38 Appendix I - C u l t u r e Media 40 ' Appendix I I - Summary Ta b l e s of Analyses of Var i a n c e 41 L i t e r a t u r e C i t e d 43 V LIST OF ILLUSTRATIONS Ta b l e To f o l l o w page 1. Average h e i g h t (cm.) of D o u g l a s - f i r s e e d l i n g s from n u r s e r y p l o t s g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments 12 2. Average ro o t volume, (cc.) of D o u g l a s - f i r s e e d l i n g s from n u r s e r y p l o t s g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments 12 3. Average number of mycorr h i z a per s e e d l i n g o c c u r r i n g i n n u r s e r y p l o t s g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments 12 k. Average h e i g h t (cm.) of D o u g l a s - f i r s e e d l i n g s grown i n pots g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments . . . . . . 13 $. Average r o o t volume (cc.) of Douglas-f i r s e e d l i n g s grown i n pots g i v e n d i f f e r e n t humus arid i n o c u l a t i o n treatments 13 6. Germination of the seed and s u r v i v a l of the s e e d l i n g s over winter i n the n u r s e r y experiment 17 7. Average measurements of s e e d l i n g s from seed-spots i n the Nanaimo R i v e r v a l l e y and c o e f f i c i e n t s o f the c o r r e l a t i o n s of these measurements • 30 8. Average number of mycorr h i z a per s e e d l i n g i n the burned and unburned p l o t s ' w i t h i n each s i t e i n the Nanaimo R i v e r v a l l e y . . . 31 LIST OF FIGURES F i g u r e To f o l l o w Page 1. S e e d l i n g s growing i n narrow-mouth, aluminum f o i l covered b o t t l e s w i t h v e r m i c u l i t e and a n u t r i e n t s o l u t i o n as the r o o t i n g medium. „ . . . 1+7 2. Mutinus sp. ( r a v e n e l l i i ?) a s s o c i a t e d with two-year-old D o u g l a s - f i r s e e d l i n g s 1+7 3. Simple, unbranched myco r r h i z a o c c u r r i n g mono-p o d i a l l y on a l a t e r a l r o o t l e t . The mycor-r h i z a have the white f u z z y type of mantle. . . . 1+8 1+. P i n n a t e l y branched m y c o r r h i z a with the opaque amber type of mantle . 1+8 5. I r r e g u l a r l y branched m y c o r r h i z a with the opaque amber type of mantle 1+9 6. a T r a n s v e r s e s e c t i o n of a m y c o r r h i z a showing the t h i c k , white f u z z y type of mantle . . . . . -$0 6. b T r a n s v e r s e s e c t i o n of a m y c o r r h i z a showing the c h a r a c t e r of the white f u z z y type of mantle and I n t e r c e l l u l a r p e n e t r a t i o n of hyphae i n t o the c o r t e x 50 7. T r a n s v e r s e s e c t i o n o f a m y c o r r h i z a showing the t h i n , opaque amber type of mantle. I n t e r -c e l l u l a r p e n e t r a t i o n Is l i m i t e d t o the o u t e r l a y e r s of the c o r t e x . 5>1 8. M y c o r r h i z a l root t i p with the b l a c k r a d i a t i n g type of mantle. Note the hyphae extending out from the mantle. 5l 9. T r a n s v e r s e s e c t i o n o f a m y c o r r h i z a showing the heavy, compact c h a r a c t e r of the b l a c k r a d i a t i n g type of mantle. I n t e r c e l l u l a r pene-t r a t i o n Is l i m i t e d t o the outer l a y e r s of the c o r t e x 52 10. Root t i p with m y c o r r h i z a l short r o o t s showing renewed non-mycorrhizal growth. The area of renewed growth has root h a i r s , i s hot swollen, and has no fungus mantle $2. 11. Short root showing renewed m y c o r r h i z a l growth. The c o n t r i c t i o n marks the p o i n t where the o r i g i n a l growth ceased 53 v i i LIST OF FIGURES (Continued) F i g u r e Page 12. Diagram i l l u s t r a t i n g the arrangement o f seed-spots w i t h i n s i t e types or unburned and burned areas i n the Nanaimo R i v e r v a l l e y t o f o l l o w page 28 13. Root system of a one-year-old D o u g l a s - f i r s e e d l i n g from the Nanaimo R i v e r v a l l e y showing well-developed m y c o r r h i z a . . . . . . . . 53 11+. Well-developed m y c o r r h i z a o c c u r r i n g on the root system of a D o u g l a s - f i r s e e d l i n g about t h r e e months o l d 51+ 15. M y c o r r h i z a with the dark f u z z y type of mantle showing c r o s s - l i k e b ranching 51+ ACKNOWLEDGEMENTS G r a t e f u l acknowledgement i s giv e n to, the N a t i o n a l Research C o u n c i l , Ottawa, f o r p r o v i d i n g a Bursary i n 1956-57,. and a St u d e n t s h i p i n 1957-58, i n support of t h i s I n v e s t i g a t i o n . The w r i t e r wishes t o express thanks t o Dr. J.E. B i e r of the Department of B i o l o g y and Botany o f the U n i v e r s i t y of B r i t i s h Columbia f o r h i s guid.ari.ee throughout t h i s study. She would l i k e t o acknowledge the a s s i s t a n c e g i v e n h e r with the s t a t i s t i c a l a n a l y s i s by Dr. J.C. Sawyer •of the F a c u l t y of A g r i c u l t u r e , and Dr. S„¥. Nash of the Department of Mathematics of the U n i v e r s i t y of B r i t i s h Columbia. She i s g r a t e f u l t o Mr. D. Mueller-Dombois and t o the Po w e l l R i v e r Company f o r p r o y i d i n g s e e d l i n g s f o r study. F i n a l l y she would l i k e t o thank the many people who helped i n d i f f e r e n t ways wit h the p r e p a r a t i o n o f t h i s t h e s i s . 1 INTRODUCTION Very l i t t l e work has been done on the mycorrhiza of D o u g l a s - f i r (Pseudotsuga m e n z i e s i i ( M i r b e l ) F r a n c o ) . W i t h i n the n a t u r a l range of D o u g l a s - f i r the o n l y i n f o r m a t i o n a v a i l a b l e on mycorrhiza i s o b s e r v a t i o n a l i n nature. As f a r as i s known, no experimental s t u d i e s have been c a r r i e d out to determine the nature and e f f e c t of D o u g l a s - f i r mycorrhiza. A d d i t i o n a l i n f o r m a t i o n on the mycorrhiza of D o u g l a s - f i r would be of value to f o r e s t n u r s e r i e s i n improving p l a n t i n g s t o c k and to f o r e s t e r s concerned w i t h e s t a b l i s h i n g vigorous p l a n t a -t i o n s on logged or burned areas. The present study was undertaken p r i m a r i l y to f i n d out i f t h e r e i s a growth response i n D o u g l a s - f i r to m y c o r r h i z a l a s s o c i a t i o n s . A nursery experiment was designed w i t h the o b j e c t of i n d u c i n g the f o r m a t i o n of mycorrhiza on s e e d l i n g s . A check experiment was c a r r i e d out on p o t t e d s e e d l i n g s . The f u n g i used i n the n u r s e r y and pot experiments were a l s o used i n pure c u l t u r e experiments as a check on t h e i r a b i l i t y to form mycorrhiza. Secondly, i t was c o n s i d e r e d d e s i r a b l e t o examine the mycorrhiza of n a t u r a l s e e d l i n g s to determine the types of mycorrhiza which occur and the age at which they are formed. F i n a l l y , the p o s s i b i l i t y of v a r i a t i o n i n the number and type of mycorrhiza as i n f l u e n c e d by s i t e f a c t o r s was c o n s i d e r e d . 2 LITERATURE REVIEW Myc o r r h i z a i n General In 1885 Frank d e f i n e d mycorrhiza: Der ganze Korper i s t a l s o weder Bsumwurzel noch P i l z a l l e i n , sondem. a h n l i c h der T h a l l u s der F l e c h t e n , eine V e r e i n i g u n g zweier v e r s c h i e d e n e r Wesen zer einem e i n h e i t l i c h e n morphologischen Organ, welches v i e l l e i c h t passend a l s P i l z w u r z e l , Mycorhiza, b e z e i c h n e t werden kann. (The whole s t r u c t u r e i s thus n e i t h e r t r e e r o o t nor , fungus alone, but analogous to the t h a l l u s of l i c h e n s , i s a union of two d i f f e r e n t organisms i n t o one homogeneous mo r p h o l o g i c a l organ, which can perhaps be f i t t i n g l y d e s i g n a t e d as fungus-root, mycorrhiza.) Hacskaylo (8) g i v e s a b r i e f , c l e a r d e s c r i p t i o n of the major m o r p h o l o g i c a l c h a r a c t e r i s t i c s of mycorrhiza. They are u s u a l l y d i v i d e d i n t o two main groups on the b a s i s of the i n t e r r e l a t i o n between the fungus hyphae and the r o o t c e l l s . The endotrophic or i n t r a m a t r i c a l type i s c h a r a c t e r i z e d by the p e n e t r a t i o n of the hyphae d i r e c t l y i n t o the .host c e l l s , where they appear as c o i l s , s w e l l i n g s or minute branches. Hyphae occur only as i n d i v i d u a l threads on the s u r f a c e of the r o o t . In e c t o t r o p h i c or e x t r a m a t r i c a l mycorrhiza the f u n g i u s u a l l y form compact mantles on the s u r f a c e of the r o o t . Subsequently the hyphae p e n e t r a t e between the epidermal and c o r t i c a l c e l l s , forming the " H a r t i g net". In n e i t h e r case i s there i n v a s i o n of the s t e t e . E c t o t r o p h i c mycorrhiza appear 1 Frank, A.B., "Ueber d i e auf Wurzelzymbiose beruhende Ernahrung gewisser Baune durch u n t e r i r d i s c h e P i l z e , " Besdeut. bof. Ges, v o l . 3 , PP« 128-145, quoted i n K e l l e y , A . P . , Mycotrophy i n P l a n t s , Waltham, Mass. C h r o n i c a B o t a n i c a Co., 1950, pToT 3 s h o r t e r and t h i c k e r than normal short r o o t s , do not develop r o o t h a i r s , and may be h e a v i l y branched. In some cases e c t o t r o p h i c mycorrhiza may show i n t r a c e l l u l a r p e n e t r a t i o n by the hyphae. T h i s i n t e r m e d i a t e form i s termed e c t e n d o t r o p h i c . Gaumann (6) suggests that i n f o r e s t t r e e s "the e c t o t r o p h i c mycorrhiza a r i s e from a primary endotrophic i n f e c t i o n by way of d i g e s t i o n , the s u c c e s s f u l p l a s m a t i c defense of the host a g a i n s t the p a r a s i t e . " I t i s i n t e r e s t i n g to note t h a t the form of the mycorrhiza, i . e . , e c t o t r o p h i c or endotrophic, i s a p p a r e n t l y s p e c i f i c f o r the higher p l a n t i n the a s s o c i a t i o n . I n most c o n i f e r s the mycorrhiza are e c t o t r o p h i c . K e l l e y expresses the r e l a t i o n s h i p as f o l l o w s : i t must be remembered t h a t mycorrhiza are formed p r i m a r i l y by the higher symbiont and t h a t t h e i r form i s determined by the v a s c u l a r p l a n t producing i t : the fungus i s of secondary s i g n i f i c a n c e . One fungus or another, or s e v e r a l f u n g i t o g e t h e r , may invade the r o o t , but the m y c o r r h i z a l form w i l l be e s s e n t i a l l y the same i n a l l cases: i t s form i s c h a r a c t e r i s t i c f o r the high e r symbiont r a t h e r than f o r the fungus.3 The nature of the r e l a t i o n s h i p of the fungus and higher p l a n t has been the s u b j e c t of c o n t r o v e r s y ever s i n c e mycorrhiza were d i s c o v e r e d . Many reviews are a v a i l a b l e , 2 Gaumann, E., P r i n c i p l e s of P l a n t I n f e c t i o n , t r a n s l . W.B. B r i e r l e y , London, Crosby Lockwood and Son, L t d . , 1950, p. 287. 