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Naturally infected root material as an inoculum source for Phellinus weirii (Murr.) Gilbertson Kellas, Jon Douglas 1979

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NATURALLY INFECTED ROOT MATERIAL AS AN INOCULUM SOURCE FOR PHELLINUS WEIRII (MURR.) GILBERTSON. by JOHN DOUGLAS KELLAS Dip. For. Creswick, 1969. B.Sc.For (Hons.), U n i v e r s i t y of Melbourne, 1975. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES i n the FACULTY OF FORESTRY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF A p r i l , © John Douglas BRITISH COLUMBIA 1979. K e l l a s , 1979. 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 a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e 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 a g r e e 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 p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . 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 n o t 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 . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 • 6 B P 7 5 - 5 1 1 E ( i i ) ABSTRACT: P h e l l i n u s w e i r l i (Murr.) G i l b e r t s o n i s an important root r o t of Douglas-f i r (Pseudotsuga m e n z i e s i i (Mirb.) Franco) i n western North America. The e f f e c t of s i t e and tree species on the growth of F_. w e i r i i along roots can be measured by i n o c u l a t i o n using n a t u r a l l y i n f e c t e d root m a t e r i a l or I>. w e i r i i c u l t u r e d on s t e r i l e wood. This t h e s i s r e p o r t s the development of an i n o c u l a -t i o n technique using n a t u r a l l y i n f e c t e d root m a t e r i a l to i n f e c t D o u g l a s - f i r and western hemlock (Tsuga h e t e r o p h y l l a (Raf.) Sarg.). I n t a c t n a t u r a l l y i n f e c t e d root s e c t i o n s of D o u g l a s - f i r used as an inocu-lum source r e s u l t e d i n JP. w e i r i i growing on approximately 87% of roots i n o c u l a t e d . Further i n o c u l a t i o n s were made using i n f e c t e d root s e c t i o n s s p l i t l o n g i t u d i n a l l y w i t h the exposed wood surface placed i n contact w i t h the host root. Subsequently an attempt was made to evaluate the i n f l u e n c e of x e r i c , submesic and h y g r i c s i t e s , w i t h i n the Demonstration area of the UBC Research For e s t , Maple Ridge, on i n o c u l a t i o n and growth of _P. w e i r i i along roots of D o u g l a s - f i r and western hemlock. Dry s o i l c o n d i t i o n s experienced during the summer of 1978 reduced the expected number of s u c c e s s f u l i n f e c t i o n s of host roots to approximately 20%, 26 weeks a f t e r i n s t a l l a t i o n . A l l i n o c u l a t i o n s of a t h i r d s e r i e s e s t a b l i s h e d a f t e r heavy r a i n s i n the l a t e summer i n f e c t e d the roots of both D o u g l a s - f i r and western hemlock, v e r i f y i n g that the technique was s u c c e s s f u l when c o n d i t i o n s were c o o l and moist. P_. w e i r i i inoculum used was c o l l e c t e d from two sources, Haney and Surrey. Laboratory s t u d i e s i n d i c a t e d i n c o m p a t i b i l i t y between the two sources when r a i s e d on agar media and f i e l d r e s u l t s i n d i c a t e d a longer r e t e n t i o n of v i a b l e P_. w e i r i i i n inoculum blocks from the Surrey source. ( i i i ) TABLE OF CONTENTS Page LIST OF TABLES i v LIST OF FIGURES v ACKNOWLEDGEMENTS v i INTRODUCTION 1 METHODS AND MATERIALS 6 F i e l d 6 T r i a l 1 6 T r i a l 2 7 T r i a l 3 10 Laboratory 10 C u l t u r a l d i f f e r e n c e s between the two sources of inoculum 10 Observation of zone l i n e s 11 RESULTS AND DISCUSSION 12 F i e l d 12 T r i a l 1 12 T r i a l 2 17 Inoculum source 19 Species i n o c u l a t e d 22 S i t e 24 T r i a l 3 26 Laboratory 27 Rate of ]?. w e i r i i growth on agar media 27 I n t e r a c t i o n between P_. w e i r i i sources on agar media . . 30 Zone l i n e s 32 CONCLUSIONS 35 BIBLIOGRAPHY 36 APPENDIX 1 38 APPENDIX 2 40 APPENDIX 3 41 ( i v ) LIST OF TABLES Page Table 1. Ecosystem u n i t s , predominant tre e cover and species i n o c u l a t e d w i t h P_. w e i r i i i n T r i a l 2, May 1978 8 Table 2. C o l o n i z a t i o n by P_. w e i r i i of roots of D o u g l a s - f i r (DF) and western hemlock (WH) nine weeks a f t e r i n o c u l a t i o n w i t h root s e c t i o n s of n a t u r a l l y i n f e c t e d m a t e r i a l , February to A p r i l , 1978 13 Table 3. Ecosystem u n i t , p l a n t a s s o c i a t i o n , s o i l type and average tre e age and height f o r each of the seven s i t e - s p e c i e s combinations used i n the second inocu-l a t i o n t r i a l l o c a t e d i n the UBC Research F o r e s t , Maple Ridge. 18 Table 4. Frequency of zone l i n e s , crustose mycelia, c o l o n i z a t i o n and n e c r o s i s at ten and twenty-six weeks f o l l o w i n g i n o c u l a t i o n of P_. w e i r i i on roots of D o u g l a s - f i r and western hemlock w i t h respect to inoculum source, UBC Research F o r e s t , May 1978 20 Table 5. Frequency of zone l i n e s , crustose mycelia, c o l o n i z a t i o n and n e c r o s i s at ten and twenty-six weeks f o l l o w i n g i n o c u l a t i o n of P_. w e i r i i on roots of D o u g l a s - f i r and western hemlock w i t h respect to species i n o c u l a t e d , UBC Research F o r e s t , May 1978 23 Table 6. Frequency of zone l i n e s , crustose m y c e l i a , c o l o n i z a t i o n and n e c r o s i s at ten and twenty-six weeks f o l l o w i n g i n o c u l a t i o n of I?, w e i r i i on roots of D o u g l a s - f i r and western hemlock w i t h respect to ecosystem u n i t used, UBC Research F o r e s t , May 1978 25 Table 7. L i n e a l growth of P_. w e i r i i from two sources r a i s e d on media c o n t a i n i n g 2% and 5% agar under c o n d i t i o n s of continuous l i g h t or dark 28 (v) LIST OF FIGURES Page Figure 1. Photographic and schematic r e p r e s e n t a t i o n of the development of P_. w e i r i i on a western hemlock root nine weeks a f t e r i n o c u l a t i o n w i t h a s e c t i o n of n a t u r a l l y i n f e c t e d D o u g l a s - f i r r o o t , (February-A p r i l , 1978) 14 Figure 2. Necrosis of a western hemlock root i n o c u l a t e d w i t h —' w e i r i i - 16 Figure 3. Comparison of a e r i a l hyphal c h a r a c t e r i s t i c s of J_. w e i r i i c o l l e c t e d from Haney and r a i s e d on 2% malt-agar f o r 16 days under c o n d i t i o n s of continuous l i g h t (upper p l a t e s ) or darkness (lower p l a t e s ) 29 Figure 4. Reaction between the advancing f r o n t of the Haney source ( l e f t of p l a t e s ) and the Surrey source ( r i g h t of p l a t e s ) of P_. w e i r i i grown on 5% agar-2% malt f o r 18 days 31 Figure 5. Reaction between the advancing f r o n t of the Haney source ( l e f t of p l a t e s ) and the Surrey source ( r i g h t of p l a t e s ) of P_. w e i r i i dark grown on 2% malt-agar f o r 18 days 33 ( v i ) ACKNOWLEDGEMENTS I wish to thank my su p e r v i s o r , Dr. B.J. van der Kamp, f o r the opportunity to study and observe f o r e s t pathology i n North America. With-out h i s guidance, patience and p e r s i s t e n c e t h i s t h e s i s would not have come to f r u i t i o n . Thanks are a l s o due to Dr. T.M. B a l l a r d and Dr. J.G. W o r r a l l f o r t h e i r p a r t i c i p a t i o n on my graduate committee. I g r a t e f u l l y acknowledge the i n c e n t i v e , f i n a n c i a l support and study leave o f f e r e d by my employer, the Forests Commission, V i c t o r i a , and the f i n a n c i a l support provided by the F a c u l t y of F o r e s t r y U.B.C. Without these generous c o n t r i b u t i o n s and support, my s t u d i e s could not have occurred. F i n a l l y , I wish to thank my w i f e , Heather, and f a m i l y , f o r t h e i r support throughout my stu d i e s and f o r t h e i r a d a p t a b i l i t y to new places and f r i e n d s . INTRODUCTION: P h e l l i n u s w e i r i i (Murr.) G i l b e r t s o n (Basidiomycetes, P o l y p o r a l e s ) , the yellow laminated root r o t , i s a f a c u l t a t i v e p a r a s i t e of s e v e r a l c o n i f e r species. P_. w e i r i i i s n a t i v e to the D o u g l a s - f i r (Pseudotsuga m e n z i e s i i (Mirb.) Franco) f o r e s t s of Oregon, Washington and B r i t i s h Columbia ( C h i l d s , 1963) and has a l s o been reported on western red cedar (Thuj a p l i c a t a Donn) i n Idaho and Alaska (Baxter, 1933). I t i s most d e s t r u c t i v e i n immature D o u g l a s - f i r stands. — m w e i r i i spreads from diseased to healthy roots p r i m a r i l y by contacts and g r a f t s . Although a r a r e occurrence, P_. w e i r i i can bridge diseased and healthy roots v i a other woody m a t e r i a l , roots of minor v e g e t a t i o n and bu r i e d rock s u r f a c e s . The fungus grows e c t o t r o p h i c a l l y on the outer bark surfaces of r o o t s . The e c t o t r o p h i c mycelia are u s u a l l y white to mauve, form a t h i c k sheath around the bark surface and may extend a few centimetres i n t o the surrounding s o i l . Mycelium seldom develops on root surfaces i n contact w i t h the organic l a y e r s or decaying l o g s . P e n e t r a t i o n and death of bark occurs some distance behind the advancing e c t o t r o p h i c f r o n t and the fungus invades the inner sapwood and outer h e a r t -wood. The advance of the fungus i n the outer heartwood i s accompanied by a red-brown s t a i n . In l a t e r stages of development the wood becomes p i t t e d and f i n a l l y laminated. Crown symptoms are o f t e n not observed u n t i l f i v e to ten years a f t e r —' w e i r i i has become e s t a b l i s h e d i n a root system. I n i t i a l l y there i s a red u c t i o n i n leader growth w i t h a subsequent t h i n n i n g and y e l l o w i n g of the f o l i a g e which i s o f t e n followed by wind throw p r i o r to death of the t r e e . Often disease symptoms are a l s o expressed by a d i s t r e s s crop of smaller - 2 -than normal cones. Pockets of P_. w e i r i i m o r t a l i t y are c h a r a c t e r i z e d by wind-thrown trees w i t h roots broken c l o s e to the root c o l l a r , producing root b a l l s . The annual m o r t a l i t y i n D o u g l a s - f i r stands i s estimated at one m i l l i o n cubic metres i n B r i t i s h Columbia ( W a l l i s , 1967) and 0.9 m i l l i o n cubic metres on the west s i d e of the Cascade Range i n Oregon and Washington (C h i l d s and Shea, 1967) which represents approximately 10% and 5% of annual increment r e s p e c t i v e l y . The r a t e of spread of P_. w e i r i i i n i n f e c t i o n centres has been estimated by two methods. One method i s to observe the spread of crown symptoms through time, the other i s to determine the r a t e of growth of mycelium along r o o t s . Nelson and Hartman (1975), us i n g a e r i a l photographs taken i n 1946 and 1972 of a mixed c o n i f e r f o r e s t east of Oakridge, Oregon i n the Cascade Range, studied ten i n f e c t i o n centres common on both s e r i e s of photographs. The r a t e of r a d i a l extension of the i n f e c t i o n centres v a r i e d from 12 to 58 centimetres per year w i t h the annual mean and standard d e v i a t i o n being 34 + 15 centimetres. I n o c u l a t i o n s t u d i e s w i t h _P. w e i r i i represent a method of observing the ra t e of spread on r o o t s . I n o c u l a t i o n s t u d i e s a l l o w a d e t a i l e d comparison of s e v e r a l f a c t o r s that may i n f l u e n c e fungal growth. These f a c t o r s i n c l u d e d i r e c t observation of spread and host r e a c t i o n as determined by t r e e age, species and e c o l o g i c a l s i t e r e s u l t i n g from root i n o c u l a t i o n w i t h one or more sources of _P. w e i r i i . E a r l y attempts to develop techniques f o r i n o c u l a t i n g healthy trees w i t h P_. w e i r i i met w i t h l i m i t e d success. I n o c u l a t i o n of D o u g l a s - f i r using wood chips or agar permeated w i t h P_. w e i r i i f a i l e d to produce i n f e c t i o n . When blocks permeated w i t h P_. w e i r i i were placed i n contact w i t h p r e v i o u s l y - 3 -damaged roots only a low percentage of roots became i n f e c t e d (Buckland &t_ a l . , 1954). Attempts by W a l l i s (1961) to i n f e c t Scots pine (Pinus s y l v e s t r i s L.) w i t h Fomes annosus (Fr.) Cke. using n a t u r a l l y I n f e c t e d root m a t e r i a l r e s u l t e d i n low l e v e l s of i n f e c t i o n of host r o o t s . However, s t e r i l e beech (Fagus  s y l v a t i c a L.) branch s e c t i o n s i n o c u l a t e d w i t h F_. annosus and placed i n contact w i t h healthy pine roots proved a s a t i s f a c t o r y i n o c u l a t i o n technique. This technique was l a t e r adapted f o r i n o c u l a t i o n of D o u g l a s - f i r w i t h P_. w e i r i i ( W a l l i s and Reynolds, 1962). Subsequent s t u d i e s have used s t e r i l e a l d e r (Alnus rubra Bong.) stem s e c t i o n s i n o c u l a t e d w i t h P_. w e i r i i as the inoculum source ( W a l l i s and Reynolds, 1965, W a l l i s , 1976a). From one of these s t u d i e s , W a l l i s and Reynolds (1962) reported average growth of mycelium away from i n o -culum blocks of deciduous hardwoods i n contact w i t h D o u g l a s - f i r r o o t s of 21.6 + 7.6 centimetres during a s i x month pe r i o d from A p r i l to October. M y c e l i a l growth over twelve months was not s i g n i f i c a n t l y d i f f e r e n t , averaging 18.8 + 4.6 centimetres. This suggests that f u n g a l . a c t i v i t y v i r t u a l l y ceases during the w i n t e r or that food reserves of the inoculum block become depleted or too d i s t a n t from the advancing m y c e l i a l f r o n t . Then i f inoculum block s i t e or d istance from the advancing f r o n t i n f l u e n c e the r e d u c t i o n of growth during the w i n t e r , then these f i g u r e s t e l l l i t t l e about the normal r a t e of spread along r o o t s . The only reported instance of the use of n a t u r a l l y i n f e c t e d m a t e r i a l as the inoculum source i s i n a mass i n o c u l a t i o n technique used f o r screening f o r r e s i s t a n c e of D o u g l a s - f i r seedlings to P_. w e i r i i ( W a l l i s and Reynolds, 1975). N a t u r a l l y i n f e c t e d stem s e c t i o n s w i t h l a t e i n c i p i e n t to early-advanced decay were placed i n c l o s e p r o x i m i t y to s e e d l i n g r o o t s . I n f e c t i o n of one and two year o l d seedlings occurred three months a f t e r establishment; m o r t a l i t y s t a r t e d a f t e r f i v e months. - 4 -This t h e s i s reports the development of a technique f o r i n o c u l a t i o n of healthy t r e e s w i t h P_. w e i r i i using n a t u r a l l y i n f e c t e d root m a t e r i a l . I n i t i a l l y , n a t u r a l l y i n f e c t e d i n t a c t root s e c t i o n s c o n t a i n i n g P_. w e i r i i were attached to healthy roots of D o u g l a s - f i r and western hemlock (Tsuga  h e t e r o p h y l l a (Raf.) Sarg.). Results suggested that high l e v e l s of i n f e c t i o n of host roots were p o s s i b l e and that i f such root s e c t i o n s were s p l i t and the i n f e c t e d root wood placed i n contact w i t h the healthy r o o t , then the pro-p o r t i o n of i n f e c t i o n s of the host roots would be greater than that achieved by using whole r o o t s . Having developed a promising i n o c u l a t i o n technique i t was decided to t e s t the e f f e c t of s i t e on the development of P_. w e i r i i . Three ecosystem u n i t types were chosen: a x e r i c , a submesic, and a h y g r i c s i t e . The major t r e e species were D o u g l a s - f i r and western hemlock. I t was p o s s i b l e to d e l i n e a t e seven s i t e - s p e c i e s combinations f o r comparison w i t h i n the Demon-s t r a t i o n area of the UBC Research F o r e s t , Maple Ridge. Subsequently 206 i n o c u l a t i o n s were made on 116 D o u g l a s - f i r and 90 western hemlock roots using inoculum c o l l e c t e d . f r o m w i t h i n the Research Forest and from an area of f o r e s t i n Surrey, B.C. Seventy i n o c u l a t i o n s were removed ten weeks a f t e r i n s t a l l a t i o n . The pr o p o r t i o n of s u c c e s s f u l i n o c u l a t i o n s was much lower than expected suggesting that the unusually dry weather c o n d i t i o n s had l i k e l y had an e f f e c t on the success of i n o c u l a t i o n , and f o l l o w i n g heavy r a i n s s h o r t l y a f t e r , a subsequent s e r i e s of i n o c u l a t i o n s were made to t e s t the i n f l u e n c e of the a l t e r e d s o i l moisture c o n d i t i o n s . Laboratory s t u d i e s included the comparison of the r a t e s of growth the two sources of inoculum on agar and some observations on zone l i n e s roots s e c t i o n s i n f e c t e d w i t h P. w e i r i i and i n excavated inoculum blocks - 6 -METHODS AND MATERIALS: FIELD: Three separate t r i a l s were e s t