3 K e l l e y , A.P., Mycotrophy i n P l a n t s , Waltham, Mass., Chr o n i c a B o t a n i c a Co., 1950, p. 116. 4 i n c l u d i n g those of Henry (12), Rayner (30), Bjorkman ( 3 ) , H a r l e y (9) and Levisohn (16). The o p i n i o n most widely accepted appears to be th a t mycorrhiza are symbiotic a s s o c i a t i o n s , the r e s u l t of c o n t r o l l e d p a r a s i t i s m , which are b e n e f i c i a l t o both the fungushand the higher p l a n t . Recent v/ork on auxins by S l a n k i s ( 3 D and on t r a n s l o c a t i o n of r a d i o a c t i v e l y - l a b e l l e d metabolic products by M e l i n and N i l s s o n (26, 27) s t r o n g l y support t h i s view. Myc o r r h i z a of: D o u g l a s - F i r K e l l e y s t a t e s , "Pseudotsuga i s not popular w i t h m y c o r r h i z a l students." 4" The r e p o r t s which are a v a i l a b l e on t h i s genus are c o n f l i c t i n g . The f i r s t mention of mycorrhiza i n D o u g l a s - f i r was by Laing (14) i n S c o t l a n d , who l i s t s i t as e c t o t r o p h i c , but notes that mycorrhiza are not produced r e a d i l y . Lewis (17) presumed t h a t an endotrophic fungus p e n e t r a t i n g a l l l i v i n g c e l l s of r o o t , stem and bud may be present i n Pseudotsuga. In Utah, McDougall and Jacobs (20) found P. mucronata had both endotrophic and e c t o t r o p h i c m y c o r r h i z a . They a t t r i -buted the s m a l l numbers of mycorrhiza to a l a c k of mushrooms. Using s o i l i n o c u l a t e d w i t h humus c o n t a i n i n g m y c o r r h i z a l f u n g i , McComb and G r i f f i t h (19) found that there were two types of sho r t r o o t s i n D o u g l a s - f i r , one t h a t was h y p e r t r o p h i e d but w i t h no fungus mantle e x t e r n a l l y v i s i b l e w i t h the microscope, 4 K e l l e y , Mycotrophy i n P l a n t s , p. 116. 5 and one that was non-hypertrophied. The former was m y c o r r h i z a l i n i n o c u l a t e d pots and non-mycorrhizal i n u n i n o c u l a t e d p o t s . The l a t t e r was n on-mycorrhizal. F e l i c l a n i and M o n t e f i o r i (5), working i n I t a l y , s t a t e that m y c o r r h i z a l f u n g i , favoured by environmental c o n d i t i o n s , f r e q u e n t l y became a c t i v e l y p a r a s i t i c and damaged the D o u g l a s - f i r s e e d l i n g s . R e c e n t l y more i n t e n s i v e i n v e s t i g a t i o n s have been c a r r i e d out on the mycorrhiza of D o u g l a s - f i r . Linnemann ( 1 8 ) , working i n Germany, gives the most comprehensive account a v a i l a b l e . She r e p o r t s that e c t o t r o p h i c mycorrhiza were the e s s e n t i a l type i n the m a t e r i a l she s t u d i e d , although i s o l a t e d i n t r a c e l l u l a r hyphae, mostly s l e n d e r and s p a r s e l y branched, were d e t e c t e d i n the c o r t i c a l parenchyma of both long and s h o r t r o o t s . The most common forms of mycorrhiza showed e i t h e r a f l a t , f e a t h e r - l i k e type of branching or a f i n e r , l o n g e r , more profuse and very i r r e g u l a r l y branched shor t root system. M y c o r r h i z a were u s u a l l y i n i t i a t e d d u r i n g the second growing season. A few s e e d l i n g s showed the beginning of e c t o t r o p h i c mycorrhiza dur i n g the f i r s t growing season, but a fungus mantle was not observed. Linnemann s t a t e s t h a t D o u g l a s - f i r of the same age under the same s i t e c o n d i t i o n s show remarkable v a r i a t i o n i n the types of r o o t s and m y c o r r h i z a l f o r m a t i o n s . W i t h i n the n a t u r a l range of D o u g l a s - f i r , McMinn (21) found two types of a b s o r p t i v e organs i n t h i s s p e c i e s , m y c o r r h i z a l r o o t l e t s and r o o t h a i r s . Root h a i r s were i n f r e q u e n t and con-s i d e r e d a r e l a t i v e l y unimportant p a r t of the absorbing system. 6 He r e p o r t s both e c t o t r o p h i c and endotrophic mycorrhiza, although from h i s d e s c r i p t i o n the presence of the endotrophic form may be d o u b t f u l . He says, Other s h o r t r o o t l e t s appeared to be e n d o t r o p h i c , f o r hand s e c t i o n s showed the presence of a H a r t i g net, and no mantle was present. These r o o t l e t s were dark brown i n c o l o u r . 5 There i s no mention of i n t r a c e l l u l a r p e n e t r a t i o n , which i s the determining f a c t o r f o r endotrophic mycorrhiza. The H a r t i g net i s u s u a l l y c o nsidered a c h a r a c t e r i s t i c of e c t o t r o p h i c mycorrhiza ( K e l l e y (13), Hacskaylo ( 8 ) , e t c . ) . I n the P a c i f i c Northwest, Wright and T a r r a n t (36) found only unbranched, e c t o t r o p h i c mycorrhiza. The number of seedlings w i t h mycorrhiza decreased w i t h the s e v e r i t y of the burn i n one study area. In the other study area there were more m y c o r r h i z a l s e e d l i n g s i n unburned than i n burned p l o t s . There was no s i g n i f i c a n t d i f f e r e n c e e i t h e r i n h e i g h t or r o o t l e n g t h between s e e d l i n g s w i t h mycorrhiza and those w i t h o u t . There are s e v e r a l r e p o r t s l i s t i n g f u n g i suspected of being m y c o r r h i z a l on D o u g l a s - f i r , but c u l t u r a l c o n f i r m a t i o n s have not been made. In New Zealand, B i r c h (1) noted t h a t Scleroderma b o y i s t a F r i e s , was a p o s s i b l e mycorrhiza-former. Rawlings (29) extended the New Zealand l i s t t o i n c l u d e B o l e t u s  p i p e r a t u s B u l l . , Boletus sp. ( v a r i e g a t u s ? ) , Amanita muscaria L., 5 McMinn, R.G., I n t e r i m Report on S t u d i e s on the C h a r a c t e r i s t i c s  and Ecology of the Root System of Second Growth Douglas Ffrl V i c t o r i a , Canada, Dept. of A g r i c u l t u r e , S c i ence S e r v i c e , D i v . of F o r e s t B i o l o g y , mimeographed, March, 1954, p. 6. 7 Tricholoma sp., Lycoperdon perlatum P e r s . , and L a c c a r i a  l a c c a t a Berk, and Br. On the North American c o n t i n e n t , S l i p p and S n e l l (32) suspected B o l e t i n u s a m a b i l i s (Peck) S n e l l of being m y c o r r h i z a l on D o u g l a s - f i r i n Idaho. In the P a c i f i c Northwest Trappe (33) l i s t e d the f o l l o w i n g as "very probably m y c o r r h i z a l " : Amanita muscaria (L.ex F r i e s ) P e r s . ex S.F. Gray, B o l e t i n u s l a k e i i (Murr.) S i n g . , C a n t h a r e l l u s  c i b a r i u s F r i e s , L a c t a r i u s d e l i c i o s u s (L. ex F r i e s ) Gray, Russula d e l i c a F r i e s , R. emetica (Schaeff ex F r i e s ) P e r s . ex F r i e s , and Gyromitra e s c u l e n t a Pres. ( s i c ) ex F r i e s . 8 EXPERIMENTAL STUDIES Nursery and Pot Experiments I n t r o d u c t i o n A n u r s e r y experiment and a p o t t e d check experiment were designed to induce the f o r m a t i o n of mycorrhiza on D o u g l a s - f i r s e e d l i n g s . These s t u d i e s were c a r r i e d out on the campus of the U n i v e r s i t y of B r i t i s h Columbia from June, 1957? to August, 1958. The treatments i n c l u d e d the use of humus and of pure c u l t u r e s of f u n g i as sources of inoculum of mycorrhiza-forming f u n g i . The d e c i s i o n to i n c l u d e humus as one of the treatments was made because: 1. Humus has p r e v i o u s l y been used as a source of inoculum f o r m y c o r r h i z a l f u n g i . M i k o l a (28) l i s t s " f o r e s t s o i l or humus" as inoculum i n experimental work. 2. I f i t was con s i d e r e d a d v i s a b l e to i n o c u l a t e n u r s e r i e s w i t h m y c o r r h i z a l f u n g i , the a d d i t i o n of humus may be more b e n e f i c i a l than the a d d i t i o n of pure c u l t u r e s . T h e r e f o r e i t was necessary to t e s t the a b i l i t y of humus to provide inoculum. 3. The a d d i t i o n of humus t o the s o i l would more c l o s e l y simulate n a t u r a l c o n d i t i o n s than the use of pure c u l t u r e s . 4. McMinn (21, 22) found t h a t there was o f t e n a p a r t i c u l a r l y r i c h development of mycorrhiza i n and j u s t below the humus l a y e r i n t r e e s under n a t u r a l c o n d i t i o n s . In one of h i s p l o t s the abundance of mycorrhiza depended upon the amount of humus p r e s e n t . Pure c u l t u r e s of f u n g i were used p r i m a r i l y because they produce e f f e c t s which can be c o n t r o l l e d and d u p l i c a t e d . 9 Methods Design of Experiment The nursery experiment c o n s i s t e d o f f i v e r e p l i c a t e s of nine treatments l a i d out i n a s p l i t - p l o t experimental d e s i g n of three rows of f i f t e e n p l o t s each. The nine t r e a t -ments were as f o l l o w s : 1. no humus added, no fungus inoculum added. 2. no humus added, i n o c u l a t e d w i t h Amanita muscaria. 3. no humus added, i n o c u l a t e d w i t h B o l e t u s sp. 4. s t e r i l i z e d humus added, no fungus inoculum added. 5. s t e r i l i z e d humus added, i n o c u l a t e d w i t h A. muscaria. 6. s t e r i l i z e d humus added, i n o c u l a t e d w i t h Boletus sp. 7. u n s t e r i l i z e d humus added, no fungus inoculum added. 8. u n s t e r i l i z e d humus added, i n o c u l a t e d w i t h A. muscaria. 9. u n s t e r i l i z e d humus added, i n o c u l a t e d w i t h Boletus sp. The f u n g i used i n pure c u l t u r e i n the nurs e r y and pot experiments were ob t a i n e d from MS. P . l . S a l i s b u r y , F o r e s t B i o l o g y D i v i s i o n , Canada Department of A g r i c u l t u r e , V i c t o r i a , B.C. They were t i s s u e c u l t u r e s from sporophores which he determined as Amanita muscaria and Boletus sp. A. muscaria was used because i t has a wide host range among the c o n i f e r s as a mycorrhiza-former ( M e l i n ( 2 4 ) , Heim (11), e t c . ) . As noted above, i t i s one of the f u n g i mentioned by Rawlings (29) and by Trappe (33) as suspected of being m y c o r r h i z a l on D o u g l a s - f i r . B oletus sp. was a l s o used because the B o l e t e s as a group are commonly m y c o r r h i z a l on c o n i f e r s . 10 Attempts were made to i s o l a t e the f u n g i from n a t u r a l l y o c c u r r i n g D o u g l a s - f i r mycorrhiza to be used as i n o c u l a . However, a l l attempts proved u n s u c c e s s f u l because of the d i f f i c u l t y of e l i m i n a t i n g other f a s t e r - g r o w i n g s o i l f u n g i which occu r r e d w i t h the mycorrhiza. The humus used i n the treatments was obtained from an immature D o u g l a s - f i r p l a n t a t i o n on the U n i v e r s i t y of B r i t i s h Columbia campus. I t was a d u f f m u l l and i n c l u d e d D o u g l a s - f i r needle l i t t e r . M y c o r r h i z a were observed to occur abundantly on the t r e e s i n the p l a n t a t i o n . The f u n g i and humus were used i n combination i n the nursery treatments because i t was f e l t the presence of the humus might a i d i n e s t a b l i s h i n g the pure c u l t u r e s of the f u n g i i n the s o i l . M e l i n (23) found t h a t f o r e s t l i t t e r e x t r a c t s f a v o u r a b l y i n f l u e n c e d the growth of m y c o r r h i z a l Hymenomyc&tes by p r o v i d i n g v/ater s o l u b l e substances which c o u l d be u t i l i z e d by the f u n g i . The use of humus w i t h the fungus inoculum would tend t o sim u l a t e n a t u r a l c o n d i t i o n s . P r e p a r a t i o n of Treatments The humus to be s t e r i l i z e d was aut o c l a v e d f o r one hour at f i f t e e n pounds p r e s s u r e on three c o n s e c u t i v e days. I t remained s e a l e d and was allowed to stand f o r s e v e r a l days b e f o r e being used. Both the s t e r i l i z e d and u n s t e r i l i z e d humus were a p p l i e d as one-inch l a y e r s over the s u r f a c e of the p l o t s b e f o r e the seed was sown. 11 The fungus inoculum was prepared i n the f o l l o w i n g manner: Amanita muscaria and Boletus sp. were grown on a n u t r i e n t agar i n p l a t e c u l t u r e s f o r f o u r weeks. Then the m y c e l i a l mats were removed and macerated i n s t e r i l e d i s t i l l e d water i n a Waring Blendor i n the i n o c u l a t i n g room u n t i l t h e r e were sma l l clumps of hyphae i n suspension. The suspensions were sprayed on the p l o t s as inoculum j u s t p r i o r to sowing the seeds. About 75 ml. of the suspension was added to each t r e a t e d p l o t . The experimental p l o t s were 2 by 1.6 f e e t , w i t h a 1.5 f o o t path between p l o t s . Ninety seeds were sown i n rows i n each p l o t on June 14, 1957• The rows were 3 inches apart and 2 f e e t l o n g . A f t e r sowing, the seeds were covered w i t h moist sand. The seed used was ob t a i n e d from Dean G. S. A l l e n of the F a c u l t y of F o r e s t r y , U.B.C. and came from a smal l area of the Nanaimo d i s t r i c t of Vancouver I s l a n d . I t was l o w - a l t i t u d e c o a s t a l D o u g l a s - f i r seed. The nursery p l o t s were watered when necessary d u r i n g the f i r s t growing season. The season was comparatively wet and c o o l , f a v o u r i n g the establishment of the s e e d l i n g s . In the f a l l , a f t e r the s e e d l i n g s were w e l l - e s t a b l i s h e d , they were watered by n a t u r a l r a i n f a l l only. Growth Measurements The s e e d l i n g s were l e f t i n the ground u n t i l the middle of J u l y , 1958, to give them the most a c t i v e p a r t of a second 6 See Appendix I f o r the formula of the n u t r i e n t agar used. 12 growing season. They were then dug c a r e f u l l y , to remove the complete r o o t systems. The r o o t s were washed w e l l to remove s o i l p a r t i c l e s . Measurements were taken f o r each s e e d l i n g of height from ground l e v e l to t e r m i n a l hud (almost a l l the s e e d l i n g s were dormant when they were dug), l e n g t h of l o n g e s t r o o t , and r o o t volume. The r o o t volume was measured by water displacement i n a graduated c y l i n d e r . The average h e i g h t of the s e e d l i n g s i n each treatment i s shown i n Table 1. Ta b l e 2 gi v e s the f i g u r e s f o r the average ro o t volume of the s e e d l i n g s i n each treatment. From each p l o t f i v e s e e d l i n g s were randomly s e l e c t e d f o r counts of the number of m y c o r r h i z a l r o o t t i p s t o gi v e an I n d i c a t i o n of the development of mycorrhiza. The counts were made under a d i s s e c t i n g microscope. The average number of mycorrhiza per s e e d l i n g i n each treatment i s g i v e n i n Table 3» An a n a l y s i s of v a r i a n c e was c a r r i e d out on the data from the measurements of h e i g h t and r o o t volume and from the counts of mycorrhiza.7 Pot Experiment An experiment was s e t up i n pots w i t h the same t r e a t -ments as were used i n the nu r s e r y as a p r e c a u t i o n a g a i n s t t o t a l l o s s of m a t e r i a l by severe c l i m a t i c c o n d i t i o n s . I t was a l s o to serve as a check on the n u r s e r y r e s u l t s . The chance of l a r g e s o i l v a r i a t i o n s was much l e s s i n the pots than i n the n u r s e r y experiment because the s o i l came from a s m a l l area, whereas the 7 See Appendix II f o r summary t a b l e s of the analyses of v a r i a n c e . to f o l l o w page 12 T a b l e 1. Average height (cm.) of D o u g l a s - f i r s e e d l i n g s from nur s e r y p l o t s g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments. TREATMENT no humus u n s t e r i l i z e d s t e r i l i z e d added humus added humus added no fungus 6.54 7.61 9.41 inoculum added i n o c u l a t e d w i t h 7.34 7.97 8.68 Amanita muscaria i n o c u l a t e d w i t h 7.41 7.22 8.71 Boletus sp. Tabl e 2. Average r o o t volume (cc.) of D o u g l a s - f i r s e e d l i n g s from nursery p l o t s g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments. TREATMENT no humus added u n s t e r i l i z e d humus added s t e r i l i z e d humus added no fungus inoculum added 0.92 1.34 1.86 i n o c u l a t e d w i t h Amanita muscaria 1.07 1.56 1.86 i n o c u l a t e d w i t h Boletus sp. 1.31 1.15 1.50 Table 3. Average number of mycorrhiza per s e e d l i n g o c c u r r i n g i n n u r s e r y p l o t s g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments. TREATMENT no humus u n s t e r i l i z e d s t e r i l i z e d added humus added humus added no fungus 129.8 263.2 144.4 inoculum added i n o c u l a t e d w i t h 126.4 232.9 I87.6 Amanita muscaria i n o c u l a t e d w i t h 116.4 88.7 143.8 Boletus sp. 13 n u r s e r y p l o t s extended over a much l a r g e r a r ea. The p o s s i b i l i t y of the spread of inoculum from treatment t o treatment was l e s s w i t h use of p o t s . The experiment was l a i d out i n a 9 by 5 randomized b l o c k d e s i g n . S i x - i n c h flower pots were f i l l e d w i t h s o i l from the n u r s e r y . The seeds were sown d i r e c t l y on the s u r f a c e of the s o i l and then the humus and fungus treatments were added. Twenty seeds were sown i n each pot on J u l y 11, 1957* The s e e d l i n g s were thinned to f i v e per pot i n the s p r i n g of 1958. The pots were sunk i n the n u r s e r y w i t h about one i n c h of the pot above the s u r f a c e of the s o i l . They were l e f t u n t i l December, when they were placed i n the greenhouse. They remained i n the greenhouse u n t i l they were analysed at the end of August, 1958. Height and r o o t volume measurements were taken on a l l of the s e e d l i n g s . The average measurements f o r each t r e a t -ment are shown i n Tables 4 and 5« Counts of the number of mycorrhiza were not made because a l l the s e e d l i n g s showed some mycorrhiza and i t was f e l t the r e s u l t s would not j u s t i f y the time r e q u i r e d . The extent of mycorrhiza development was graded s u b j e c t i v e l y f o r each pot. An a n a l y s i s of v a r i a n c e was c a r r i e d out f o r the he i g h t and the r o o t volume measurements to i n d i c a t e whether or not the s e e d l i n g s v a r i e d as a r e s u l t of the d i f f e r e n t treatments which were a p p l i e d . Summary t a b l e s are g i v e n i n Appendix II. to f o l l o w page 13 Table 4. Average height (cm.) of D o u g l a s - f i r s e e d l i n g s grown i n pots g i v e n d i f f e r e n t humus and i n o c u l a t i o n treatments. TREATMENT no humus u n s t e r i l i z e d s t e r i l i z e d added humus added humus added no fungus inoculum added 9.67 11.26 9.87 i n o c u l a t e d w i t h 9.84 11.98 11.75 Amanita muscaria i n o c u l a t e d w i t h 10.13 10.04 12.41 Boletus sp. Table 5. Average r o o t volume (cc.) of D o u g l a s - f i r s e e d l i n g s grown i n pots given d i f f e r e n t humus and I n o c u l a t i o n treatments. TREATMENT no humus u n s t e r i l i z e d s t e r i l i z e d added humus added humus added no fungus 1.13 1.26 1.33 inoculum added i n o c u l a t e d w i t h 1.18 1.49 1.26 Amanita muscaria i n o c u l a t e d w i t h 1.10 1.39 1.46 Boletus sp. 14 R e s u l t s of Nursery and Pot Experiments Main E f f e c t s A l l the s e e d l i n g s from each of the p l o t s i n the nursery, i n c l u d i n g c o n t r o l p l o t s , had well-developed mycorrhiza at the time of d i g g i n g . Two forms of mycorrhiza were pres e n t . The most common one was c h a r a c t e r i z e d by a prominent t h i c k , white mantle. The other had a t h i n h y a l i n e mantle which d i d not change the c o l o u r of the r o o t . Both forms showed well-developed branching. A more d e t a i l e d d e s c r i p t i o n of the mycorrhiza i s giv e n i n the s e c t i o n on morphology. The presence of mycorrhiza on a l l the s e e d l i n g s i n d i c a t e s the presence of a m y c o r r h i z a l fungus (or f u n g i ) i n the nursery s o i l b e f o r e treatments were added. I f t h i s i s the case, attempts to use some method of i n o c u l a t i o n were u n s u i t a b l e f o r the area. Neither the use of pure c u l t u r e s of Amanita  muscaria and Boletus sp. nor the use of humus can be c o n s i d e r e d to have been t e s t e d as a means of i n o c u l a t i n g an area l a c k i n g m y c o r r h i z a l f u n g i . The establishment of A. muscaria and B o l e t u s sp. i n the s o i l as a r e s u l t of adding pure c u l t u r e s i s problem-a t i c a l . C o n c l u s i v e proof of establishment would be the appearance of the sporophores of these f u n g i i n the n u r s e r y , which has not occurr e d . The humus as used had no s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on the growth of the s e e d l i n g s . The primary c o n s i d e r a t i o n i n i t s use was to provide an inoculum of m y c o r r h i z a l f u n g i . The 15 humus e f f e c t , per se, was secondary. Thus c o n s i d e r a t i o n s of amount, depth, method of a p p l i c a t i o n , e t c . , were perhaps not weighed to best b r i n g out n u t r i t i o n a l or other e f f e c t s not d i r e c t l y connected w i t h mycorrhiza. G e n e r a l i z a t i o n s cannot be made, t h e r e f o r e , on the e f f e c t of humus except as a source of inoculum. The a n a l y s i s of v a r i a n c e showed no s i g n i f i c a n t d i f f e r e n c e s between treatments i n h e i g h t , r o o t volume, or the number of mycorrhiza per s e e d l i n g . In a s p l i t - p l o t d e s i g n the whole-plot e f f e c t i s not measured as s e n s i t i v e l y as the sub-p l o t e f f e c t because there are fewer r e p l i c a t i o n s of the whole-p l o t treatments. D i f f e r e n c e s must be l a r g e r between the whole-p l o t s than between the s u b - p l o t s to be s i g n i f i c a n t . Thus the t e s t of the humus e f f e c t s was not as s e n s i t i v e as the t e s t s of the fungus e f f e c t s . However, i t i s f e l t t h a t d i f f e r e n c e s between treatments i n t h i s type of work should be r e a d i l y demonstrable to j u s t i f y making recommendations f o r changing e x i s t i n g p r a c t i c e s . Wilde's (34) o b s e r v a t i o n of "once f o r e s t s o i l , always Q f o r e s t s o i l " w i t h regard t o the presence of m y c o r r h i z a l f u n g i seems t o have been upheld. The nursery i s s i t u a t e d i n an area which was o r i g i n a l l y covered w i t h a c o n i f e r o u s f o r e s t , i n c l u d i n g D o u g l a s - f i r . Wilde f e e l s t h a t the e x t e r m i n a t i o n of m y c o r r h i z a l f u n g i i n f o r e s t s o i l s r e s u l t s o n l y from r a d i c a l changes i n s o i l 8 Wilde, S.A., " M y c o r r h i z a l F u n g i : T h e i r D i s t r i b u t i o n and E f f e c t on Tree Growth," S o i l S cience, V o l . 