a b l i s h e d i n the UBC Research Forest, Maple Ridge, B.C. The t r i a l s were l o c a t e d w i t h i n the C o a s t a l Western Hemlock b i o g e o c l i m a t i c zone ( K l i n k a , 1976). T r i a l 1: The i n i t i a l i n o c u l a t i o n study was e s t a b l i s h e d i n a submesic ecosystem u n i t ( K l i n k a , 1976) i n and adjacent to a 22-year-old p l a n t a t i o n of Douglas-f i r . Inoculum was c o l l e c t e d from the roots of r e c e n t l y dead D o u g l a s - f i r trees l o c a t e d w i t h i n the p l a n t a t i o n . The presence of P_. w e i r i i i n the inoculum was confirmed by the presence of brown s e t a l hyphae seven to ten days a f t e r c u l t u r i n g of samples on 2% malt-agar. I n t a c t root s e c t i o n s 1.5 cm to 7 cm i n diameter were cut i n t o s e c t i o n s up to 30 cm i n length and cleaned of surface s o i l and loose bark. Twenty-two roots on a t o t a l of seven D o u g l a s - f i r trees were c a r e f u l l y excavated about one metre from the base of the t r e e , brushed v i g o r o u s l y to remove surface s o i l and loose bark t a k i n g care not to i n j u r e the root bark. A piece of i n f e c t e d root m a t e r i a l was bound onto the healthy root w i t h twine and the i n o c u l a t i o n was covered w i t h p l a s t i c sheeting to exclude s o i l and organic matter. The s o i l and/or organic matter was then replaced to i t s o r i g i n a l depth. Three western hemlock trees were s i m i l a r l y i n o c u l a t e d on a t o t a l of eight roots at a second submesic s i t e where western hemlock was i n t e r s p e r s e d w i t h D o u g l a s - f i r . A l l 30 i n o c u l a t i o n s were removed and inspected nine weeks a f t e r establishment. The presence and extent of P_. w e i r i i mycelium on the inoculum blocks and host r o o t s , n e c r o s i s of the host roots and the host root diameter were recorded. - 7 -T r i a l 2: The r e s u l t s of the f i r s t t r i a l i n d i c a t e d that n a t u r a l l y i n f e c t e d root m a t e r i a l was a favourable inoculum source f o r P_. w e i r i i , and that by modifying the technique, r e s u l t s could be improved. I t was apparent that when the i n f e c t e d root wood was exposed and placed i n contact w i t h the host r o o t , a more copious m y c e l i a l growth occurred c r e a t i n g a greater chance of c o l o n i z a t i o n of the host r o o t . To t e s t the e f f e c t of s i t e and species combinations on i n o c u l a t i o n , seven s i t e - s p e c i e s combinations were s e l e c t e d w i t h i n the Demonstration area of the UBC Research Forest. These are described i n Table 1. Root s e c t i o n s of D o u g l a s - f i r n a t u r a l l y i n f e c t e d w i t h P_. w e i r i i were c o l l e c t e d from two sources -for use as inoculum. One source of inoculum was from the Haney l o c a t i o n used i n T r i a l 1, and the other was from an area of D o u g l a s - f i r f o r e s t near the j u n c t i o n of Scott Road and Highway 10 i n Surrey, B.C., which i s i n the Coastal D o u g l a s - f i r B i o g e o c l i m a t i c zone ( K r a j i n a , 1965). Root s e c t i o n s s e l e c t e d f o r use showed early-advanced decay w i t h w e l l d i s t r i b u t e d decay pockets on 20-50% of cut surfaces. The inoculum blocks were prepared by c u t t i n g root s e c t i o n s i n t o s i x to nine centimetre lengths and then s p l i t t i n g them l o n g i t u d i n a l l y . P a i r e d roots of f i f t e e n t r e e s from each s i t e - s p e c i e s combination were i n o c u l a t e d w i t h both sources of inoculum. Roots to be i n o c u l a t e d were c a r e f u l l y excavated about one metre from the root c o l l a r and brushed f r e e of s o i l and/or organic matter. Care was taken not to damage the l i v i n g bark. The wood of f r e s h l y prepared inoculum blocks was trimmed i n order to achieve c l o s e contact w i t h the root - 8 -to be i n o c u l a t e d and held i n place w i t h polypropylene rope. P l a s t i c sheet-in g to the width of the inoculum block covered the s i t e of i n o c u l a t i o n . The excavated s o i l and/or organic matter were replaced to t h e i r o r i g i n a l depth. A l l i n o c u l a t i o n s were completed i n the l a t t e r two weeks of May 1978. I t was only p o s s i b l e to i n o c u l a t e 13 s u i t a b l e D o u g l a s - f i r t r e e s growing on the predominantly western hemlock submesic s i t e . TABLE 1. Ecosystem u n i t s , predominant t r e e cover and species i n o c u l a t e d w i t h P. w e i r i i i n T r i a l 2, May 1978. S i t e 1 Predominant Tree Cover Species Inoculated X e r i c D o u g l a s - f i r D o u g l a s - f i r Submesic D o u g l a s - f i r D o u g l a s - f i r Submesic D o u g l a s - f i r Western hemlock Submesic Western hemlock D o u g l a s - f i r Submesic Western hemlock Western hemlock Hygric D o u g l a s - f i r / D o u g l a s - f i r / Western hemlock Western hemlock The s i t e s s e l e c t e d and t h e i r ecosystem u n i t were based on the c l a s s i f i c a t i o n by K l i n k a (1976). - 9 -The number of i n o c u l a t i o n s can be summarized thus: S i t e - s p e c i e s combinations 7 Inoculum source 2 Trees i n o c u l a t e d on each s i t e 15 210 Less those i n o c u l a t i o n s not p o s s i b l e 4 T o t a l number of i n o c u l a t i o n s 206 Ev a l u a t i o n of i n o c u l a t i o n s was made twice. I n i t i a l l y i n o c u l a t i o n s from f i v e trees on each s i t e - s p e c i e s combination were removed and inspected ten weeks a f t e r establishment. The balance of the i n o c u l a t i o n s were removed and inspected 26 weeks a f t e r establishment. For each i n o c u l a t i o n , the extent of P_. w e i r i i mycelium on the surface of the i n o c u l a t e d root and the presence and extent of root n e c r o s i s were recorded i n the f i e l d . The i n o c u l a t e d root was considered c o l o n i z e d i f P_. w e i r i i mycelium was present on the root surface. Roots showing n e c r o s i s were taken i n t o the l a b o r a t o r y and c u l t u r e s were made on 2% malt-agar from dead and d i s c o l o u r e d bark and wood to deter-mine the presence and l o c a t i o n of P_. w e i r i i . Inoculum blocks were a l s o examined i n the l a b o r a t o r y f o r the presence of v i s i b l e surface crustose mycelia, and s p l i t to observe the presence of zone l i n e s . To t e s t the s u r v i v a l of P_. w e i r i i i n each inoculum b l o c k , small pieces of each block were placed on 2% malt-agar and incubated at room temperature. P_. w e i r i i could be i d e n t i f i e d a f t e r seven to ten days m i c r o s c o p i c a l l y or by the presence of c h a r a c t e r i s t i c brown s e t a l hyphae. - 10 -T r i a l 3: The degree of s u c c e s s f u l i n o c u l a t i o n i n T r i a l 2 was much l e s s than i n T r i a l 1. Observations suggested that unusually dry weather c o n d i t i o n s had reduced the expected s u c c e s s f u l number of c o l o n i z a t i o n s of host r o o t s . To t e s t t h i s , a f u r t h e r 20 i n o c u l a t i o n s were e s t a b l i s h e d at the s i t e used i n T r i a l 1 a f t e r above average r a i n s f e l l i n August. Ten D o u g l a s - f i r and ten western hemlock roots were i n o c u l a t e d using only inoculum from the Haney source. The blocks and i n o c u l a t i o n s were pre-pared as i n T r i a l 2, and were removed a f t e r seven weeks. C o l o n i z a t i o n and ne c r o s i s of host roots were recorded. LABORATORY: C u l t u r a l D i f f e r e n c e s Between the Two Sources of Inoculum: The inoculum c o l l e c t e d from both l o c a t i o n s was from r e c e n t l y dead D o u g l a s - f i r trees from a s i n g l e i n f e c t i o n centre at each s i t e . These i n o c u l a were c h a r a c t e r i z e d by observing the r a t e of r a d i a l growth using 2% malt-2% agar and 2% malt-5% agar i n continuous l i g h t or darkness. The d i f f e r i n g amounts of agar i n the media were used to observe the e f f e c t of higher matrix p o t e n t i a l associated w i t h higher agar content on m y c e l i a l growth P_. w e i r i i n a t u r a l l y grows under dark c o n d i t i o n s and l i g h t and dark treatments were included to observe any d i f f e r e n c e s of m y c e l i a l growth on the malt-agar s u b s t r a t e s . Cores 4 mm i n diameter were taken from the advancing zone of c u l t u r e s of the two sources p r e v i o u s l y r a i s e d on 2% malt-agar and t r a n s f e r r e d to the edge of the f r e s h l y made media i n s t e r i l e p e t r i dishes. The treatments r e q u i r i n g continuous l i g h t were placed on a l a b o r a t o r y bench at room - 11 -temperature and I l l u m i n a t e d continuously by f l u o r e s c e n t c e i l i n g l i g h t s . Dark treatments were placed i n a cupboard f o r the du r a t i o n of the growth pe r i o d . The temperature of a l l treatments was near constant at room temperature (22° + 2°C). R a d i a l growth of s i x r e p l i c a t e s of each treatment was measured a f t e r 14 days. The slower growing treatments r a i s e d under continuous l i g h t were remeasured at 18 days. Four m i l l i m e t r e cores taken from the advancing f r o n t s of c u l t u r e s of each source were placed approximately three centimetres apart on a malt-agar p l a t e to observe i n t e r a c t i o n between the mycelia from the two sources. This study was r e p l i c a t e d on 2% malt-media w i t h e i t h e r 2% or 5% agar i n continuous l i g h t or dark. Observations were made p e r i o d i c a l l y . Observation of Zone L i n e s : Zone l i n e s were f r e q u e n t l y observed i n inoculum blocks from T r i a l 2. Selected blocks were b o i l e d i n water f o r one hour and allowed to c o o l . Thin s e c t i o n s of areas c o n t a i n i n g zone l i n e s were made using a razor blade or a s l i d i n g wedge microtome. Sections were s t a i n e d w i t h phloxene, observed and photographed using a L e i t z Orthoplan l a r g e - f i e l d l i g h t microscope. Other inoculum blocks were s p l i t l o n g i t u d i n a l l y and r a d i a l l y to expose f r e s h P_. w e i r i i i n f e c t e d wood and placed over a water bath i n a con-stant temperature cabinet (20° + 2°C) f o r s e v e r a l week. These c o n d i t i o n s approach 100% r e l a t i v e humidity, c o n d i t i o n s under which zone l i n e formation has been reported (Lopez-Real, 1975). P e r i o d i c observations were made of m y c e l i a l development on the wood surfaces of of zone - l i n e formation. - 12 -RESULTS AND DISCUSSION: FIELD: T r i a l 1: An i n o c u l a t i o n was considered s u c c e s s f u l when mycelia from the i n f e c t e d root s e c t i o n had extended on to and adhered to the host r o o t . In t h i s s i t u a t i o n , I considered that P_. w e i r i i had c o l o n i z e d the host root surface. Of the 30 i n o c u l a t i o n s made using n a t u r a l l y i n f e c t e d root p i e c e s , 26 c o l o n i z e d host r o o t s . N e c r o t i c bark under the mycelia was observed i n 15 instances. —• w e i r i i could o f t e n be i s o l a t e d from the n e c r o t i c bark, i n d i c a t i n g penetra-t i o n of the host. There was l i t t l e d i f f e r e n c e i n the p r o p o r t i o n of roots c o l o n i z e d or showing n e c r o s i s between western hemlock and D o u g l a s - f i r (Table 2). Hyphae developed from the i n f e c t e d root s e c t i o n s both from the bark and from the exposed wood at the ends of the s e c t i o n s . In many cases the hyphal growth was profuse, w i t h hyphae, extending 2 or 3 cm away from the contact w i t h the host root. The mycelia were o f t e n white i n colour at the advancing edge w i t h the o l d e r mycelia developing the c h a r a c t e r i s t i c brown coloured s e t a l hyphae. Figure 1 i l l u s t r a t e s the m y c e l i a l development on a western hemlock root. This type of m y c e l i a l development on host roots i s not t y p i c a l of the n a t u r a l i n f e c t i o n . In n a t u r a l i n f e c t i o n s the e c t o t r o p h i c mycelia are grey-white to l i g h t mauve i n colour and do not extend very f a r out from the root surface. The p r o f u s i o n of mycelia observed i n Figure 1 i s probably due to the f r e e space provided between the i n o c u l a t i o n and the covering p l a s t i c sheet. In subsequent t r i a l s the amount of p l a s t i c used was j u s t s u f f i c i e n t to cover the width of the inoculum block. Those i n o c u l a t i o n s where c o l o n i z a t i o n f a i l e d to occur g e n e r a l l y showed - 13 -TABLE 2. C o l o n i z a t i o n by P_. w e i r i i of roots of D o u g l a s - f i r (DF) and western hemlock (WH) nine weeks a f t e r i n o c u l a t i o n w i t h root s e c t i o n s of n a t u r a l l y i n f e c t e d m a t e r i a l , February to A p r i l , 1978. Species Number in o c u l a t e d wexrix on host roots N e c r o t i c bark at i n o c u l a t i o n DF WH 22 20 6 11 4 T o t a l 30 26 15 - 14 -F i g u r e l a . Fi g u r e l b . Fi g u r e 1. Photographic and schematic r e p r e s e n t a t i o n of the development of P_. w e i r i i on a western hemlock root nine weeks a f t e r i n o c u l a t i o n w i t h a s e c t i o n of n a t u r a l l y i n f e c t e d D o u g l a s - f i r r o o t , ( F e b r u a r y - A p r i l 1978). - 15 -that there was very l i t t l e contact between the inoculum block and the host root surface. In subsequent t r i a l s the inoculum used was s p l i t to expose the i n f e c t e d root wood and a l s o pared to f i t the host root. Table 2 shows that there was l i t t l e d i f f e r e n c e between c o l o n i z a t i o n of D o u g l a s - f i r and western hemlock r o o t s . Although the number of observa-t i o n s i s s m a l l , the r e s u l t i s i n t e r e s t i n g . M o r t a l i t y i n western hemlock due to P_. w e i r i i i n pure stands i s not s i g n i f i c a n t , b u t the disease develop-ment i n mixed stands of western hemlock and D o u g l a s - f i r i s more s e r i o u s . W a l l i s (1976b) reports that although e c t o t r o p h i c growth of P_. w e i r i i i s s i m i l a r on roots of both s p e c i e s , death of roots i s l e s s common i n western hemlock. Two roots of western hemlock tr e e s i n o c u l a t e d w i t h P_. w e i r i i developed a d i s t i n c t zone surrounding the n e c r o t i c bark t i s s u e . In Figure 2, the dark brown areas were n e c r o t i c regions from which P_. w e i r i i was i s o l a t e d . The narrow l i g h t e r coloured zone surrounding the n e c r o s i s , and the l i v i n g bark, were f r e e of P_. w e i r i i . Several anthocyanidin ( c y a n i d i n and p e l a r g o n i d i n ) and non-anthocyanidin pigments have been i s o l a t e d from secondary periderms formed i n western hemlock stem bark ( M u l l i c k , 1969, M u l l i c k and Jensen, 1973). The term necro-p h y l a c t i c periderm has been a p p l i e d to these secondary periderms. The ne c r o p h y l a c t i c periderm forms an impervious b a r r i e r between l i v i n g and a d j a -cent dead bark c e l l s as a n o n - s p e c i f i c response to agents causing i n j u r y , b i o t i c and a b i o t i c , and als o i n the absence of i n j u r y or disease ( M u l l i c k and Jensen, 1973, M u l l i c k , 1975). N e c r o p h y l a c t i c periderms have been observed on stems of many c o n i f e r s and deciduous hardwoods (Soo, 1977), and p r e l i m i n a r y observations have al s o i n d i c a t e d involvement of.host r e a c t i o n - 16 -Figure 2. Necrosis of a western hemlock root i n o c u l a t e d w i t h J?. w e i r i i . The fungus was i s o l a t e d from the n e c r o t i c regions (N). Note ( i ) the d i s t i n c t zone (S) surrounding the n e c r o s i s which d i d not c o n t a i n P_. w e i r i i and ( i i ) the darker pigmented boundary (P) between the s t e r i l e zone and the healthy bark (H). - 17 -i n c l u d i n g n e c r o p h y l a c t i c periderm formation i n the i n t e r a c t i o n between P. w e i r i i and D o u g l a s - f i r ( M u l l i c k , 1977). I suggest that the narrow s t e r i l e coloured zone surrounding the n e c r o t i c area i s p o s s i b l y a host h y p e r s e n s i t i v e r e a c t i o n to fungal i n v a s i o n and that the d i s t i n c t , darker l i n e surrounding t h i s zone could conceivably be a n e c r o p h y l a c t i c periderm. T r i a l 2: Species composition, coverage and the number of stems per hectare of the tree species f o r the x e r i c , submesic and h y g r i c s i t e are presented i n Appendix 1. Table 3 presents the ecosystem c l a s s i f i c a t i o n and s o i l types as described by K l i n k a (1976) and the average age and height of f i v e t r e e s at each s i t e - s p e c i e s combination. The t r e e age was computed from r i n g counts of increment borings at 1.3 metres w i t h c o r r e c t i o n f o r estimated e a r l y t r e e growth to reach that height. The c o r r e c t