78, No. 1, 1954, p. 28. 16 environmental c o n d i t i o n s . Apparently such changes have not occurred i n the s o i l at the U.B.C. nu r s e r y . Secondary E f f e c t s of Treatments There was a remarkable v a r i a t i o n i n the germination of seeds i n the three d i f f e r e n t humus treatments i n the nu r s e r y experiment. Germination was h i g h e s t i n the p l o t s w i t h no humus added and lowest i n the p l o t s w i t h s t e r i l i z e d humus, as shown by the number of s e e d l i n g s per p l o t at the end of August, 1957* (Table 6.) T h i s was a t t r i b u t e d to l a c k of s u f f i c i e n t water f o r germination. The humus, p a r t i c u l a r l y the s t e r i l i z e d humus, d r i e d out on the s u r f a c e very q u i c k l y . Seeds that were not plant e d on the s u r f a c e of the s o i l , but remained i n the humus, d i d not r e c e i v e s u f f i c i e n t moisture f o r germination. The s t e r i l i z i n g process seemed to a l t e r the humus, lowering i t s water-holding c a p a c i t y . Germination was much more uniform i n the pots . The seed was p l a n t e d d i r e c t l y on the s u r f a c e of the s o i l b e f o r e the humus was added. In t h i s way a l l the seeds had a s i m i l a r s u b s t r a t e and moisture d i d not become a d i f f e r e n t i a t i n g f a c t o r . During the f i r s t growing season some s e e d l i n g s were l o s t when seed-eating b i r d s nipped o f f the tops before the seed coats were shed. T h i s l o s s was evenly d i s t r i b u t e d over the whole experiment. Although the w i n t e r of 1957-58 was very m i l d , t h e r e were s e v e r a l f r o s t s during the season which caused some f r o s t -heaving of the s e e d l i n g s . Damage was s l i g h t i n p l o t s which had 17 been t r e a t e d w i t h humus, e i t h e r s t e r i l i z e d or u n s t e r i l i z e d . Loss was more severe i n the s o i l p l o t s . F i g u r e s given i n Table 6 show the per cent of the s e e d l i n g s a l i v e i n the f a l l which s u r v i v e d the w i n t e r . T a b l e 6. Germination of the seed and s u r v i v a l of the s e e d l i n g s over w i n t e r i n the nur s e r y experiment. Average number of s e e d l i n g s percent of Percent of TRE ATI/IE NT per p l o t seed pro- s e e d l i n g s Aug.28, 1957 F i n a l number ducing s u r v i v i n g s e e d l i n g s w i n t e r  no humus 67.3 34.7 74.8$ 51.7$ u n s t e r i l i z e d 31.6 20.7 35.1$ 65.8$ humus s t e r i l i z e d 24 .5 19.7 27.2$ 80.4$ humus 18 Pure C u l t u r e Techniques to Induce M y c o r r h i z a Formation I n t r o d u c t i o n Pure c u l t u r e experiments were c a r r i e d out i n attempts to induce m y c o r r h i z a l f o r m a t i o n i n the l a b o r a t o r y as proof of the a b i l i t y of a fungus t o form mycorrhiza. Knowledge of a proven m y c o r r h i z a l fungus would g r e a t l y f a c i l i t a t e any f u r t h e r s t u d i e s of the mycorrhiza of D o u g l a s - f i r . The seed used i n a l l experiments was s t r a t i f i e d t o give a uniform germination. Seed used f o r pure c u l t u r e work was sur f a c e s t e r i l i z e d i n 0,1% b i c h l o r i d e of mercury f o r nine minutes, w i t h a s m a l l amount of Lux l i q u i d detergent added as a w e t t i n g agent. Some of the seeds were damaged at t h i s exposure, but sh o r t e r p e r i o d s d i d not e l i m i n a t e s u r f a c e contamination. The seeds were r i n s e d i n three changes of s t e r i l e d i s t i l l e d water. They were then placed on malt agar i n p e t r i p l a t e s to germinate. When the r a d i c l e was at l e a s t 5 nun. long the seed was t r a n s f e r r e d to the prepared c u l t u r e medium. Only seeds i n p l a t e s showing no contamination were used. Methods The f i r s t method was a m o d i f i c a t i o n of that d e s c r i b e d by Hacskaylo (7) using v e r m i c u l i t e as a s u b s t r a t e w i t h a n u t r i e n t s o l u t i o n i n erlenmeyer f l a s k s . The n u t r i e n t s o l u t i o n used was that developed by M e l i n and N i l s s o n . The a c i d i t y was a d j u s t e d Q to pH4 before a u t o c l a v i n g . T w o - l i t r e wide-mouth erlenmeyer 9 See Appendix I f o r formula f o r n u t r i e n t s o l u t i o n . 19 f l a s k s were used, w i t h 1 0 0 0 cc. of v e r m i c u l i t e and 7 5 0 ml. of n u t r i e n t s o l u t i o n i n each. The f l a s k s were plugged w i t h c o t t o n and autoclaved at 1 5 pounds pressure f o r 2 0 minutes. A germinated seed was placed i n each f l a s k . The f l a s k s were i n o c u l a t e d w i t h 1 cm. sq. p i e c e s from the advancing zone of the fungus mycelium grown i n p l a t e c u l t u r e s . 1 0 Amanita muscaria and B oletus sp. were the f u n g i t e s t e d . T h i s method was not s u c c e s s f u l because the f u n g i f a i l e d t o develop on the v e r m i c u l i t e and a f t e r about fo u r months the s e e d l i n g s d i e d . The reason the s e e d l i n g s d i e d was thought to be a l a c k of s u f f i c i e n t t r a n s p i r a t i o n caused by the h i g h humidity i n the f l a s k s . I f t h i s was the cause, then the remedy appeared to be a method i n which the crown of the s e e d l i n g remained ou t s i d e the c o n t a i n e r , w h i l e the r o o t s were kept under . a s e p t i c c o n d i t i o n s . To f u l f i l t h i s c o n d i t i o n s m a l l narrow-mouth b o t t l e s were used and the tops were covered w i t h aluminum f o i l , which was depressed i n t o the openings. Each b o t t l e was f i l l e d w i t h v e r m i c u l i t e and 1 0 0 ml. of n u t r i e n t s o l u t i o n was added. The b o t t l e s were autoclaved w i t h the aluminum f o i l l o o s e l y i n p l a c e . In the i n o c u l a t i n g room under a s e p t i c c o n d i t i o n s the aluminum f o i l was l i f t e d to allow the a d d i t i o n of the fungus inoculum. Some of the b o t t l e s were i n o c u l a t e d w i t h a 5 mm* cube from c u l t u r e s of the fungus i n p e t r i p l a t e s . The remainder were i n o c u l a t e d w i t h 10 ml. of a m y c e l i a l suspension which had been prepared i n a Waring Blendor. 10 See Appendix I f o r formula f o r n u t r i e n t agar. 20 Another fungus was used i n a d d i t i o n t o Amanita  muscaria and Boletus sp. i n t h i s method. A s t i n k h o r n had been found among two-year-old D o u g l a s - f i r s e e d l i n g s which were h i g h l y m y c o r r h i z a l ( F i g u r e 2 ) . The fungus was determined by the author as Mutinus sp. ( r a v e n e l l i i ? ) . S u c c e s s f u l t i s s u e c u l t u r e s were made on the n u t r i e n t agar. T h i s fungus was t e s t e d i n pure c u l t u r e work because i t was suspected of being a fungus symbiont on the s e e d l i n g s w i t h which i t was a s s o c i a t e d . A seed which had been germinated a s e p t i c a l l y was p l a c e d i n each b o t t l e . By the time the seed: coat and e p i c o t y l were r a i s e d above the ground ( g e r m i n a t i o n i n D o u g l a s - f i r i s epigeous) the r a d i c l e had penetrated w e l l i n t o the v e r m i c u l i t e . At t h i s stage the aluminum f o i l was punctured and the seed coat and e p i c o t y l brought up a s e p t i c a l l y through the f o i l . T h i s allowed the cotyledons to develop o u t s i d e the b o t t l e . The hole i n the f o i l was s e a l e d w i t h a v a s e l i n e - p a r a f f i n mixture to keep the root medium a s e p t i c . F i g u r e 1 shows s e e d l i n g s growing i n these b o t t l e s . The s e e d l i n g s were kept i n b o t t l e s at room temperature f o r about e i g h t months. During the summer the b o t t l e s were pl a c e d at a window w i t h a northwest exposure. In the f a l l they were p l a c e d under continuous f l u o r e s c e n t l i g h t . The s e e d l i n g s grew w e l l f o r about f o u r months and then growth ceased. The f u n g i showed some development i n the b o t t l e s i n o c u l a t e d w i t h m y c e l i a l suspensions. However, no mycorrhiza were formed, perhaps because the s e e d l i n g s stopped growing b e f o r e i n v a s i o n took p l a c e . In the b o t t l e s i n o c u l a t e d with a cube of 21 agar c o n t a i n i n g a p i e c e of m y c e l i a l mat, the fungus d i d not spread throughout the v e r m i c u l i t e . The t h i r d method proved to be the most s u c c e s s f u l , w i t h regard to s e e d l i n g growth. I t was an a d a p t a t i o n of the vented cap method f o r the r e g u l a t i o n of a e r a t i o n i n wood s o i l c o n t a c t c u l t u r e technique developed by Eades and Roff (4) . In t h i s method 16 f l u i d oz. j a r w i t h domestic two-piece type screw l i d s were used. A 4 cm. long p i e c e of 6 mm. (O.D.) g l a s s tubing was set i n t o the i n n e r f l a t cover at an angle of 45° and s e a l e d i n p l a c e w i t h de Khotinsky cement. Nursery s o i l was used as the c u l t u r e medium. I t had p r e v i o u s l y been au t o c l a v e d f o r one hour at 1(5 pounds p r e s s u r e on three c o n s e c u t i v e days and had remained s e a l e d a f t e r auto-c l a v i n g f o r some time. In each j a r was p l a c e d 150 c c . of s o i l and 60 ml. of d i s t i l l e d water. The amount of water was enough to wet the s o i l thoroughly without l e a v i n g an excess i n the bottom of the j a r . The j a r s were covered w i t h paper t o w e l l i n g and a u t o c l a v e d f o r 45 minutes at 15 pounds p r e s s u r e . Because the cement c o u l d not be a u t o c l a v e d , the l i d s were s t e r i l i z e d i n b o i l i n g water and p l a c e d i n j a r s i n the i n o c u l a t i n g room. Mutinus sp. ( r a v e n e l l i i ? ) was grown i n p e t r i p l a t e s c o n t a i n i n g n u t r i e n t agar. The m y c e l i a l mat from a four-week-old c u l t u r e was added to each of two j a r s . Two j a r s remained as c o n t r o l s . The mat was cut up and mixed w i t h the s u r f a c e s o i l w i t h a long i n o c u l a t i n g needle. Seed had been s u r f a c e - s t e r i l i z e d and germinated as 22 d e s c r i b e d p r e v i o u s l y . A germinated seed was p l a c e d i n each of the two b o t t l e s i n o c u l a t e d w i t h Mutinus sp. and the two c o n t r o l b o t t l e s . The s e e d l i n g s were allowed to grow f o r f i v e months and then were dug and examined. The s e e d l i n g s grew w e l l i n the vented cap j a r s . Dormancy oc c u r r e d , f o l l o w e d by renewed growth, a phenomenon which d i d not take p l a c e i n the other method. By the time the seedlings were dug they had grown to the tops of the j a r s , a d i s t a n c e of about 8 cm. from the s o i l . The development of the fungus In the s o i l i s p r o b l e m a t i c a l . The mycelium continued growth on the p i e c e s o f agar which were i n c l u d e d w i t h the inoculum. However, i t i s qu e s t i o n a b l e i f the hyphae spread throughout the s o i l . No mycorrhiza were found on the r o o t systems of the s e e d l i n g s , although there were more short r o o t s produced by the s e e d l i n g s i n t h i s method than by the s e e d l i n g s i n e i t h e r of the other methods. Apparently Mutinus sp. d i d not develop to the p o i n t of inv a d i n g the short r o o t s . D i s c u s s i o n The whole problem of i n d u c i n g the fo r m a t i o n of mycorrhiza on D o u g l a s - f i r s e e d l i n g s i n pure c u l t u r e i s one of unknowns. One of the most d i f f i c u l t aspects i s p r o v i d i n g s u i t a b l e a s e p t i c c o n d i t i o n s f o r the growth of the s e e d l i n g s . L i t t l e i s known about the p h y s i o l o g y of D o u g l a s - f i r s e e d l i n g s . F a c t o r s such as a e r a t i o n , temperature, l i g h t and moisture, as w e l l as chemical composition of the r o o t i n g medium, must be 23 c o n s i d e r e d , e s p e c i a l l y when the s e e d l i n g s are r e q u i r e d to develop under a s e p t i c c o n d i t i o n s . The medium used f o r growing s e e d l i n g s should a l s o he s u i t a b l e f o r the development of the fungus. As there i s no proven m y c o r r h i z a l fungus on D o u g l a s - f i r , there i s no s a t i s f a c t o r y e s t a b l i s h e d c o n t r o l f o r t e s t i n g the medium being used. I f mycorrhiza are not produced, i t i s d i f f i c u l t to know i f i t i s because the c o n d i t i o n s are not s u i t a b l e or because the fungus i s not capable of forming mycorrhiza on D o u g l a s - f i r . S e v e r a l p o i n t s of i n t e r e s t a r i s e from these s t u d i e s . The most s a t i s f a c t o r y method of i n o c u l a t i o n was the use of a m y c e l i a l suspension. The fungus was i n i t i a l l y d i s t r i b u t e d throughout the medium, and d i d not r e q u i r e time to spread by growth. In g e n e r a l , m y c o r r h i z a l f u n g i grow very slowly i n c u l t u r e . The c o n t a i n e r s used should be l a r g e enough to a l l o w the s e e d l i n g s to develop f o r at l e a s t s i x months. The methods employing e i t h e r the aluminum-foil covered b o t t l e s or the vented cap j a r s may prove to be s a t i s f a c t o r y i f l a r g e r c o n t a i n e r s are used. Both methods hol d promise f o r the p r o d u c t i o n of mycorrhiza. The development of a s u c c e s s f u l technique f o r i n d u c i n g the formation of mycorrhiza on D o u g l a s - f i r s e e d l i n g s i n pure c u l t u r e i s e s s e n t i a l before knowledge of the mycorrhiza i n t h i s s p e c i e s can be anything but e m p i r i c a l i n nature. 24 DESCRIPTIVE STUDIES Morphology of S e e d l i n g M y c o r r h i z a The s t r u c t u r e of the mycorrhiza from the v a r i o u s types of s e e d l i n g s grown e x p e r i m e n t a l l y and from those c o l l e c t e d from n a t u r a l areas was s t u d i e d . The gross morphology was examined w i t h the a i d of a d i s s e c t i n g microscope. Trans-verse s e c t i o n s were made to determine m i c r o s c o p i c s t r u c t u r e . Types of Branching The mycorrhiza of the s e e d l i n g r o o t s were of s e v e r a l types w i t h regard to the branching p a t t e r n s . The branching was commonly i n one plane o n l y , producing a f l a t t e n e d mycorrhiza, because the r o o t of D o u g l a s - f i r i s d i a r c h . One type was a simple, unbranched m y c o r r h i z a l s h o r t r o o t , d e s c r i b e d by McMinn (21) as d i g i t a t e . In most cases examined, s e v e r a l of t h i s type were developed i n a monopodial arrangement along a l a t e r a l r o o t l e t as shown i n F i g u r e 3« T h i s may be the form d e s c r i b e d by K e l l y (13) as racemose. I t showed t y p i c a l e c t o t r o p h i c f e a t u r e s , being swollen, stunted and without r o o t h a i r s . They were a p p a r e n t l y caused by s e v e r a l d i f f e r e n t f u n g i because the mantles v a r i e d i n c o l o u r and t e x t u r e . Another type found was of the form d e s c r i b e d by Linnemann (18) as f e d e r a r t i g ( f e a t h e r l i k e ) . T h i s appears to be the most c h a r a c t e r i s t i c form f o r D o u g l a s - f i r . The shor t r o o t s were monopodially arranged on the l a t e r a l r o o t and were themselves monopodially branched, producing a pinnate form 25 ( F i g u r e 4 ) . I n some specimens of t h i s type the branching was very dense and the mantle was t h i c k , making i t d i f f i c u l t to d i s t i n g u i s h i n d i v i d u a l branches. O c c a s i o n a l l y a number were formed along one root to produce an extremely h e a v i l y m y c o r r h i z a l area. Both the simple and the pinnate forms of mycorrhiza were very iSommon. A l e s s common form was h e a v i l y and i r r e g u -l a r l y branched ( F i g u r e 5.) Except f o r the branching i t appeared s u p e r f i c i a l l y l i t t l e d i f f e r e n t from a non-mycorrhizal r o o t . The mantle was ve r y t h i n and h y a l i n e , producing only s l i g h t s w e l l i n g . I t was smooth and about the same c o l o u r as a normal r o o t . S e c t i o n i n g showed that i t was d e f i n i t e l y a mycorrhiza, w i t h i n t e r c e l l u l a r p e n e t r a t i o n of the fungus i n t o the c o r t e x . In no case was there c o r a l l o i d f o r m a t i o n of mycorrhiza, as has been r e p o r t e d i n the p i n e s . Dichotomous branching was r a r e , o c c u r r i n g i n only one or two specimens. A l l the mycorrhiza examined showed t y p i c a l e c t o t r o p h i c s t r u c t u r e , w i t h a mantle and a H a r t i g net pr e s e n t . There was no evidence of i n t r a c e l l u l a r i n v a s i o n i n any of the s e c t i o n s s t u d i e d . Hyphae n e i t h e r p e n e t r a t e d between the endodermal c e l l s nor i n t o the s t i l e . Mantle Formation The mantle v a r i e d i n c o l o u r , t e x t u r e and t h i c k n e s s , but was always present i n some form. The two most common types of mantles were t h i c k , w i t h a f u z z y appearance. One was white 26 and cottony, and the other was dark, almost b l a c k . I t i s p o s s i b l e t h a t the dark form i s an o l d e r stage of the same type as the white one, although they n o r m a l l y d i d not occur on the same r o o t system. M i c r o s c o p i c a l l y , the mantle c o n s i s t e d of uniform hyphae which appeared as i n t e r t w i n e d threads ( F i g u r e 6 . ) . Hyphae pe n e t r a t e d between the c e l l s of the c o r t e x as f a r as the endodermis. Deepest p e n e t r a t i o n of the r o o t o c c u r r e d w i t h t h i s t y p e .of mantle. Both c o l o u r s occurred i n e i t h e r simple or pinnate mycorrhiza. A l e s s common type of mantle was smooth, w i t h an opaque, amber appearance. I t was t h i n n e r than the f u z z y types, o c c a s i o n a l l y o n l y a few strands t h i c k . The amber col o u r was caused by the r o o t being apparent through the h y a l i n e hyphae. I n t e r c e l l u l a r p e n e t r a t i o n was u s u a l l y con-f i n e d to the outer one or two l a y e r s of the c o r t e x ( F i g u r e 7 ) . T h i s type of mantle oc c u r r e d most commonly i n i r r e g u l a r l y and h e a v i l y branched m y c o r r h i z a , although i t was a l s o present i n both the simple and p i n n a t e forms. A type of mantle o c c u r r i n g r a t h e r r a r e l y was dense and b l a c k , w i t h s t i f f hyphae r a d i a t i n g out from i t ( F i g u r e 8 ) . M i c r o s c o p i c a l l y , i t was very t h i c k , w i t h dark hyphae c l o s e l y compressed ( F i g u r e 9 ) . I n t e r c e l l u l a r p e n e t r a t i o n was a g a i n c o n f i n e d to the outer l a y e r s of the c o r t e x . Most of the mycorrhiza having t h i s type of mantle were unbranched, but a few were branched once. 27 Renewed Growth Renewed growth of m y c o r r h i z a l s h o r t r o o t s was observed on s e v e r a l o c c a s i o n s . In some cases the mantle was broken when the r o o t s t a r t e d to grow again and the fungus d i f l not form a mantle over the new growth. ( F i g u r e 10.) In other words, the new growth was n o n - m y c o r r h i z a l . Root h a i r s were present and the r o o t was not swollen. I n other i n s t a n c e s the fungus a l s o renewed i t s growth and the e n t i r e s h o r t r o o t was m y c o r r h i z a l . The evidence of renewed growth was a con-s t r i c t i o n at the p o i n t where the o r i g i n a l growth ceased and o c c a s i o n a l l y a s l i g h t c o l o u r change ( F i g u r e 11). Neither type of renewed growth was observed on one-year-old s e e d l i n g s , although i t may be a s e a s o n a l phenomenon r a t h e r than an annual one. Occurrence of M y c o r r h i z a on D o u g l a s - F i r S e e d l i n g s Under N a t u r a l C o n d i t i o n s S e e d l i n g s i n the Nanaimo R i v e r V a l l e y I n t r o d u c t i o n A t o t a l of 620 one-year-old D o u g l a s - f i r s e e d l i n g s grown by Mr. D i e t e r Mueller-Dombois i n the Nanaimo R i v e r v a l l e y were examined f o r mycorrhiza. The seed was sown i n p l o t s i n burned and unburned logged-over areas i n c l u d i n g a range of D o u g l a s - f i r s i t e s . In each p l o t t h e r e was a seed-spot exposed to s u n l i g h t and a seed-spot under n a t u r a l shade. 