i o n s were obtained from the F o r e s t r y Handbook f o r B r i t i s h Columbia (Anon, 1975). The three month pe r i o d of May to J u l y was unusually dry. A t o t a l of 16.46 cm of r a i n f e l l , and when compared to the 19-year average of 27.31 cm, was the t h i r d d r i e s t f o r the period. Only 8.71 cm f e l l during the f i r s t ten weeks a f t e r i n o c u l a t i o n . The dry s o i l c o n d i t i o n s were r e f l e c t e d i n the behaviour of the i n o c u l a t i o n s removed at ten and twenty-s i x weeks. For the balance of the t r i a l p e riod a f u r t h e r 61.73 cm of r a i n f e l l . The p e r i o d May to November was a l s o the t h i r d d r i e s t f o r the 19 years that records have been kept at the UBC Research Forest. Seventy i n o c u l a t i o n s were removed and examined ten weeks a f t e r establishment. The i n o c u l a t i o n data f o r each s i t e - s p e c i e s combination are - 18 -TABLE 3. Ecosystem u n i t , p l a n t a s s o c i a t i o n , s o i l type and average t r e e age and height f o r each of the seven s i t e - s p e c i e s combinations used i n the second i n o c u l a t i o n t r i a l l o c a t e d i n the UBC Research F o r e s t , Maple Ridge. X e r i c S i t e : 1 2 Ecosystem u n i t : ' Gaultheria-WH-DF. Soil:''' sandy loam M i n i Humo-Ferric Podzol w i t h mor humus. 3 Average tre e age: DF 106. 3 Average dominant tre e height: 24 m. Submesic S i t e : Ecosystem u n i t : Moss-WH. S o i l : loamy sand M i n i Humo-Ferric Podzol w i t h mor humus. Predominantly western hemlock. Average t r e e age: WH 96: DF 96. Average dominant t r e e height: 37 m. Predominantly D o u g l a s - f i r . Average tre e age: DF 103: WH 97. Average dominant tre e height: 33 m. Hygric S i t e : Ecosystem u n i t : Rubus-Polystichum-WRC. S o i l : sandy loam "Gleyed M i n i Humo-Ferric Podzol w i t h m u l l humus. Average tre e age: DF 97: WH 91. Average dominant tre e height: 50 m. '"Ecosystem u n i t s and s o i l types from K l i n k a (1976). 2 A d e s c r i p t i o n of ve g e t a t i o n and stem numbers per hectare i s presented i n Appendix 1. 3 Average of the f i v e trees inspected at ten weeks a f t e r i n o c u l a t i o n . - 19 -presented i n Appendix 2, and are summarized f o r inoculum source, species i n o c u l a t e d and ecosystem u n i t i n Tables 4, 5, and 6. Using a r a t i o estima-t i o n technique f o r comparing proportions (Choi, 1978, p 101.), t e s t s of s i g n i f i c a n c e were computed. Twenty-six weeks a f t e r establishment, 136 i n o c u l a t i o n s were removed and inspected. Observations and r e s u l t s of i n o c u l a t i o n s f o r each s i t e -species combination are presented i n Appendix 3 and summarized i n Tables 4, 5, and 6. Inoculum Source: Results are summarized i n Table 4. (a) Ten weeks: Three d i s t i n c t d i f f e r e n c e s between the two inoculum sources were evident a f t e r ten weeks. A l l blocks from the Haney source developed a dark brown m y c e l i a l c r u s t on the exposed wood surfaces. Only 60% of blocks from the Surrey source showed the crustose mycelia. The second d i f f e r e n c e between the two sources was i n the p r o p o r t i o n of i n o c u l a t i o n s c o l o n i z i n g the host root surface. The p r o p o r t i o n f o r the Haney source was double that of the Surrey source. Zone l i n e s were common i n a l l b locks but there was a s i g n i f i c a n t l y lower p r o p o r t i o n of blocks from the Haney source c o n t a i n i n g v i a b l e P_. w e i r i i . (b) Twenty-six weeks: S i g n i f i c a n t d i f f e r e n c e s between the two inoculum sources were observed f o r the development of crustose mycelia and the presence of v i a b l e P_. w e i r i i i n the blocks examined. The crustose mycelia developed on the m a j o r i t y of the blocks from the Haney source, but were absent on TABLE 4. Frequency of zone l i n e s , crustose mycelia, c o l o n i z a t i o n and nec r o s i s at ten and twenty-six weeks f o l l o w i n g i n o c u l a t i o n . o f P_. w e i r i i on roots of D o u g l a s - f i r and western hemlock w i t h respect to inoculum source, UBC Research F o r e s t , May 1978. Number of inoculum blocks w i t h : C o l o n i z a t i o n of host roots N e c r o t i c bark 1 2 Zone Crustose V i a b l e m , V i a b l e inoculum at i n o c u l a t i o n , . , . -j T o t a l , , . lxnes mycelia inoculum blocks TEN WEEKS: Haney ( 3 5 ) 3 31a 4 (34) 34a (33) 19c 17a 9b 3 Surrey (35) 33a 21c (26) 25a 8b 7a 5 1 TWENTY-SIX WEEKS: Haney (68) 64a 55b 37c 14b 13a 7 Surrey (68) 67a 30c 55b 12b 12a 8 Crustose mycelia always a s s o c i a t e d w i t h zone l i n e s , except i n one instance. Blocks c o n t a i n i n g v i a b l e _P. w e i r i i a l s o contained zone l i n e s , except i n one instance. 'Number of observations unless r e s u l t s accompanied by a number i n brackets. Values followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l (Comparison of pr o p o r t i o n s , Choi, 1978, pp. 101). One inoculum block was not teste d f o r the presence of P_. w e i r i i . - 21 -more than h a l f the blocks from the Surrey source. 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 i n the frequency of zone l i n e s between the two sources, but s i g n i f i c a n t l y more blocks from the Surrey source contained v i a b l e P_. w e i r i i . A t h i r d d i f f e r e n c e between the sources noted at ten weeks was the p r o p o r t i o n of root c o l o n i z e d . No s i g n i f i c a n t d i f f e r e n c e was observed at twenty-six weeks. (c) Comparison of r e s u l t s between ten and twenty-six weeks. The comparison of v i a b i l i t y of inoculum blocks at ten and twenty-six weeks showed a s i g n i f i c a n t l y lower p r o p o r t i o n of blocks from the Surrey source contained v i a b l e P_. w e i r i i at the second observation w h i l e the p r o p o r t i o n of blocks c o n t a i n i n g v i a b l e V_. w e i r i i from the Haney source remained unchanged. However, note that at 26 weeks the percentage of v i a b l e blocks from the Surrey source was s t i l l s i g n i f i c a n t l y higher than that f o r the Haney source. Zone l i n e s r e a d i l y formed i n inoculum blocks from both sources. However, i t would appear that the e f f e c t i v e n e s s of the zone l i n e s to maintain v i a b i l i t y i s greater i n the Surrey blocks than i n the Haney source. I t i s p o s s i b l e that the Surrey source of P_. w e i r i i has a greater a b i l i t y to s u r v i v e under dry c o n d i t i o n s such as were encountered during T r i a l 2 than the Haney source. Average annual p r e c i p i t a t i o n near where the Surrey inoculum was c o l l e c t e d i s approximately 125 centimetres compared to the Haney average of approximately 225 centimetres. Heavy r a i n s i n the months of August and September d i d not enhance c o l o n i z a t i o n of host r o o t s . In f a c t the p r o p o r t i o n of c o l o n i z a t i o n s was s i g n i f i c a n t l y lower at twenty-six weeks than at ten weeks, p r i m a r i l y due to a s i g n i f i c a n t l y lower p r o p o r t i o n of c o l o n i z a t i o n s observed a s s o c i a t e d - 22 -wi t h blocks from the Haney source. There was no evidence on or i n any inoculum block that _P. w e i r i i had re-invaded the areas beyond a zone l i n e . In s e v e r a l cases a second zone l i n e had formed i n s i d e the f i r s t as the P_. w e i r i i r e t r e a t e d i n t o the centre of the blocks. Cultures made from blocks i n the l a b o r a t o r y f a i l e d to detect the presence of P_. w e i r i i beyond the innermost zone l i n e . I t should be noted that zone l i n e s very r a r e l y formed i n the wood under a surface that supported any crustose mycelia. In f a c t the two s t r u c t u r e s merged to form a continuous b a r r i e r . * Species Inoculated: Results are presented i n Table 5. (a) Ten weeks: Zone l i n e formation, inoculum block v i a b i l i t y and c o l o n i z a -t i o n of host roots were not s i g n i f i c a n t l y d i f f e r e n t between the two species i n o c u l a t e d , D o u g l a s - f i r and western hemlock. However, crustose mycelia were observed more f r e q u e n t l y on blocks a s s o c i a t e d w i t h western hemlock. The nature of r o o t i n g i n western hemlock i s such that most roots i n o c u l a t e d occurred i n the organic l a y e r s or near the soil/humus i n t e r f a c e and these l o c a t i o n s may i n i t i a l l y favour m y c e l i a l development of P_. w e i r i i , compared to the environment o f f e r e d i n the mineral s o i l l a y e r s where most of the i n o c u l a t i o n s were made on D o u g l a s - f i r . (b) Twenty-six weeks: No s i g n i f i c a n t d i f f e r e n c e s were observed between i n o c u l a t i o n s on D o u g l a s - f i r and western hemlock f o r block v i a b i l i t y , crustose m y c e l i a l development, zone l i n e formation of c o l o n i z a t i o n of host roots a f t e r twenty-s i x weeks. TABLE 5. Frequency of zone l i n e s , crustose mycelia, c o l o n i z a t i o n and nec r o s i s at ten and twenty-six weeks f o l l o w i n g i n o c u l a t i o n of J?. w e i r i i on roots of D o u g l a s - f i r and western hemlock w i t h respect to species i n o c u l a t e d , UBC Research Forest, May 1978. Number of inoculum blocks with: Zone C r u s t o s e x V i a b l e ^ l i n e s - mycelia inoculum C o l o n i z a t i o n of host roots T o t a l N e c r o t i c bark V i a b l e inoculum at i n o c u l a t i o n blocks TEN WEEKS: D o u g l a s - f i r Western Hemlock (30) ( 4 0 ) 3 35a 4 29a (39) 27b 28a (33) 23a (26) 21a 12ab 13ab 7c 9bc~ TWENTY-SIX WEEKS: Do u g l a s - f i r (76) 73a Western Hemlock (60) 58a 47b 38b 48a 44a 13a 13a 12ab 13a 1 Crustose mycelia always a s s o c i a t e d w i t h zone l i n e s , except i n one instance. Blocks c o n t a i n i n g v i a b l e P_. w e i r i i a l s o contained zone l i n e s , except i n one instance. ^Number of observations unless r e s u l t s accompanied by a number i n brackets. +Values followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l (Comparison of proportions, Choi, 1978, pp. 101). One inoculum block not teste d f o r the presence of P. w e i r i i . - 24 -(c) Comparison of r e s u l t s between ten and twenty-six weeks. The only c h a r a c t e r i s t i c that was s i g n i f i c a n t l y d i f f e r e n t when the two s e r i e s of observations were compared was the s i g n i f i c a n t r e d u c t i o n i n the p r o p o r t i o n of western hemlock roots c o l o n i z e d at twenty-six weeks compared to ten weeks. The m a j o r i t y of western hemlock roots i n o c u l a t e d i n T r i a l 2 were l o c a t e d i n the organic l a y e r or at the i n t e r f a c e between the organic l a y e r and the mineral s o i l s urface. This zone, at the mineral s o i l s u r f a c e , supports one of the most di v e r s e populations of l i v i n g organisms of a l l e c o l o g i c a l niches (Spurr and Barnes, 1973) and contains many saprophytes that could compete w i t h P_. w e i r i i f o r occupation of the surface of the host r o o t s , and p o s s i b l y replace P_. w e i r i i a f t e r i t had become e s t a b l i s h e d . S i t e : R e s u lts are presented i n Table 6. (a) Ten weeks: V i a b i l i t y of inoculum blocks a f t e r ten weeks i n the s o i l 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 s i t e s . However, the frequency of crustose mycelia on blocks on the x e r i c s i t e was s i g n i f i c a n t l y lower than that on the other two s i t e s . The same trend was observed f o r c o l o n i -z a t i o n of host r o o t s . Without m y c e l i a l development away from the surface of the inoculum b l o c k , no contact can be made between the fungus and the root surface and hence no c o l o n i z a t i o n occurs. (b) Twenty-six weeks: No s i g n i f i c a n t d i f f e r e n c e s were observed f o r the c h a r a c t e r i s -t i c s measured at twenty-six weeks i n T r i a l 2 between the three s i t e s used. TABLE 6. Frequency of zone l i n e s , crustose mycelia, c o l o n i z a t i o n and necrosis at ten and twenty-six weeks f o l l o w i n g i n o c u l a t i o n w i t h J?. w e i r i i on roots of D o u g l a s - f i r and western hemlock w i t h respect to ecosystem u n i t used, UBC Research F o r e s t , May 1978. Number of inoculum blocks w i t h : 1 „. 2 Zone l i n e s Crustose mycelia V i a b l e inoculum C o l o n i z a t i o n of host roots T o t a l V i a b l e inoculum blocks N e c r o t i c bark at i n o c u l a t i o n TEN WEEKS: X e r i c Submesic Hygric (10)" (40) (20) 10a 34b 20a 5c (39) 33a 17ab (8) 5a (32) 22a (19) 17a l b 15a 9a l a 8b" 7a TWENTY-SIX WEEKS: X e r i c Submesic Hygric (20) (76) (40) 20a 7 lab 40a 10c 49c 26bc 16a 48a 28a 3b 15b 8ab 2a 15a 8a 0 9 6 Crustose mycelia always a s s o c i a t e d w i t h zone l i n e s , except i n one instance. Blocks c o n t a i n i n g v i a b l e P_. w e i r i i a l s o contained zone l i n e s , except i n one instance. Number of observations unless r e s u l t s accompanied by a number i n brackets. Values f o l l o w i n g by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l (Comparison of pr o p o r t i o n s , Choi, 1978, pp. 101). One inoculum block was not t e s t e d f o r the presence of P_. w e i r i i . - 26 -(c) Comparison of r e s u l t s between ten and twenty-six weeks: There was a s i g n i f i c a n t r e d u c t i o n i n the number of c o l o n i z a -t i o n s observed on the submesic s i t e (5% l e v e l ) and the h y g r i c s i t e (10% l e v e l ) between ten and twenty-six weeks a f t e r establishment of the i n o c u l a -t i o n s . V i a b i l i t y of inoculum blocks a s s o c i a t e d w i t h c o l o n i z a t i o n of host roots d i f f e r e d a l s o between the two observation p e r i o d s . At twenty-six weeks a l l but one of the inoculum blocks a s s o c i a t e d w i t h c o l o n i z a t i o n of roots were v i a b l e , whereas only 16 of 24 inoculum blocks a s s o c i a t e d w i t h c o l o n i z a t i o n at ten weeks were v i a b l e . Blocks i n which v i a b l e P_. w e i r i i could not be detected, o r i g i n a t e d from the Haney source. I t would appear that f o r s u c c e s s f u l colonization^^tl^at the inoculum must remain v i a b l e u n t i l the fungus has become e s t a b l i s h e d i n the host r o o t . The twenty i n o c u l a t i o n s were removed seven weeks a f t e r establishment. A l l had developed mycelia onto the host r o o t s . The amount v a r i e d from one or two small pockets two m i l l i m e t r e s i n diameter to instances where the inoculum b l o c k was f i r m l y attached t o the host root along the e n t i r e l e n g t h and width of the block. N e c r o t i c bark was observed on three D o u g l a s - f i r and f i v e western hemlock r o o t s . T r i a l 3: This t r i a l showed f u r t h e r , that given s u i t a b l e moisture c o n d i t i o n s , e x c e l l e n t r e s u l t s can be obtained using the i n o c u l a t i o n procedure used throughout t h i s study. LABORATORY: Rate of P_. w e i r i i growth on agar media: The r a d i a l growth of mycelia c u l t u r e d from the Haney and Surrey sources was measured on media c o n t a i n i n g 2% malt and e i t h e r 2% or 5% agar, r a i s e d i n l i g h t or dark c o n d i t i o n s . The r e s u l t s are presented i n Table 7. The r a d i a l growth of c u l t u r e s grown i n the dark was s i g n i f i c a n t l y greater f o r both sources than those c u l t u r e s r a i s e d i n continuous l i g h t , and there were al s o d i f f e r e n c e s i n appearance. Dark grown c u l t u r e s e x h i b i t e d r a i s e d , evenly textured white to cream coloured a e r i a l hyphae w h i l s t mycelia on the l i g h t grown c u l t u r e s were not elevated and showed a mottled appearance due to the presence of brown s e t a l hyphae (Figure 3). There i s no ex p l a n a t i o n f o r the d i f f e r e n c e s i n m y c e l i a l appearance other than a response to l i g h t i nducing the production of s e t a l hyphae. 