28 There were two or three r e p l i c a t i o n s of p l o t s i n some of the s i t e s , to give a t o t a l of 27 spots. The arrangement of the seed-spots i s i l l u s t r a t e d i n F i g u r e 12. The s i t e s were c l a s s i f i e d by Mr. Mueller-Dombois 11 a c c o r d i n g to the system used by Dr. V.J. K r a j i n a . They were c h a r a c t e r i z e d as s a l a l - l i c h e n s i t e s , s a l a l s i t e s , moss s i t e s and swordfern s i t e s . They r e p r e s e n t a range of c o n d i t i o n s under which D o u g l a s - f i r w i l l grow i n the area, w i t h the s a l a l - l i c h e n s i t e being the poorest and the swordfern s i t e the b e s t . ^ The seed used i n the experiment was from one seed-t r e e , to minimize the e f f e c t of g e n e t i c v a r i a t i o n . The seed was sown i n l a t e September, 1956, and the s e e d l i n g s grew f o r one complete season. The s e e d l i n g s were g i v e n to the author by Mr. Mueller-Dombois a f t e r they were dug. Method of Examination The r o o t systems were thoroughly washed and examined under a d i s s e c t i n g microscope. A count was made of the number of mycorrhiza per s e e d l i n g to give an o b j e c t i v e b a s i s f o r comparison of m y c o r r h i z a l development. Notes were kept of the types of mycorrhiza found i n each s e e d l i n g . Measurements of height and r o o t volume had been taken f o r each s e e d l i n g by Mr. Mueller-Dombois. F i g u r e s are given f o r the average number of mycorrhiza per s e e d l i n g , the average h e i g h t and the average r o o t volume of the s e e d l i n g s i n each p l o t i n T a b l e 7. 11 K r a j i n a , V . J . , Unpublished data. 12 More d e t a i l e d i n f o r m a t i o n on the s i t e c h a r a c t e r i s t i c s w i l l be a v a i l a b l e i n the t h e s i s to be presented by Mr. Mueller-Dombois f o r the degree of Doctor of Philosophy at the U n i v e r s i t y of B r i t i s h Columbia. unburned logged land burned logged land salal- l ichen salal moss swordfern s a l a l - l i c h e n salal moss swardfern site site site site site site site site exposed shaded exposed shaded exposed shaded exposed shaded exposed shaded exposed shaded exposed shaded exposed shaded spot spot spot spot spot spot spot spot spot spot spot spot spot spot spot spot F i g . 1 2 . Diagram illustrating the arrangement of seed-spots within site types on unburned and burned areas in the N a n a i m o River valley. c+ O K> O H H O Z Ti CW CD ro CD 2 9 R e s u l t s a) M y c o r r h i z a f o r m a t i o n Only 24 s e e d l i n g s (out of a t o t a l of 620 examined) r e p r e s e n t i n g f o u r seed-spots were l a c k i n g mycorrhiza. A l l the r e s t showed some m y c o r r h i z a l development, although i t v a r i e d c o n s i d e r a b l y i n c h a r a c t e r . A l l of the types d e s c r i b e d i n the s e c t i o n on morphology were p r e s e n t . I t i s i n t e r e s t i n g to note t h a t mycorrhiza were so common and so well - d e v e l o p e d i n one-year o l d s e e d l i n g s . F i g u r e 13 shows one r o o t system i l l u s t r a t i n g the extent of m y c o r r h i z a l development. M y c o r r h i z a o c c u r r e d i n a l l s i t e s and under both c o n d i t i o n s of burning and exposure. M y c o r r h i z a o c c u r r e d i n a l l seed spots. b) Height growth Because mycorrhiza are b e l i e v e d t o be b e n e f i c i a l , i t was thought t h a t the number of mycorrhiza might i n f l u e n c e the heig h t and r o o t volume of the s e e d l i n g . Analyses were made f o r a l l the s e e d l i n g s i n each p l o t to c o r r e l a t e the number of mycorrhiza w i t h h e i g h t , the number of mycorrhiza w i t h r o o t volume, and the height w i t h r o o t volume. The c o e f f i c i e n t s of c o r r e l a t i o n are g i v e n i n Tab l e 7» Only the a n a l y s i s of the number of mycorrhiza w i t h r o o t volume r e g u l a r l y showed a s i g n i f i c a n t c o r r e l a t i o n . In 18 of 27 p l o t s the c o r r e l a t i o n was s i g n i f i c a n t to at l e a s t the 5$ l e v e l . I t was a p o s i t i v e c o r r e -l a t i o n , i . e . , the number of mycorrhiza i n c r e a s e d as the r o o t volume i n c r e a s e d . 30 c) Root Volume There are s e v e r a l ways of i n t e r p r e t i n g the c o r r e l a t i o n of the number of mycorrhiza per s e e d l i n g w i t h the r o o t volume of the s e e d l i n g . I t may be a d i r e c t dependent r e l a t i o n s h i p i n which an i n c r e a s e i n the number of mycorrhiza enables the s e e d l i n g to compete more f a v o u r a b l y and, t h e r e f o r e , produce a l a r g e r r o o t system. However, i t seems more probable that the number of mycorrhiza formed depends on the a b i l i t y of the s e e d l i n g to produce a l a r g e r o o t system. I t may be simply t h a t the more short r o o t s t h e r e are, the more p o s s i b i l i t i e s there are f o r mycorrhiza. The a b i l i t y to produce a l a r g e r o o t system may depend on other f a c t o r s as s o i l moisture, s o i l a e r a t i o n , e t c . , which a r e , i n e f f e c t , l i m i t i n g f a c t o r s . I t would then be independent of the number of mycorrhiza. The l a t t e r i n t e r p r e t a t i o n would seem.more l i k e l y when d e a l i n g w i t h one-year-old s e e d l i n g s . I t i s some time before the r o o t system of a young s e e d l i n g i s developed to the stage of producing the short r o o t s which are s u s c e p t i b l e to m y c o r r h i z a l i n f e c t i o n . The time remaining i n one growing season would h a r d l y seem s u f f i c i e n t f o r the mycorrhiza to have a s i g n i f i c a n t e f f e c t on the r o o t development f o r t h a t y e a r . d) E f f e c t s of S i t e S t a t i s t i c a l analyses were c a r r i e d out using the mean number of mycorrhiza per s e e d l i n g to d e t e c t d i f f e r e n c e s between seed-spots which were a t t r i b u t a b l e to the e f f e c t s of s i t e , burning or l i g h t . to f o l l o w page 30 Table 7» Average measurements of s e e d l i n g s from seed-spots i n the Nanaimo R i v e r v a l l e y and c o e f f i c i e n t s of the c o r r e l a t i o n s of these measurements. S i t e P l o t No. of Ave.no.of Ave.root Ave. C o e f f i c i e n t of C o r r e l a t i o n No. seed- M y c o r r h i z a volume of height l i n g s per •• s e e d l i n g of seed- Mycorr- Mycorr- Root s e e d l i n g (cc.) l i n g (cm.) h i z a X r o o t volume h i z a X height volume X h e i g h t S a l a l - 67E 30 30.4Q .287 4.65 .553 # _ # .404 $ .199 l i c h e n 573 (un- 0 b u r n e d ) a 9 E 31 34.23 .219 4.16 . 5 1 3 ^ .251 .367 # 23 27.35 .254 3.21 .554## .051 .409 # 89s 20 13.50 .150 3.59 .387 .196 .352 S a l a l - 82E 28 22.18 l i c h e n 82S 30 34.84 (burned) 8 g E 2 Q 38.52 .274 .304 .267 4.26 4.22 3.03 ,436 # .321 434 # 101 294 .217 .446 * .334 S a l a l 16E 12 2.67 (un- 16S 25 13.64 burned)25E 29 13.79 25S 30 11.37 .126 .196 .261 .223 3.35 3.44 3.47 3.35 ' 7 4 8 J f , 5 8 5 ^ ,552## ,205 741 051 352 128 JIM-WW S a l a l 17E 27 21.22 .373 6.07 .478 # 'Churned) 17s 3 16.67 .343 4.33 -.802 68E 6 6.17 1.468 12.65 .615 68S 28 23.61 .248 3.47 .644## 41E 26 40.96 .281 3.05 .783## 41S 31 30.87 .241 3.33 .528## 40S 23 39.43 .216 4.18 .409 046 812 030 145 424 # 282 214 . 8 4 8 ^ .023 . 6 3 9 ^ .295 .999 # .730 .099 .538## .260 .521 Moss X8E Cun- 18s burnedj 28 9 20.39 8.22 .187 .057 3.59 3.84 .571 .838 241 687 # .533 .419 Moss 20E 8m 30.50 (burned)20S 24 18.50 .349 •_283, 3.95 4.46 .780 #  .586 # 467 236 •.691 _____ Sword-f e r n 14E (un 1 4 S  burned)  28 28 23.64 18.93 .406 .301 4.67 4.54 .272 .233 330 292 .452 # .374 # Sword- 13E 29 35.86 f e r n 13s 12 14.25 (burned) 35S 23 43.87 .370 .336 .439 3.89 3.97 3.89 .617## .170 .463# 039 002 558## .274 • .119 .425 # # S i g n i f i c a n t ## S i g n i f i c a n t to the 5$ l e v e l to the l i l e v e l 31 The d i f f e r e n c e s between s e e d l i n g s , and between r e p l i c a t i o n s , w i t h i n any one s i t e were so great that c o n s i s t e n t v a r i a t i o n s between s i t e s were impossible to d e t e c t . T h i s agrees w i t h Linnemann (18) who found i t remarkable t h a t D o u g l a s - f i r of the same age growing immediately s i d e by s i d e could vary so g r e a t l y i n t h e i r r o o t f o r m a t i o n , e s p e c i a l l y w i t h regard to t h e i r s h o r t r o o t s . She noted that t h i s v a r i a b i l i t y was present i n very young p l a n t s as w e l l as i n o l d e r t r e e s . She was unable to r e c o g n i z e any c o n s i s t e n t c o r r e l a t i o n of mycorrhiza w i t h s i t e c o n d i t i o n s . One of the reasons f o r the l a c k of a c o r r e l a t i o n i n the present study may have been the use of a s i t e c l a s s i f i c a t i o n that was too broad. When a s m a l l c o n c e n t r a t e d sample i s used, the s i t e must be analysed down to the micro-environment of the area i n v/hich the s e e d l i n g s are growing. The only a l t e r n a t i v e i s to use a l a r g e number of r e p l i c a t e s w i t h i n a s i t e t o compensate f o r v a r i a t i o n w i t h i n the s i t e . e) E f f e c t s of Burning W i t h i n any one s i t e t here was a s i g n i f i c a n t l y g r e a t e r average number of mycorrhiza per s e e d l i n g on p l o t s which had been burned as compared to p l o t s which had not been burned. Ta b l e 8 shows the average number of mycorrhiza per s e e d l i n g i n the burned and unburned p l o t s w i t h i n each s i t e . I t should be s t r e s s e d t h a t very few s e e d l i n g s were non-mycorrhizal. The f a c t o r being analysed i s the number of mycorrhiza per s e e d l i n g r a t h e r than the number of m y c o r r h i z a l s e e d l i n g s compared to t o f o l l o w page 31 Table 8. Average number of mycorrhiza per s e e d l i n g i n the burned and unburned p l o t s w i t h i n each s i t e i n the Nanaimo R i v e r v a l l e y . ^ SITE UNBURNED BURNED S a l a l - L i c h e n 32.32 20.43 28.51 S a l a l 8.16 12.58 14.89 18.93 35.92 Moss 14.31 24.50 Swordfern 21.29 25.06 # A t t e s t showed a d i f f e r e n c e between the unburned and burned p l o t s which i s s i g n i f i c a n t to the 1% l e v e l . 32 the number of non-mycorrhizal s e e d l i n g s . T h i s i s emphasized because Wright and T a r r a n t (36) found t h a t the number of m y c o r r h i z a l D o u g l a s - f i r s e e d l i n g s i n t h e i r study areas i n the P a c i f i c Northwest was r e l a t e d t o whether or not the s o i l had burned. There were more m y c o r r h i z a l s e e d l i n g s on the unburned areas. However, they note t h a t the abundance of mycorrhiza, as determined s u b j e c t i v e l y , was not a s s o c i a t e d w i t h the degree of burning. There are s e v e r a l p o s s i b l e e x p l a n a t i o n s f o r the occurrence of more mycorrhiza i n the burned than i n the unburned areas. One i s t h a t m y c o r r h i z a l f u n g i may be able to reinvade a burned area more e a s i l y than the l i t t e r - d e s t r o y i n g f u n g i because they are not dependent on the humus as the only source of carbohydrates. A f t e r a burn i t may be some time b e f o r e the humus i s developed enough to have i t s normal complement of saprophytes. The m y c o r r h i z a l f u n g i would t h e r e f o r e be i n a more f a v o u r a b l e c o m p e t i t i v e p o s i t i o n i n a burned area than they would be i n an unburned area. T h i s i s s p e c u l a t i o n , but i t i s supported by the f i n d i n g s of Wright and T a r r a n t (35) i n t h e i r study of the numbers of s o i l mirroorganisms a f t e r l o g g i n g and s l a s h - b u r n i n g . They found t h a t i n the upper 1-|- inches of s o i l there were twice as many f u n g i i n unburned s o i l as t h e r e were i n l i g h t l y or s e v e r e l y burned s o i l . At a depth of 3 inches the s e v e r e l y burned s o i l s t i l l had only h a l f as many f u n g i as the unburned or l i g h t l y burned s o i l . Another p o s s i b l e e x p l a n a t i o n i s that i f there i s a 33 decrease i n n u t r i e n t l e v e l s i n the burned areas, mycorrhiza could be expected to be more p l e n t i f u l than i n unburned areas where the n u t r i e n t l e v e l i s h i g h e r . S e v e r a l workers, i n c l u d i n g Hatch (10) and Bjorkman ( 3 ) , note that the abundance of mycorrhiza v a r i e s i n v e r s e l y w i t h s o i l f e r t i l i t y , e s p e c i a l l y w i t h the amounts of n i t r o g e n , phosphorus and potassium a v a i l a b l e . T h i s i s an aspect of the e f f e c t s of burning which bears f u r t h e r i n v e s t i g a t i o n . f ) E f f e c t s of L i g h t The a n a l y s i s of the e f f e c t of l i g h t using p a i r e d seed-spots (exposed and shaded) w i t h i n a s i t e i n d i c a t e d t hat there was no s i g n i f i c a n t d i f f e r e n c e i n the number of my c o r r h i z a per s e e d l i n g a t t r i b u t a b l e to l i g h t . In t h i s study there was no measurement of the percent r a d i a t i o n to g i v e accurate estimates of the amount of l i g h t . Bjorkman (2) found t h a t the m y c o r r h i z a l development i n a l l the s o i l s he used was l a r g e l y dependent upon the i n t e n s i t y of l i g h t up to a maximum at 49$ r a d i a t i o n . The i n f o r m a t i o n a v a i l a b l e on the l i g h t i n t e n s i t y i n the seed-spots i n the present study i s not d e t a i l e d enough to draw c o n c l u s i o n s about the e f f e c t of l i g h t on the abundance of mycorrhiza on D o u g l a s - f i r s e e d l i n g s . S e e d l i n g s i n Other C o a s t a l Areas The mycorrhiza of s e e d l i n g s growing under n a t u r a l c o n d i t i o n s were examined to give an i n d i c a t i o n of the development th a t could be expected i n young s e e d l i n g s , and the v a r i a t i o n i n 34 the mycorrhiza according to s i t e . Samples were taken from good s i t e s and from poor s i t e s . U n i v e r s i t y F o r e s t Area T h i r t y - t w o s e e d l i n g s were c o l l e c t e d from the U n i v e r s i t y of B r i t i s h Columbia f o r e s t at Haney, B.C., d u r i n g the e a r l y summer of 1957. They were c o l l e c t e d under a mixed D o u g l a s - f i r -Western hemlock-Western red cedar stand. None of the s e e d l i n g s was more than f o u r months o l d . Twenty-two were m y c o r r h i z a l and ten were d o u b t f u l or without mycorrhiza. The youngest s e e d l i n g examined, about three months o l d , showed c h a r a c t e r i s t i c mycorrhiza, w i t h prominent fungus mangles ( F i g u r e 14). T h i s i s the e a r l i e s t f o r m a t i o n of mycorrhiza r e c o r d e d i n D o u g l a s - f i r s e e d l i n g s . A previous r e p o r t (Linnemann (18)) i n d i c a t e d t hat f o r m a t i o n of mycorrhiza d u r i n g the f i r s t growing season was d o u b t f u l . In the course of the present i n v e s t i g a t i o n i t became apparent t h a t i n the c o a s t a l r e g i o n of B r i t i s h Columbia, D o u g l a s - f i r s e e d l i n g s commonly form well-developed e c t o t r o p h i c mycorrhiza dur i n g the f i r s t growing season. Only a v e r y few of the l a r g e number of one-year-old s e e d l i n g s examined showed no mycorrhiza. Powell R i v e r Area In October, 1957? 10 one-year-old and 2 two-year-old s e e d l i n g s were c o l l e c t e d from a good s i t e on Powell R i v e r Co. la n d near S t i l l w a t e r , B.C. A l l twelve s e e d l i n g s showed w e l l -developed mycorrhiza. The most common form was the dark f u z z y type, although there were a few of the white f u z z y type p r e s e n t . Most of the mycorrhiza were unbranched, but some were b e g i n n i n g to show pinnate branching, w i t h a c r o s s - l i k e appearance ( F i g u r e 15). Gu l f I s l a n d s Area A number of s e e d l i n g s c o l l e c t e d by Powell R i v e r Co. f o r e s t e r s from the Gulf I s l a n d s i n October, 1958, were examined. A l l the areas sampled were c o n s i d e r e d to be poor D o u g l a s - f i r s i t e s . The s e e d l i n g s v a r i e d from one to s e v e r a l years i n age. The r o o t systems were not completely removed from the s o i l , but a l l the s e e d l i n g s showed some m y c o r r h i z a l development. The most common type of mycorrhiza encountered was the b l a c k r a d i a t i n g one, w i t h some of the white f u z z y type a l s o p r e s e n t . 36 CONCLUSIONS S e v e r a l p o i n t s are r a i s e d from the experiments and obs e r v a t i o n s made i n t h i s study. The f i r s t i s t h a t i n order to o b t a i n c o n c l u s i v e r e s u l t s on the e f f e c t s of mycorrhiza on D o u g l a s - f i r s e e d l i n g s , long-term t e s t i n g i n an area l a c k i n g i n mycorrhiza i s necessary. E x p e r i m e n t a t i o n i n an area on a poor s i t e i n n o n - f o r e s t land would accentuate d i f f e r e n c e s a t t r i b u t a b l e to m y c o r r h i z a l development. While d i f f e r e n c e s were s l i g h t i n the one- and two-year-old s e e d l i n g s examined i n t h i s study, d i f f e r e n c e s may become g r e a t e r a f t e r s e v e r a l years to the p o i n t where they become s i g n i f i c a n t . E f f e c t s which may be important over an extended p e r i o d of time may be undetectable d u r i n g the f i r s t or second growing season. Another aspect which bears i n v e s t i g a t i n g i s the p o s s i b i l i t y of d i f f e r e n t i a l e f f e c t s of the v a r i o u s types of mycorrhiza. In D o u g l a s - f i r there are s e v e r a l types of mycorrhiza, some of which may be more e f f e c t i v e than o t h e r s . Levisohn (15) found that Amanita muscaria and Boletus scaber on young b i r c h and Norway spruce produced unequal e f f e c t s , which were a s s o c i a t e d w i t h the s o i l c o n d i t i o n s under which the p l a n t s were growing. She s t r e s s e s the need f o r c a u t i o n i n s e l e c t i n g m y c o r r h i z a l f u n g i f o r i n o c u l a t i n g purposes and i n choosing n u r s e r y stock f o r p l a n t i n g . An e s s e n t i a l p r e r e q u i s i t e to experimentation of the type suggested i s the development of a s u c c e s s f u l technique f o r i n d u c i n g the for m a t i o n of mycorrhiza on D o u g l a s - f i r 37 s e e d l i n g s i n pure c u l t u r e . U n t i l more knowledge of the fungus p a r t n e r s i s a v a i l a b l e , experimentation can be o n l y e m p i r i c a l . T h i s phase of the work should be one of the primary o b j e c t i v e s f o r f u t u r e experimentation. I f f u r t h e r i n v e s t i g a t i o n of the v a r i a t i o n of mycorr-h i z a a s s o c i a t e d w i t h s i t e c o n d i t i o n s i s c o n s i d e r e d d e s i r a b l e , care must be taken i n choosing an experimental d e s i g n which i s s u i t a b l e f o r the study. There should be s u f f i c i e n t r e p l i c a t i o n to compensate f o r w i t h i n - s i t e h e t e r o g e n e i t y . The s i t e l o c a t i o n s must be analysed to the micro-environment when d e a l i n g w i t h young s e e d l i n g s . I t i s f e l t t h a t work on the s i t e r e l a t i o n s h i p s of mycorrhiza should be accompanied by s i n g l e - f a c t o r experiments, i n order to gi v e a b a s i c explan-a t i o n f o r d i f f e r e n c e s which may be encountered between s i t e s . In c o n c l u s i o n i t should be noted t h a t a l l the seed-l i n g s s t u d i e d were c o a s t a l D o u g l a s - f i r . No attempt was made to sample the whole range of D o u g l a s - f i r . Some of the s p e c i f i c i n f o r m a t i o n gained may, t h e r e f o r e , be a p p l i c a b l e only to the p a r t i c u l a r areas sampled. However, mycorrhiza were present i n a l l the s i t e s and c o n d i t i o n s examined, i n d i c a t i n g t h a t probably there i s a wide-spread occurrence of mycorrhiza i n young D o u g l a s - f i r s e e d l i n g s . I f t h i s i s so, the a p p l i c a t i o n of work on mycorrhiza i n D o u g l a s - f i r i n the c o a s t a l r e g i o n s of B r i t i s h Columbia may be much l e s s important than i n n o n - f o r e s t r e g i o n s or i n f o r e s t r e g i o n s where D o u g l a s - f i r has not p r e v i o u s l y been grown. 38 SUMMARY 1. A nu r s e r y experiment and a potted experiment were conducted at the U n i v e r s i t y of B r i t i s h Columbia to determine i f the presence of m y c o r r h i z a l a s s o c i a t i o n s a f f e c t e d the growth of D o u g l a s - f i r s e e d l i n g s . Seed was sown i n June, 1957, and the s e e d l i n g s were dug i n l a t e J u l y , 1958. Treatments i n c l u d e d the use of s t e r i l i z e d and u n s t e r i l i z e d humus and of pure c u l t u r e s of Amanita muscaria and Bol e t u s sp. A l l the s e e d l i n g s were m y c o r r h i z a l , because of the presence of an unknown m y c o r r h i z a l fungus (or f u n g i ) i n the nursery s o i l . There was no s i g n i f i c a n t d i f f e r e n c e between the treatments as i n d i c a t e d by the h e i g h t , the r o o t volume or the number of mycorrhiza of the s e e d l i n g s . 