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 r a t e of r a d i a l growth between the two sources of mycelia when r a i s e d on the same medium i n the dark. However, when r e s u l t s are compared between the two media used, growth on the 5% agar was s i g n i f i c a n t l y greater than on 2% agar. The trends observed f o r r a d i a l growth of l i g h t grown c u l t u r e s d i f f e r e d from the dark grown ones. Growth on 5% agar was s i g n i f i c a n t l y l e s s than that observed on 2% agar. Also s i g n i f i c a n t was that the r a t e of growth of the Haney source was greater on both media than that of the Surrey source. The reason f o r the r e v e r s a l of trends f o r r a d i a l growth on the two media f o r dark versus l i g h t c o n d i t i o n s i s not obvious. S i m i l a r l y , why the Haney source should grow more r a p i d l y than the Surrey source on the same medium i n the l i g h t i s unknown. - 28 -TABLE 7. L i n e a l growth of P_. w e i r i i from two sources r a i s e d on media co n t a i n i n g e i t h e r 2% or 5% agar under c o n d i t i o n s of continuous l i g h t or dark. Source of P. w e i r i i Agar L i n e a l growth i n m i l l i m e t r e s per day medium Dark grown L i g h t grown 1 Haney Haney Surrey Surrey 2% 5% 2% 5% 4.73b 5.29a 4.70b 5.16a 3.07c 2.35e 2.79d 2.09f Mean of s i x r e p l i c a t e s . ^Means followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l (Duncan's M u l t i p l e Range Tes t ) . Figure 3. Comparison of the a e r i a l hyphal c h a r a c t e r i s t i c s of P. w e i r i i c o l l e c t e d from Haney and r a i s e d on 2% malt-agar f o r 16 days under c o n d i t i o n s of continuous l i g h t (upper p l a t e s ) or darkness (lower p l a t e s ) . - 30 -The reason f o r using media with two agar concentrations was to com-pare the e f f e c t of moisture a v a i l a b i l i t y from the s u b s t r a t e on the growth of P_. w e i r i i . The only c o n c l u s i o n i s that under dark c o n d i t i o n s both sources are s t i m u l a t e d on the medium con t a i n i n g the higher agar concentra-t i o n and retarded on the media c o n t a i n i n g the lower agar c o n c e n t r a t i o n , and that the reverse occurs i n l i g h t c o n d i t i o n s . Edgecombe (1941) examined the r a t e of growth of s i x wood-inhabiting basidiomycetes on media composed of sucrose, potato e x t r a c t , sodium c h l o r i d e and peptone together w i t h agar concentrations of one, two and three percent. Edgecombe found that the fungal growth increased as agar c o n c e n t r a t i o n was reduced, and was independent of the l i g h t i n g regime. R e s u l t s presented here f o r l i g h t r a i s e d c u l t u r e s agree w i t h Edgecombe's f i n d i n g s but disagree w i t h respect to the e f f e c t of l i g h t i n g . The relevance of exposure to l i g h t i s questionable. Under normal c o n d i t i o n s P_. w e i r i i grows i n the dark and i s not v i s i b l e above ground except when f r u i t i n g . I n t e r a c t i o n between P_. w e i r i i sources on agar media: A r e a c t i o n between the two inoculum sources when grown i n c l o s e p r o x i m i t y was evident. C u l t u r e s r a i s e d i n the l i g h t e x h i b i t e d a c l e a r zone devoid of hyphae between the advancing f r o n t s from each source. The r e a c t i o n observed i n the dark was more pronounced. In c u l t u r e s c o n t a i n i n g 5% agar, the advancing f r o n t s of each source stopped ab r u p t l y when they met. A t h i n l i n e separated the two sources and a d i s t i n c t brown l i n e was evident when the p l a t e s were viewed from beneath (Figure 4). The r e a c t i o n between the two sources was more pronounced on c u l t u r e s r a i s e d on media c o n t a i n i n g 2% agar. The a e r i a l hyphae at the boundary of Figure 4b. Bottom view. Figure 4. Reaction between the advancing f r o n t of the Haney source ( l e f t of p l a t e s ) and the Surrey source ( r i g h t of p l a t e s ) of P_. w e i r i i dark grown on 5% agar-2% malt f o r 18 days. Note that no reac-t i o n occurs when two samples of the Haney source (top l e f t ) or the Surrey source (top r i g h t ) were r a i s e d together. - 32 -the two sources were brown and microscopic examination showed that these were s e t a l hyphae. As shown i n Figure 5a, s e t a l hyphae of t e n extended over the e n t i r e surface of the a e r i a l hyphae from the Haney source. The under s i d e of the p l a t e s showed a more d i s t i n c t boundary between the two sources (Figure 5b) than was observed on the medium c o n t a i n i n g 5% agar. S e t a l hyphae were i d e n t i f i e d i n the boundary zone submerged i n the agar. These r e s u l t s i n d i c a t e i n c o m p a t i b i l i t y between the two sources. When cores of the same source were placed on the same medium no r e a c t i o n occurred when the advancing f r o n t s met (Figures 4 and 5). D i f f e r e n c e s between sources of P_. w e i r i i have been p r e v i o u s l y reported (Buckland et^ a l . 1954, C h i l d s , 1963). I t should be noted that these types of i n t e r -a c t i o n s on c u l t u r e media can be observed between P_. w e i r i i i n o c u l a from i n f e c t i o n centres i n c l o s e p r o x i m i t y to each other ( C h i l d s , 1963). Zone L i n e s : Zone l i n e s are composed of thickened hyphae accompanied by dark pigments i n the c e l l s of wood i n f e c t e d by many basidiomycetes and some ascomycetes. They are formed by the fungus to act as a b a r r i e r between i t s e l f and the e x t e r n a l environment. Zone l i n e s can form r a p i d l y . For instance when A r m i l l a r i a mellea (Vahl.) Quel, i s r a i s e d i n sawdust c u l t u r e , zone l i n e s form i n two to three days (Lopez-Real, 1975). Zone l i n e s i n wood i n f e c t e d by P_. w e i r i i can be induced by s e v e r a l f a c t o r s i n c l u d i n g temperature, s o i l moisture and s o i l m i c r o f l o r a (Nelson, 1973). Zone l i n e s were found i n many inoculum b l o c k s and at the c o n c l u s i o n of T r i a l 2 a l l blocks that contained P_. w e i r i i showed zone l i n e s . However, zone l i n e s do not guarantee r e t e n t i o n of v i a b i l i t y . In many in s t a n c e s , P_. w e i r i i could not be detected from blocks c o n t a i n i n g zone l i n e s . Another f e a t u r e - 33 -Figure 5a. Top view. Figure 5b. Bottom view. Figure 5. Reaction between the advancing f r o n t of the Haney source ( l e f t of p l a t e s ) and the Surrey source ( r i g h t of p l a t e s ) of P_. w e i r i i dark grown on 2% agar-2% malt f o r 18 days. Note that no reac-t i o n occurs when two samples of the Haney source (top l e f t ) or the Surrey source (top r i g h t ) were r a i s e d together. - 34 -was that i n a l l but one instance where c o l o n i z a t i o n occurred at twenty-six weeks, the inoculum blocks contained zone l i n e s and v i a b l e P_. w e i r i i . F r e s h l y exposed surfaces of root s e c t i o n s and inoculum bl o c k s con-t a i n i n g J?. w e i r i i placed over water i n a constant temperature c a b i n e t , developed e i t h e r crustose mycelium or zone l i n e s j u s t below the surface i n such a manner that the two s t r u c t u r e s formed a continuous l a y e r . No instances were observed where mycelia developed beyond a p r e v i o u s l y formed zone l i n e . This observation was a l s o noted i n the f i e l d and r a i s e s questions about the r e - i n f e c t i o n of new stands of D o u g l a s - f i r growing on o l d P_. w e i r i i i n f e c t i o n centres. P_. w e i r i i can remain v i a b l e i n dead root systems f o r at l e a s t 50 years (Buckland e_t a l . ^ 1954, W a l l i s and Reynolds, 1965) but the mycelia are o f t e n surrounded by sound wood h e a v i l y impregnated w i t h r e s i n , w i t h no i n d i c a t i o n that P_. w e i r i i could invade t h i s zone. In a d d i t i o n the margin of v i a b l e P_. w e i r i i i s f r e q u e n t l y marked by zone l i n e s (Hansen, 1976), probably making the J?. w e i r i i w i t h i n these b a r r i e r s i n e f f e c t i v e i n c r e a t i n g new i n f e c t i o n s i n the regenerating f o r e s t . Hence, the p o t e n t i a l f o r new i n f e c t i o n s would depend on the l o n g e v i t y of e c t o t r o p h i c mycelia a s s o c i a t e d w i t h the bark. This period can exceed 20 years (Hansen, 1976). The spread of _P. w e i r i i i n the absence of e c t o t r o p h i c mycelia i s d o u b t f u l . Roots from the advancing regeneration would have to penetrate the wood and zone l i n e s surrounding the r e s i d u a l P_. w e i r i i or produce an exudate that would s t i m u l a t e the advance of J?. w e i r i i towards the healthy r o o t . In both i n s t a n c e s , the zone l i n e b a r r i e r surrounding v i a b l e J?. w e i r i i would be broken a l l o w i n g p o s s i b l e replacement by other saprophytes and l i m i t i n g the p o s s i b i l i t y of new i n f e c t i o n . - 35 -CONCLUSIONS: 1. Root m a t e r i a l n a t u r a l l y i n f e c t e d w i t h P_. w e i r i i i s a s a t i s f a c t o r y inoculum f o r a r t i f i c i a l i n o c u l a t i o n of healthy roots of Douglas-f i r and western hemlock. High r a t e s of i n f e c t i o n w i t h such inoculum were only achieved during moist c o o l weather as experienced during the s p r i n g and autumn of 1978 at the UBC Research F o r e s t , Maple Ridge. D i f f e r e n c e s i n behavior were observed between P_. w e i r i i c o l l e c t e d from Haney and Surrey. D i f f e r e n c e s included i n c o m p a t i b i l i t y when the two sources were r a i s e d together on agar media and longer r e t e n t i o n of v i a b l e P_. w e i r i i i n inoculum b l o c k s from the Surrey source. - 36 -BIBLIOGRAPHY: Anon. 1975. F o r e s t r y handbook f o r B r i t i s h Columbia. 