2. Attempts were made to induce the fo r m a t i o n of mycorrhiza on D o u g l a s - f i r s e e d l i n g s using pure c u l t u r e techniques. S e v e r a l methods were t r i e d . The f u n g i used were Amanita muscaria. Boletus sp. and Mutinus sp. ( r a v e n e l l i i ? ) . A l l attempts were u n s u c c e s s f u l and no mycorrhiza were formed. 3. Only e c t o t r o p h i c mycorrhiza, showing t y p i c a l mantles and H a r t i g n e t s , were found i n D o u g l a s - f i r s e e d l i n g s . The v a r i o u s types of branching ahd of mantle f o r m a t i o n of the mycorrhiza are d e s c r i b e d and i l l u s t r a t e d . 4. M y c o r r h i z a were found .-. to occur i n D o u g l a s - f i r s e e d l i n g s i n a range of s i t e s . The abundance of the mycorrhiza c o u l d not be c o r r e l a t e d w i t h v a r i a t i o n i n s i t e . 5« Well-developed e c t o t r o p h i c mycorrhiza were found i n n a t u r a l s e e d l i n g s as e a r l y as three months a f t e r seed germination. Most of the s e e d l i n g s examined showed w e l l -developed mycorrhiza by the end of the f i r s t growing season. 6. D o u g l a s - f i r s e e d l i n g s grown e x p e r i m e n t a l l y i n the Nanaimo R i v e r v a l l e y of B.C. by a graduate student i n the Department of Botany, U.B.C., were examined f o r mycorrhiza. S t a t i s t i c a l a n a l y s i s showed a p o s i t i v e c o r r e l a t i o n of the number of mycorrhiza per s e e d l i n g w i t h the r o o t volume of the s e e d l i n g . There was no c o r r e l a t i o n of the number of mycorrhiza w i t h the h e i g h t of the s e e d l i n g nor of the h e i g h t w i t h the r o o t volume. There were no s i g n i f i c a n t d i f f e r e n c e s between the samples of s e e d l i n g s which were a t t r i b u t a b l e to the e f f e c t s of s i t e or l i g h t . The s e e d l i n g s on burned p l o t s had a s i g n i f i c a n t l y l a r g e r number of mycorrhiza than the s e e d l i n g s on unburned p l o t s . 40 APPENDIX I - CULTURE MEDIA 1. N u t r i e n t agar glucose 10 g NH4 t a r t r a t e 1 g KH2PO4 1 g MgSO4.7H.2O 0 . 5 g f e r r i c i t r a t e 5 mg ZnS04.7H20 4.4 mg MnS04.4H20 5 mg CaCl2 55.5 mg V i t a m i n B l 40 / agar 15 g d i s t i l l e d water 1000 ml (formula obtained from Dr. V. S l a n k i s , l e t t e r to Dr. J.E. B i e r , 23 October, 1956.) 2. N u t r i e n t s o l u t i o n used w i t h v e r m i c u l i t e K 2HP0 4 0 . 5 g C a C l 2 0 .05 g NaCl 0.025 g MgS0 4.7H 2 0 0 .15 g (NH 4) 2HP0 4 0 .25 g f e r r i c i t r a t e (1$ s o l u t i o n ) 1 .2 ml glucose 2.5 g th i a m i n 15 Y d i s t i l l e d water 1000 ml (formula obtained from M e l i n , E., and N i l s s o n , H., "T r a n s f e r of R a d i o a c t i v e Phosphorus to Pine S e e d l i n g s by Means of M y c o r r h i z a l Hyphae," P h y s i o l o g i a Plantarum, V o l . 3 , 1950, p.89.) 41 APPENDIX I I - SUMMARY TABLES OF ANALYSES OF VARIANCE 1. Data from the nursery experiment ( s p l i t - p l o t design) a) A n a l y s i s of v a r i a n c e of number of mycorrhiza per s e e d l i n g . Source of v a r i a t i o n d.f. Sum of Mean C a l c . Tabled F at Squares Square F. P = .05 Whole-plot t o t a l 14 6,927,563 494,826 1.516 3.23 Blocks 4 3,377,783 938,687 844,446 2.587 3.84 Humus 2 469,344 326,387 1.437 4.46 E r r o r 8 2,611,093 Sub-plot t o t a l 30 10,033,016 334,434 1.043 1.94 Fungus 2 1,038,762 519,381 1.620 3.40 FxH i n t e r a c t i o n 4 1,303,881 7,690,373 325,970 1.017 2.78 E r r o r 24 320,432 T o t a l 44 16,960,579 b) A n a l y s i s of v a r i a n c e of average h e i g h t of s e e d l i n g s Source of v a r i a t i o n d.f. Sum of Mean C a l c . Tabled F at Squares Square F. P - .05 Whole-plot t o t a l 14 10,157.37 725.53 0.951 3.23 Blocks 4 1,341.72 335.43 0.439 3.84 Humus 2 2,711.84 1,355.92 1.777 4.46 E r r o r 8 6,103.81 762.98 Sub-plot t o t a l 30 13,402.52 446.75 1.061 1.94 Fungus 2 36.13 18.06 0.043 3.40 FxH i n t e r a c t i o n 4 3,257.11 814.28 1.933 2.78 E r r o r 24 10,109.28 421.22 T o t a l 44 23,559.89 c) A n a l y s i s of va r i a n c e of average r o o t volume of s e e d l i n g s Source of v a r i a t i o n d . f . Sum of Mean C a l c . T a b l e d F at Squares Square F. p 9 .05 Whole-plot t o t a l 14 1,323.11 94.51 1.588 3.23 Blocks 4 547.60 136.90 2.300 3.84 Humus 2 299.40 149.70 2.515 4.46 E r r o r 8 476.1- 59.51 Sub-plot t o t a l 30 1,037.88 345.96 1.393 1.94 Fungus 2 22.19 11.09 0.045 3.40 FxH i n t e r a c t i o n 4 419.64 104.91 0.422 2.78 E r r o r 24 596.05 248.35 T o t a l 44 2,360.99 42 APPENDIX I I (Cont.) 2. Data from the pot experiment (randomized b l o c k design) a) A n a l y s i s of v a r i a n c e of he i g h t of s e e d l i n g s Source of v a r i a t i o n d . f . Sum of Squares Mean Square C a l c . F T a b l e d F at p = .05 Treatments R e p l i c a t i o n s E r r o r T o t a l 8 110,394 4 58,223 32 1,422,764 45 1,591,380 13,799.2 14,555.7 44,461 .4 0.310 0.327 3.08 5.74 b) A n a l y s i s of v a r i a n c e of r o o t volume of s e e d l i n g s Source of v a r i a t i o n d.f. Sum of Squares Mean Square C a l c . F T a b l e d F at p m .05 Treatments R e p l i c a t i o n s E r r o r T o t a l 8 1,666.4 4 801.6 32 242 ,254.0 45 244,722 208.31 200.39 7,570.44 0.028 0.026 3.08 5.74 43 LITERATURE CITED 1. B i r c h , T.T.C. "A Synopsis of Forest Fungi of S i g n i f i c a n c e i n New Zealand." New Zealand J o u r n a l of F o r e s t r y , V o l . 4 , No. 1, 193&7 PP- 109-125. 2. Bjorkman, E. 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" A r t i f i c i a l I n o c u l a t i o n of M y c o r r h i z a l F u n g i . " Archlvum S o c i e t a t i s Z o o l o g i c a l B o t a n i c a l Fennicae 'Vanamo', V o l . 9: s u p p l . , 1955, pp. 197-201. 2 9 . Rawlings, G.B. "The Mycorrhizas of Trees i n New Zealand F o r e s t s . " New Zealand F o r e s t S e r v i c e , F o r e s t Research I n s t i t u t e , F o r e s t Research Notes, V o l . 1, No. 3 , 1951, PP- 15-17. 3 0 . Rayner, M.C. "The M y c o r r h i z a of C o n i f e r s : A Review." J o u r n a l of Ecology, V o l . 22, No. 1, 1934, pp. 308-312, 31. S l a n k i s , V. "The Role of Auxin and Other Exudates In M y c o r r h i z a l Symbiosis of F o r e s t T r e e s . " i n Thimann, K.V., ed., The P h y s i o l o g y of F x e s t T r e e s , New York, The Ronald P r e s s , 1958, pp. 427-443. 32. S l i p p , A.W., and S n e l l , W.H. "Taxonomic-Ecologic S t u d i e s of the Boletaceae i n Northern Idaho and Adjacent Washington." L l o y d i a , V o l . 7, 1944, pp. 1-66. 46 33« Trappe, J.M. "Some Probable M y c o r r h i z a l A s s o c i a t i o n s i n the P a c i f i c Northwest." Northwest Sci e n c e , V o l . 31, No. 4, 1957, PP. iW^lWT 34. Wilde, S.A. " M y c o r r h i z a l F u n g i : T h e i r D i s t r i b u t i o n and E f f e c t on Tree Growth." S o i l S c i e n c e , V o l . 78, No. 1, 1954, pp. 2 3 - 3 1 . 35. Wright, E. and T a r r a n t , R.F. " M i c r o b i o l o g i c a l S o i l P r o p e r t i e s a f t e r Logging and S l a s h Burning." U.S.D.A., F o r e s t S e r v i c e , P a c i f i c Northwest F o r e s t and Range Experiment S t a t i o n , Research Note No. 157, 1957,. PP. 1-5. 3 6 . "Occurrence of Mycorrhizae A f t e r Logging and S l a s h Burning i n the D o u g l a s - F i r F o r e s t Type." U.S.D.A., F o r e s t S e r v i c e , P a c i f i c Northwest F o r e s t and Range Experiment S t a t i o n , Research Note No. 160, 1958, pp. 1-7. PLATE I P i g . 1 S e e d l i n g s growing i n narrow-mouth, aluminum f o i l c o v ered b o t t l e s w i t h v e r m i c u l i t e and a n u t r i e n t s o l u t i o n as t h e r o o t i n g medium., xj-. P i g . 2 M u t i n u s s'p. ( r a v e n ' - e l l i i ?) a s s o c i a t e d w i t h two-y e a r - o l d D o u g l a s - f i r s e e d l i n g s . X 1 / 3 • P L A T E I F i g . 2 kQ PLATE I I P i g . 3 S i m p l e , unbranched m y c o r r h i z a o c c u r r i n g mono-p o d i a l l y on a l a t e r a l , r o o t l e t „ The m y c o r r h i z a have t h e w h i t e f u z z y t y p e o f m a n t l e . X2. P i g . k P i n n a t e l y branched m y c o r r h i z a w i t h t h e opaque amber t y p e o f m a n t l e . Xi+. P i g . k i+9 PLATE I I I P i g . $ I r r e g u l a r l y branched m y c o r r h i z a w i t h t h e opaque amber t y p e of mantle„ Xl|. PLATE III F i g . 5 5 o PLATE IV P i g i 6a Transverse s e c t i o n of a m y c o r r h i z a showing the t h i c k , white f u z z y type of rnant l e i X125. P i g . 6b T r a n s v e r s e s e c t i o n of a m y c o r r h i z a showing the c h a r a c t e r of the white f u z z y type of mantle and i n t e r c e l l u l a r p e n e t r a t i o n of hyphae i n t o the c o r t e x . Xl+ 5 0 . P i g . 6b PLA.TE V P i g . 7 T r a n s v e r s e s e c t i o n of a m y c o r r h i z a showing the t h i h , opaque amber t y p e . o f mantle,, I n t e r c e l l u l a r p e n e t r a t i o n i s l i m i t e d t o the o u t e r l a y e r s of the c o r t e x . X600, P i g . 8 M y c o r r h i z a l root t i p with the b l a c k radi.s.ting type of mantle. Note the hyphae'extending out from the mantle. PLATE V F i g . 8 52 PLATE V I P i g . 9 T r a n s v e r s e s e c t i o n o f a m y c o r r h i z a showing t h e heavy, compact c h a r a c t e r o f t h e b l a c k r a d i a t i n g t y p e o f m a n t l e . I n t e r c e l l u l a r p e n e t r a t i o n Is l i m i t e d t o t h e o u t e r l a y e r s o f t h e c o r t e x . X600. P i g . 10 Root t i p w i t h m y c o r r h i z a l s h o r t r o o t s showing renewed n o n - m y c o r r h i z a l growth. The a r e a o f renewed growth has r o o t h a i r s , i s not s w o l l e n , and has no fungus m a n t l e . Xk. PLATE VI Pig.10 53 PLATE VII F i g . 11 Short root showing renewed m y c o r r h i z a l growth. The co'ntric t i o r i marks the p o i n t where the o r i g i n a l growth ceased. X20. F i g . 13 Root system of a one-year-old D o u g l a s - f i r s e e d l i n g from the Nanaimo R i v e r v a l l e y showing well-developed m y c o r r h i z a . X •§•. PLATE VII 5k PLATE V I I I P i g . 111. W e l l - d e v e l o p e d m y c o r r h i z a o c c u r r i n g on t h e r o o t system of a D o u g l a s - f i r s e e d l i n g about t h r e e months o l d . X P i g . 15 M y c o r r h i z a w i t h t h e dark f u z z y t y p e o f m a n t l e showing c r o s s - l i k e b r a n c h i n g . Xl(.. PLATE VI I I P i g . 15 

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