3rd Ed. The F o r e s t r y Club, UBC. Baxter, D.V. 1933. Some resupinate polypores from the region of the Great Lakes. V. Pap. Mich. Acad. S c i . , A r t s , and L e t t e r s 19: 305-332. Buckland, D.C, Molnar, A.C. and W i l l i s , G.W. 1954. Yellow laminated root r o t of D o u g l a s - f i r . Can. J . Bot. 32: 69-81. C h i l d s , T.W. 1963. P o r i a w e i r i i root r o t . Phytopath. 53: 1124-1127. : and Shea, K.R. 1967. Annual l o s s e s from disease i n P a c i f i c Northwest f o r e s t s . USDA For. Serv. Resource B u l l . PNW 20. Choi, S.C. 1978. Introductory a p p l i e d s t a t i s t i c s i n science. P r e n t i c e - H a l l , New Jersey. Edgecombe, A.E. 1941. The growth of s e v e r a l wood-inhabiting f u n g i . Phytopath. 31: 825-831. Hansen, E.M. 1976. Twenty-year s u r v i v a l of P h e l l i n u s (Poria) w e i r i i i n D o u g l a s - f i r stumps. Can. J . For. Res. 6: 123-128. K l i n k a , K. 1976. Ecosystem u n i t s , t h e i r c l a s s i f i c a t i o n , i n t e r p r e t a t i o n and mapping i n the U n i v e r s i t y of B r i t i s h Columbia Research Forest. Ph. D. Thesis, F a c u l t y of F o r e s t r y , UBC. K r a j i n a , V.J. 1965. B i o g e o c l i m a t i c zones and biogeocoenoses of B r i t i s h Columbia. Ecology of Western North America. 1: 1-17. Lopez-Real, J.M. 1975. Formation of p s e u d o s c l e r o t i a ("zone l i n e s " ) i n wood decayed by A r m i l l a r i a mellea and Stereum hirsutum. I. Morphological aspects. Trans, of the B r i t . Mycol. Soc. 64: 465-471. M u l l i c k , D.B. 1969. Reddish purple pigments i n the secondary periderm t i s s u e of western North American c o n i f e r s . Phytochem 8: 2205-2211. 1975. A new t i s s u e e s s e n t i a l to n e c r o p h y l a c t i c periderm formation i n the bark of four c o n i f e r s . Can. J . Bot. 53: 2443-2457. 1977. The n o n - s p e c i f i c nature of defense i n bark and wood during wounding i n s e c t and pathogen a t t a c k . Recent Adv. i n Phytochem Vo l 11: 395-441. and Jensen, G.D. 1973. C r y o f i x a t i o n r e v e a l s uniquemess of reddish-purple sequent periderm and equivalence between brown f i r s t brown sequent periderms of three c o n i f e r s . Can. J . Bot. 51: 135-143. Nelson, E.E. 1973. Observations on the formation of zone l i n e s i n wood by P o r i a w e i r i i . USDA For. Serv. Res. Note. PNW 210. - 37 -. and Hartman, T. 1975. Est i m a t i n g spread pf P o r i a w e i r i i i n a h i g h - e l e v a t i o n , mixed c o n i f e r stand. J of F o r e s t r y 73: 141-142. Soo, B.V.L. 1977. General occurrence of ex o p h y l a c t i c and n e c r o p h y l a c t i c periderm and non-suberized t i s s u e s i n wood p l a n t s . Ph. D. F a c u l t y " of F o r e s t r y , UBC. Spurr, S.H. and Barnes, B.V. 1973. Forest Ecology. 2nd Ed. Ronald. New York. W a l l i s , G.W. 1961. I n f e c t i o n of Scots pine roots by Fomes annosus. Can. J . Bot. 39: 109-121. 1967. P o r i a w e i r i i root r o t . In: Important f o r e s t i n s e c t s and diseases of mutual concern to Canada, the United States, and Mexico. Dept. For e s t , and Ru r a l Devel., Canada, Publ. 1180, pp. 204-206. 1976a. Growth c h a r a c t e r i s t i c s of P h e l l i n u s (Poria) w e i r i i i n s o i l and on root and other surfaces. Can. J . For. Res. 6: 229-232. 1976b. P h e l l i n u s (Poria) w e i r i i root r o t . D e t e c t i o n and manage-ment proposals i n D o u g l a s - f i r stands. Environment Canada, For. Serv., F o r e s t r y Tech. Rep. 12. and Reynolds, G. 1962. I n o c u l a t i o n of D o u g l a s - f i r r o o t s w i t h P o r i a w e i r i i . Can. J. Bot. 40: 637-645. 1965. The i n i t i a t i o n and spread of P o r i a w e i r i i root r o t of D o u g l a s - f i r . Can. J . Bot. 43: 1-9. 1975. Mass i n o c u l a t i o n of D o u g l a s - f i r seedlings w i t h P h e l l i n u s (Poria) w e i r i i (Murr.) G i l b e r t s o n . Can. J . For. Res. 5: 741-742. - 38 -APPENDIX 1. TABLE 1. Vegetation d e s c r i p t i o n of s i t e s used i n T r i a l 2 f o r i n o c u l a t i o n study, UBG Research F o r e s t , Maple Ridge. Ecosystem u n i t Species X e r i c Submesic Submesic Hygric Predominantly Predominantly D o u g l a s - f i r Western hemlock TREES Pseudotsuga m e n z i e s i i 8 8 5 7 Thuja p l i c a t a 4 4 4 4 Tsuga h e t e r o p h y l l a 5 5 8 8 SHRUBS AND HERBS Acer c i r c i n a t u m 3 Oplopanax h o r r i d u s 1 Vaccinium p a r v i f o l i u m 1 1 1 1 G a u l t h e r i a s h a l l o n 8 5 6 Polystichum munitum 1 3 Vaccinium o v a l i f o l i u m 1 Rubus s p e c t a b i l i s 1 Dry o p t e r i s a u s t r i a c a 1 Goodyera o b l o n g i f o l i a 1 T r i l l i u m ovatum 1 Achlys t r i p h y l l a 1 Menziesia f e r r u g i n e a 1 BRYOPHYTES Hylocomium splendens 5 4 4 3 Rhizomnium glabrescens 1 1 1 Plagiothecium undulatum 3 3 3 3 Rhytidiadelphus loreus 1 1 1 S t o k e s i e l l a oregana 3 3 3 3 Isothecium s t o l o n i f e r u m 1 1 Dicranum fuscescens 1 1 Species s i g n i f i c a n c e , t o t a l f o r s i t e : 1, sparse, .3-1%; 2, 1-2%; 3, 2-5%; 4, 5-10%; 5, 10-25%; 6, 25-33%; 7, 33-50%; 8, 50-75%; 9, 75+%. - 39 -APPENDIX 1. TABLE 2. Stem numbers per hectare f o r D o u g l a s - f i r , western hemlock and western red cedar on s i t e s used i n T r i a l 2, f o r i n o c u l a t i o n study, UBC Research Fo r e s t , Maple Ridge. Figures c a l c u l a t e d from 30m x 10m p l o t s at each s i t e . Stems per hectare Species X e r i c Submesic Submesic Hygric Predominantly Predominantly D o u g l a s - f i r Western hemlock D o u g l a s - f i r dominant-codominant 1000 suppressed 100 Western hemlock dominant-codominant 100 suppressed 0 Western red cedar dominant-codominant 433 intermediate 900 suppressed 800 TOTALS 3333 634 467 367 0 0 0 100 767 300 100 133 167 133 133 67 0 0 0 233 67 33 1200 1567 934 APPENDIX 2. Extent of c o l o n i z a t i o n and root n e c r o s i s , and inoculum block c h a r a c t e r i s t i c s of seventy i n o c u l a -t i o n s made on D o u g l a s - f i r (DF) and western hemlock (WH), ten weeks a f t e r i n o c u l a t i o n w i t h P h e l l i n u s w e i r i i on x e r i c , submesic and hyg r i c s i t e s i n the UBC Research Forest, Maple Ridge. C o l o n i z a t i o n of host roots S i t e - s p e c i e s Inoculum Zone l i n e s Crustose V i a b l e V i a b l e N e c r o t i c combinations source present i n mycelia inoculum T o t a l inoculum bark at blocks on blocks blocks blocks i n o c u l a t i o n X e r i c DF Haney 5 5 3 1 1 1 Surrey 5 0 2 ( 3 ) 1 0 0 0 Submesic DF Haney 2 5 2 2 0 0 Surrey 5 3 2 2 2 0 Submesic DF Inoculated WH Haney 5 5 2 1 0 1 Surrey 4 4 3 1 1 0 Submesic WH Haney 5 5 5 3 3 0 Surrey 5 5 3 (3) 2 I 2 0 Submesic WH Inoculated DF Haney 4 4 (4) .2 4 1 1 Surrey 4 (4) 2 3 0 0 0 Hygric DF Haney 5 5 4 (4) 3 3 0 Surrey 5 3 5 0 0 0 Hygric WH Haney 5 5 3 3 1 0 Surrey 5 4 5 3 3 1 TOTALS 64 55 44 25 16 4 '''Number of observations i f l e s s than f i v e . 2 One inoculum block not tested f o r presence of P . . w e i r i i . APPENDIX 3. Extent of c o l o n i z a t i o n and root n e c r o s i s and inoculum block c h a r a c t e r i s t i c s of 136 i n o c u l a t i o n s made on D o u g l a s - f i r (DF) and western hemlock (WH), twenty-six weeks a f t e r i n o c u l a t i o n w i t h P h e l l i n u s w e i r i i on x e r i c , submesic and h y g r i c s i t e s i n the UBC Research Forest, Maple Ridge. S i t e - s p e c i e s Inoculum Zone l i n e s Crustose V i a b l e C o l o n i z a t i o n of host roots N e c r o t i c combinations source present i n mycelia inoculum T o t a l inoculum bark at blocks on blocks blocks blocks i n o c u l a t i o n X e r i c DF Haney 10 8 7 2 1 0 Surrey 10 2 9 1 1 1 Submesic DF Haney 7 4 0 0 0 0 Surrey 10 5 10 6 6 5 Submesic DF Inoculated WH Haney 9 6 1 0 0 0 Surrey 9 4 10 1 1 1 Submesic WH Haney 10 10 9 3 3 2 Surrey 10 7 7 3 3 1 Submesic WH 1 8 Inoculate DF Haney (8) 8 6 2 2 0 Surrey (8) 8 5 5 0 0 0 Hygric DF Haney 10 10 5 2 2 1 Surrey 10 5 6 0 0 0 Hygric WH Haney 10 9 9 5 5 4 Surrey 10 2 8 1 1 1 TOTALS 131 85 92 26 25 15 Number i n brackets represents number of observations i f l e s s than ten. 

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